MICROBIOLOGY

Lecture 1:
Introducti
on
HISTORY
Name of Scientist Discovery
Alexander Fleming ✔ Discoverer of antibiotic Penicillin from penicillium notatum, an extract
from a mold known as Penicillium chrysogenum
✔ Observed zone of clearing
✔ Penicillin was first used for the inhibition of laboratory bacteria and
after 30 years it is used as an antibiotic
Anton Von Leeuwenhoek ✔ Father of Protozoology and Bacteriology
✔ Discovered and observed “animalcules” – small moving organism
✔ Develop the first simple microscope
✔ “Animalcules” were observed from rainwater samples, feces, and
teeth scrapings
✔ “Spontaneous Generation Theory” – forms of life could arise
spontaneously from nonliving matter
Robert Hooke (1665) ✔ Observation of small boxes known as cells
✔ “Cell Theory” – all living things are composed of cells
Aristotle ✔ Responsible for uncoiling the spontaneous generation theory

Spontaneous generation theory – organisms originated from non-

living things which came from the observation of a lake
o during dry season when the lake is empty, it contains no fishes
and during rainy season, small fishes starts to live.
o Observed a file of garbage, small flies were flying on top of
garbage file and he believed that the flies are from the file of
garbage

✔ invertebrates arise from spontaneous generation

Carlos Juan Finlay (1881) ✔ Discovered the transmission of Yellow Fever through Aedes aegypti
Edward Jenner ✔ Prophylaxis for small pox via injecting an individual with the plasma of
infected cow with small pox
✔ (May 4, 1796)
✔ Variola minor and Variola major
Elie Metchnikoff (1884) ✔ Discovered phagocytosis
Emil von Bering ✔ Vaccination for Diphtheria and Tetanus
Fanny Hesse (1882) ✔ Use of agar as culture media to replace gelatin
Fransesco Redi ✔ disproved spontaneous theory
o according to him, fly do not originate from non-living object
o proved that fly comes from the eggs laid by mother fly harboring
on the file of garbage

“Meat Broth Experiment”

a. Prepare two jars with decaying meat
b. First jar is left unsealed, flies laid eggs in the meat which
 developed into larva
c. Second jar is sealed, flies could not lay eggs on the meat

“Modified Meat Broth Experiment”

a. Prepare two jars with decaying meat
b. First jar is left unsealed, flies laid eggs in the meat which
developed into larva
c. Second jar is covered with fine gauze, flies could not lay eggs on
the meat
Fransesco Stelluti ✔ Observed veins of bees and wasps in their wings using a microscope by
Galileo
Ignaz Semmelweis (1840s) ✔ Father of Handwashing
John Needham ✔ “Vital Force Theory”
o Living objects originate from other carbon-containing sources

✔ Meat broth experiment

John Tyndall ✔ Tyndallization – method of fractional sterilization
o Fractional sterilization – series of sterilization technique
o Performed with the use of Arnold’s sterilizer
Joseph Lister ✔ Antiseptic technique
✔ Introduce the first antiseptic material – carbolic acid, replaced by
merthiolate
o Both carbolic acid and merthiolate contains a very large amount of
mercury, these two were removed from the market
Lazzaro Spalanzani ✔ Improved the heating broth experiment of John Needham
Louis Pasteur ✔ Air contamination
- “Swan-neck Flask or S Flask” – allowed air to pass through the
flask, but any organism present in air was trapped
✔ Mircobes are also present in non-living organisms
✔ Vaccination
✔ Discover the presence of a contaminant
✔ Microorganism such as pathogenic organism may also be seen in non-
living objects which are called fomites
✔ Able to purify plasma to be converted into a vaccine
✔ Discovered “fermentation”
- Conversion of sugars into alcohol due to the absence of air by
yeasts
a. Pasteurization” – heating of beer and wine just to kill bacteria
causing spoilage ; wet sterilization
Lucretius and Fracastoro ✔ Invisible organisms
✔ These two lived prior to the discovery and development of compound
microscope
Paul Ehrlich ✔ Salvarsan (Syphilis)
o Arsenic extends the life of syphilitic patients
o Discovered the “magic bullet/ Salvarsan/606 drug”, a drug used
to treat syphilitic patients.
o “Salvarsan” – stands for Salvation from syphilis with Arsenic
Rebecca Lancefield (1934) ✔ Streptococcal antigens to classify Streptococcaceae
Richard Petri ✔ One of the assistant of Robert Koch
 ✔ Petri Dish – glassware as a vessel for agar or culture media prior to
inoculation ; contains a larger cover to allow the entry but prevent the
entry of air contaminant
- Before a culture dome was utilized
Robert Koch ✔ Bacteria causes diseases
✔ Discovered Mycobacterium tuberculosis which causes pulmonary
tuberculosis via Koch’s Postulate
“Koch’s Postulates” – framework for the study of infectious diseases
1. The same pathogen must be present in every case of disease
2. The pathogen must be isolated from the diseased host and grown
in pure culture
3. The pathogen from the pure culture must cause the disease when
it is inoculated into a healthy, susceptible laboratory animal
4. The pathogen must be isolated from the inoculated animal and
must be shown to be the original organism
o Discovered that diseases actually originate to certain
organisms
o A specific bacterial cell may cause a specific infection
✔ First to culture organisms from potato, gelatin and meat
✔ Utilize a naturally derived media → cornstarch and gelatin
- Agar: a solidifying component in a media
✔ Developed culture media for isolated organisms
✔ Discoverer of Bacillus anthracis, a zoonotic pathogen infecting cattle

2 Bacteria That are Spore forming:

a. Bacillus – aerobic spore-forming ; fried rice
b. Clostridium – non-aerobic spore-forming ; can good
Rudolph Virchow ✔ Challenged biogenesis theory
“Biogenesis Theory” – cells arise only from pre-existing level cells
Theodore Schwann ✔ Flaming – heating of the mouth of a test tube prior to inoculation to
avoid the entry of air contaminant

TAXONOMY – Orderly classification and grouping of organisms into categories (taxa)

Classification - Is a method for organizing microorganisms into groups or taxa based on similar
morphologic, physio- logic, and genetic traits. he hierarchical classification system consists of the
following taxa designations:
Levels of Classification:
1. Domain: Bacteria
2. Kingdom: Bacteria or Monera
3. Division/Phylum: Proteobacteria
4. Class: Gammaproteobacteria
5. Order: Enterobacterales
6. Family Enterobacteriaceae
7. Tribe: Escherichia
8. Genus: Escherichia
9. Species: coli
10. Subspecies or Serotypes (e.g.: Escherichia coli)

Rules in naming Bacteria:

1. The family name is capitalized and has “-aceae” ending
Eg.: Micrococcaceae, Streptococcaceae, Enterobacteriaceae
- One exception to the rule in microbiology is the family Enterobacteriaceae; it is named after
the “enteric” group of bacteria rather than the type species Escherichia coli.
2. The genus name is capitalized followed by the species starting with a lowercase letter
Eg. : Streptococcus pyogenes, Streptomyces erytheus, Escherichia coli
- Each species within a genus differs sufficiently to maintain its status as an individual species.
Placement of a species within a particular genus is based on various genetic and phenotypic
characteristics shared among the species.
3. The genus and species must be italicized (printed) or underlined when written
Eg. Streptococcus pyogens, Staphylococcus aureus
- Nomenclature is the naming of microorganisms according to established rules and
guidelines set forth in the International Code of Nomenclature of Bacteria (ICNB) or the
Bacterio- logical Code (BC).
- As more information is gained regarding organism classification and identification, a
particular species may be moved to a different genus or assigned a new genus name. he
rules and criteria for these changes are beyond the scope of this chapter, but such changes
are documented in the International Journal of Systemic and Evolutionary Microbiology.
4. The organism can be abbreviated using the first letter, or with the first syllable when two or more
genera begins in the same letter
Eg. S. aureus (Staphylococcus aureus), Strep. pyogenes (Streptococcus pyogenes)

Lecture 2:
Eukaryote
and
Prokaryot
e

Cell Division
Characteristic Eukaryote Prokaryote
Eu → True ; Karyo → nut Released prior to the development
Contains a true nucleus attributed to of a nuclear membrane
the development of a nuclear First organisms (cyanobacteria)
membrane Pro→before
 Nuclear body ✔ Nucleus is bound to a ✔ Nucleoid is not bound to
membrane any membrane
✔ Contains DNA and Histones ✔ Has a single chromosome
only
Cell division ✔ Mitosis ✔ Binary fission
Cell wall ✔ No cell wall on animal cells ✔ Has a cell wall made up of
✔ Contains cellulose and chitin for peptidoglycan layer except for
plants and fungi mycoplasma and ureaplasma
- Chitin can be stained using - No distinct membrane
periodic acid Schiff or
hematoxylin
Cytoplasmic membrane ✔ Fluid phospholipid bilayer with ✔ Fluid phospholipid bilayer
protein and sterol without protein and sterol
except for ureaplasma it
contains a sterol
Cell organelle ✔ Present ✔ absent
Site of energy production ✔ Mitochondria ✔ Cytoplasmic membrane
Site of protein synthesis ✔ Rough endoplasmic ✔ Free ribosome
reticulum

CELL DIVISION
1) Mitosis – a process where a cell divides into identical two daughter
cells
a. Interphase – copying of DNA in preparation to cell division
producing two identical sets of chromosomes; microtubules
extend from the centrosome, which is made up of a pair of
centrioles
b. Prophase – condensing of chromosomes into X-shaped structures,
visible under the microscope
c. Metaphase – chromosome line up end-to-end along the equator of the cell
d. Anaphase – sister chromatids are pulled apart by mitotic spindle to end poles
- Mitotic spindles are the ones that become as the Cabot rings
 e. Telophase – each poles contains a full set of chromosome. A
membrane then creates a new nucleus around each set of
chromosome

2) Binary Fission – self generation ; no make-up necessary

a. Replication of the prokaryotic chromosome
b. Bacterial cell begins to elongate
c. Separation of duplicated chromosome continue to move
away from each other towards opposite ends of the cell
d. Formation of a septum at the center of the plasma
membrane
e. Pinching of bacterial cells into two

Note: the location of mesosome, septum is also observed

Note: palisade occurs because there is no cytoplasmic pulling
off

Parts of a Bacterial Cell

1. Cell Wall
− The function of a cell wall is to provide rigidity and strength to
the exterior of the cell. Most eukaryotic cells do not have cell
walls. However, fungi have cell walls principally made of
polysaccharides, such as chitin, mannan, and glucan. Chitin is a
distinct component of fungal cell walls.
− It is about 10 to 25 nm in thickness and shares 20 to 30 percent
of dry weight of the cells.
− Multi-layered structure comprising 20% of the total bacterial
weight
− Responsible for the shape of the cell and staining property of
the cell
− Prevents rupture of cell when the when the water pressure inside is greater than the outside of
the cell
− Point of anchor of the flagella
− Composed of “peptidoglycan/murein”
− Determinant of gram positive (thick peptidoglycan) and negative (thin peptidoglycan) or acid
fast staining or not based on the lipid component (e.g.: tubercular mycobacterium,
rhodococcum and legionella contains high lipid cell wall)
− Responsible for the antigenic determination (e.g.: S. aureus containing protein A)
Note: majority of organism contains cell wall but in the absence of cell wall, the laboratory may
create an artificial cell membrane using lysozymes and penicillin

Gram Positive Gram Negative

Peptidoglycan Peptidoglycan
Teichoic acid Lipoprotein
Phospholipid
Lipopolysaccharide
GRAM POSITIVE CELL WALL:
• The peptidoglycan layer of Gram-positive bacteria is much thicker (15 to 25 nm) than that in Gram
negative (10 to 15 nm).
• Specific components of Gram-positive cell wall include significant amount of teichoic and
teichuronic acids which are soluble polymer containing ribotol or glycerol polymers and maintain a
level of divalent cations outside cell mem- brane. The teichoic acids constitute major surface
antigens of Gram-positive bacteria.

GRAM NEGATIVE CELL WALL:

• Lipoprotein layer which connects outer membrane to peptidoglycan.
• Outer membrane is a phospholipid bilayer containing specific proteins. These specific proteins form
porins and hydrophilic molecules are transported through these porins. Other proteins are target
sites for phages, antibiotics and bacteriocins.
• Lipopolysaccharide consists of a complex lipid, called lipid A, to which is attached a polysaccharide.
Lipopolysaccharide is the endotoxin of Gram negative bacteria—The toxicity is associated with lipid
portion (lipid A) and the polysaccharide represents a major surface antigen, the O antigen.

✔ Abnormal Cell Walls:

o Protoplast – Gram positive bacteria; produced by lysozyme in hypotonic solution
✔ Unstable structures.
✔ Very sensitive to the influence of osmotic pressure, mechanical action and
aberration.
✔ Unable to synthesize the component parts of cell wall (diaminopimelic and
muramic acids).
✔ Resistant to phage infection.
✔ Not capable of active motility.
o Spheroplast – Gram negative bacteria; produced by penicillin in hypertonic solution

**Protoplast and spheroplast are spherical regardless of original shape of the bacterium.
Such organisms might have a role in certain persistent chronic infections such as
pyelonephritis

2. Cytoplasmic Membrane
Functions:
o Site of energy production
o Act as a staining determinant of a bacterial cell
o Anchors the flagella → pertains to a bacterial cell
o Act as an antigen
o It is thin semipermeable membrane which lies just beneath the cell wall. The whole bacterial
cytoplasm is bound peripherally by a very thin, elastic and semipermeable cytoplasmic
membrane also known as cell membrane.
o It is 5 to 10 nm in width.
o Electron microscope membrane shows the presence of three layers constituting a unit
membrane structure.
o Chemically the membrane consists of phospholipid with small amount of protein.
o Sterol is absent except in mycoplasma

3. Mesosomes
 − Point of attachment for the chromosome; also an indicator to where bacterial cell will divide
− Where septum formation occurs
− E.g.: a bacilli and a chromosome 🡪 during reproduction, the cell needs to divide identically.
During the process of binary fission, the chromosome material will attached itself to the
mesosoma

4. Inclusions – food reserves / storage

Common Bacterial Inclusions

Metachromatic Reserves of C. diphtheriae
granules / Babes-Ernst inorganic • Utilizes Pai media which is rich in inorganic
granules / volutin Phosphates phosphate to observe the babes-ernst granules
granules • Burke’s Modification of Gram stain may also be
used
Polysaccharide Contains glycogen P. aeruginosa
granules and starch • PAS, hematoxylin and carbohydrate stains such
as mucicarmine, carmine may be used for
observation
Lipids Bacillus, Mycobacterium, Spirillum, Azotobacter
Sulfur granules Sulfur for energy Thiobacillus, Nocardia, Actinomyces
Carboxysomes Ribulose Cyanobacterium, Thiobacillus
carboxylase
Gas vacuoles Cyanobacteria, Halobacteria
Magnetosome Iron oxide Magnetospirillum

5. Endospore
− Aka asexual spore → seen in a bacilli
− For protection of microorganism specially its genetic material from the harsh environment via
elongation
− Commonly seen in Gram positive organisms, Clostridia and Bacillus
➔ Coxiella burnetiii (Gram negative)
o Has an endospore-like structure that protects the organism from heat
− Made up of Calcium dipicolinate
− Seen in Clostridia and Bacillus
o Clostridium: anaerobic Bacillus: aerobic

− Stains include: Shaeffer and Fulton, Dorrer’s, Wortz and Conklin

o Location of Spores:
▪ Terminal Spore – C. tetani
▪ Subterminal Spore – C. botulinum
▪ Central Spore – B. anthracis

• Formation of endospore by Sporulation:

1. Spore septum begins to isolate newly replicated DNA
2. Plasma membrane starts to surround DNA
3. Spore septum surrounds isolated portion forming a forespore
4. Formation of spore coat
5. Endospore is freed

Note: the spore of a bacterial cell is not use as a means of reproduction but use as a
means of protection

Spore formation:
e.g.: bacilli → a bacilli contains a single chromosome.
During autoclaving or sterilization, the bacterial cells are
exposed in a very hot environment and the very first
reaction of bacterial cells is to protect themselves with
the use of a spore.
How do they protect, using a spore?
e.g.: mother chromosome is shorter. The mother
chromosome produces a daughter chromosome which
attracts calcium dipicholinate from its environment.
calcium dipicholinate will solidify and forms a spore
which is seen inside the bacterial cell. Inside the
bacterial cell spore is where the daughter chromosome
will be located. Once the daughter chromosome has
been protected and exposed into a hot temperature, the
mother chromosome will die but the daughter chromosome is still intact inside the
calcium dipicholinate shell and the entire bacilli will be protected

For short: formation of endospore originate from the heating of a bacterial cell →
upon heating, the bacterial cell chromosome will be triggered to produce a daughter
chromosome → daughter chromosome attracts calcium dipicholinate from the
environment → daughter chromosome will isolate inside the calcium dipicholinate
→ upon exposure to a high temperature, daughter chromosome will not be affected
but the mother chromosome will die and the bacterial cell will not be harmed
 Note: Intoxication occurs upon ingestion of a spore-forming bacteria resulting to
gastrocnemius caused by Bacillus cereus

6. Capsule (K antigen)
a. Mistaken as a slime layer
o Capsule → seen among pathologic meningitis organism
o Slime layer → seen in all microorganisms whether pathologic or not
b. Organized material attached to cell wall
c. Prevents phagocytosis
d. Immunogenic therefore, tested with Quellung reaction or capsular swelling (H. influenza, N.
meningitidis, S. pneumonia) by Dr. Fred Newfeld
o Quellung reaction or capsular swelling – immunologic test, wherein antibodies are used
against capsular antigen. When antibodies becomes attracted to the capsular antigen, the
capsule antigen will swell
Principle:
Anti-capsular antibodies present in the serum reacts with carbohydrate present in the
pneumococcal capsule, causing a microprecipitin reaction on the surface of the capsulated
organism. This antigen-antibody reaction causes a change in the refractive index of the
capsule so that it appears “swollen” and more visible.
e. Stained using Hiss stain and India ink
● India Ink – negative stain or background stain

OTHER FUNCTION OF CAPSULE:

- Capsulated bacteria are usually nonmotile as flagella remains un functional in the presence of
capsule.
- Development of capsule is dependent on the existence of favorable environmental conditions such
as presence of high sugar concentration, blood serum or growth in a living host.
- The capsules which are thinner than true capsules are called microcapsule, e.g. Meningococci,
Streptococcus pyogenes and Haemophilus influenzae. It may be composed of complex
polysaccharide (pneumococci and klebsiella) or polypeptide (Bacillus anthracis) or hyaluronic acid
(Streptococcus pyogenes).
- Capsules have no affinity for dyes and so they are not seen in stained preparations.

7. Slime Layer
a. Unorganized material
b. Present among all types of bacterial cell

8. Pili (Vi antigen)

- Fimbriae (fringes) are filamentous, short, thin, straight, hair-like appendage 0.1 to 1.5 µ long and
less than 4 to 8 nm thick.
- They are also called pili (hair).
- Fimbriae are seen only in some Gram-negative bacteria
- They are best developed in freshly isolated strains and in liquid culture.
- They tend to disappear following subcultures on solid media.
- Fimbriae are composed of protein known as pillin (molecular weight 18000 daltons).
a. Small appendages that can be found on the cell wall of bacterial cell
b. An immunogenic structure of bacterial cell
 Two Types:
a. Somatic pili/ ordinary pili for adhesion
● Present in the membrane of cells responsible for the adhesion to a host cell which is
attached in a mucous membrane (S. pyogenes)

b. Fertilizing/Sex pili/ Gene Transfer Pili

● Not responsible for sexual activities but is utilized for gene transfer of a certain
component from one bacterial cell to another
● They are associated with fertility (F+) and help in bacterial conjugation processes.
● They are longer (20 µ length) than common and Col I pili.
o E.g.: S. aureus vs. MRSA → both bumped into each other. S. aureus will
communicate with MRSA. The pili of MRSA and S. aureus will join and the
chromosome of MRSA will migrate from MRSA to S. aureus and will integrate to S.
aureus and S. aureus will become MRSA too. MRSA will detach from S. aureus and
MRSA will look for another S. aureus for another sharing.

Difference between the two:

e.g.: S. aureus vs. MRSA → MRSA is similar with S. aureus but differs with the MRSA
gene

Difference between a Flagella and Pili

Flagella: longer, for motility Pili: small

● Types of common fimbriae:

Type 1 fimbriae relatively thick and involved in hemagglutinating activity. They can be inhibited
by mannose (mannose sensitive).
Type 2 fimbriae are like type 1 fimbriae but they are non-hemagglutinating.
Type 3 fimbriae are thin and are mannose resistant. They confer hemagglutin ating activity on
parent cells.
Type 4 fimbriae thinner than type 3 fimbriae and are mannose resistant with hemagglutinating
activity on fresh red blood cells.
Type 5 fimbriae are monopolar which have been found only in one species of pseudomonas.
Type 6 fimbriae are very long and few of them are seen in one species of Klebsiella

9. Flagella (H antigen)
- Makes the cell motile ; seen in bacilli organisms only
- Organ of locomotion in bacterial cell
- Contains a protein known as flagellin
- Motility is seen in 25oC
- 37C is inhibitory for Lestiria and Yersinia
- can be stained using Leifson, Gray, Fisher and Conn
- These are extremely thin 12 to 30 nm, helical shaped structure of uniform diameter throughout
their length. These are 3 to 20 µ long.
- Each flagellum consists of hook and basal body. It originates in a spherical body (basal granule)
located just inside cell wall.
- These are antigenic and are composed of protein called flagellin which has properties of fibrous
 protein, keratin and myosin.
Part of a Flagella
1. Hook
2. Basal Body
3. Filament

Tests for detecting Flagella

1. Swarming phenomenon – observe among proteus organisms
2. SIM Media
3. Hanging Drop Method
4. Dark-field microscopy
5. Electron microscopy

Flagellar Arrangements
● Atrichous – no flagella
● Monotrichous – Flagellum in one pole
Eg. Pseudomonas aeruginosa & Vibrio cholerae

● Amphitrichous – single flagellum in each pole ;

Eg. Aquaspirillum serpens

● Lophotrichous – tuft of flagella at one or both sides

Eg. Pseudomonas fluorescens

● Peritrichous – flagella all over the organism

Eg. Proteus mirabilis, Escherichia coli & Salmonella typhi

● Periplasmic flagella/axial filament/Endoflagella for spirochete – modified flagella

Eg. Campilo bacter

Note: spirochete and spirilla is not the same, they differ on the type. Spirochete are larger
while spirilla do not usually harm human but are found in soil that harms the plants
Flagella Pili
Size Large because it is only one and it needs to push itself Small
Origin Cell membrane Cell wall
Locomotion + -
Adhesion - +
Conjugation - +

● Types of Movement: either a clockwise or a counterclockwise movement, depending on the cell’s

generation of energy.
❖ Run or Swim - counterclockwise rotation; one movement
❖ Tumble- clockwise rotation; periodic abrupt random movement changes
● Flagellar receptors
❖ Flagella moves towards an environment it has sensed; compare or sense the chemical
environment (temporal sensing)
❖ Absence of gradient - counterclockwise movement
 ❖ Presence of attractant gradient - changes motility; moves up the gradient; clockwise
movement
❖ Attractant - chemotactic signal allow the movement of the flagella
✓ Chemotaxis - movement towards a source of nutrients
✓ Phototaxis - movement towards a source of light
✓ Aerotaxis - movement towards a source of oxygen
❖ Phase variation - after the expressed antigenic type of flagella that they produce, making
detection and identification difficult.

10. Virulence Factor

- Components that will stimulate or cause the infection
- Different parts or substances of a bacterial cell which may cause an infection
‐ Pathogen- produced structure that allows the bacterial cell to invade or evade the immune
system and possibly cause disease.
❖ Adherence of bacteria to cells or mucosal surfaces
❖ Resist phagocytosis
❖ Resistance to bactericidal action
❖ Biofilms
▪ Protects the cells underneath
▪ Facilitates communication among microorganisms found within the biofilm
▪ Survival of the cells by attaching to various surfaces in its natural environment.
-
E.g.: Flagella→Enterobacteriaceae Slime layer→meningitis
a. Toxins
Characteristic Endotoxin Exotoxin
Source ✔ Gram negative ✔ Gram positive and Gram negative
Release ✔ Lysis of the cell ✔ Living cell therefore more toxic or
infectious
Composition ✔ Lipids ✔ Proteins/ peptides
Heat Stability ✔ Heat stable ✔ Heat labile except Staphylococcus
Immunogenecity ✔ Not converted to toxoids; can ✔ Converted to toxoids
be easily neutralized with ✔ Easily neutralized
antibodies
Toxicity ✔ Low toxicity ✔ Highly toxic
Lethal Dose ✔ Larger ✔ Smaller
Disease Associated ✔ UTI, Typhoid ✔ Tetanus and botulinum

● Limulus Lysate test – uses Horse shoe crab serum (Limulus polyphemus), with the clumping
of blood as a positive result ; a coagulation test
o Coagulates the blue blood of a horse shoe. A clump will indicate a positive for
endotoxin

Why horse shoe crab produces a blue color? Because instead of iron, it contains
 copper

Pathogenesis of Bacteria
A. Invasiveness
- Manifested by inflammation
- Ability of bacteria to remain

Factors for Invasiveness

1. Enzymes
- Speed up reaction or break down substances

a. Collagenase / Hyaluronidase
- Spreading factor ; destroys connective tissue thus causing spread of disease
- E.g.: S. pyogenes

b. Coagulase
- Catalyzes the conversion of fibrinogen to fibrin
- The fibrin formation provides protection to microorganism (S. aureus)

c. IgA Protease
- Degrades the secretory IgA (saliva, sweat, urine, breast milk)
- E.g.: Neisseria gonorrheae, H. influenzae, S. pneumoniae

d. Leukocidins
- Group of enzymes that kill lymphocytes
- Microorganism persist in the tissue
- Kills bacteria

2. Capsule
- Prevents phagocytosis ; antiphagocytic (S. pneumoniae and N. gonorrheae)

3. Cell Wall Proteins of Gram + cocci

a. M protein – seen in Group A Streptococci (pyogenes)
b. Protein A – seen in staphylococcus

B. Toxins
1. Exotoxins
- With high toxicity
- Seen in both gram (-) and (+) bacteria
- Secreted from the cell membrane
- Composed of polypeptides
- With various clinical effects
- Induces high titer antibodies
- Toxoids are produced as vaccines
- Most are heat labile
- Peptides and needs antibodies
a. Diphtheria Toxin
- Produced by Corynebacterium diphtheriae
- Produces diphtheria toxin which attaches ribose to ADP leading to death of cells to lack
protein synthesis
- Action: causes ADP ribosylation of EP2 (elongation factor needed for protein synthesis)

Note: when bacteria dies, P. seudomembranes accumulate to the respiratory system

resulting to death

- EF2 + NAD 🡪 EF2-ADP-ribose + nicotinamide + H+ (inactivate substances)

→ Stops protein synthesis
→ Result: composed of dead cells and debris

Pseudomembranes – composed of dead cells and debris

b. Tetanus Toxin
- Inhibits glycine by clostridium tetani
- A neurotoxin which affects the nerves
- Glycine inhibits acetylcholine for muscle to relax but without acetylcholine it contracts
- Action: inhibits action of the neurotransmitter glycine
- Effect: spasms occur because toxins inhibits the inhibitory transmitter glycine (e.g.:
sardonic smile)

c. Botulinum Toxin
- In clostridium botulinum
- The most potent toxin ; inhibits acetylcholine leading to paralysis
- Action: inhibits action of the facilitatory transmitter acetylcholine at the synapse
- Effect: patient experiences paralysis like difficulty in swallowing and breathing

d. Exotoxin – contains adenylate cyclase

i. Heat Labile
- In E. Coli causing diarrhea to the host
- Adenylate cyclase 🡪 C-AMP 🡪 Na+Cl- 🡪 H2O 🡪 watery stool
- Action: Toxin stimulates adenylate cyclase which increases the amount of cyclic-
AMP
- Effects:
1. Hypersecretion of Cl ions out of the cell, together with Na and H2O
2. outpouring of electrolytes happen in the intestines resulting to a watery stool
also in V. cholerae but more severe

ii. Heat stable

- In Bacillus cereus
- Guanylate cyclase is activated
- Guanylate cyclase 🡪 C-AMP 🡪 Na+Cl- → H2O 🡪 watery stool

e. Pertussis Toxin
- In Bordetella pertussis
- Causes whooping cough 🡪 whistling sound
 - Promotes ribosylation of ADP causing cell death and promotes lymphocytosis

f. Anthrax Toxin
- Used as a biological weapon ; Bacillus anthracis
- Can exist in spores but dies using autoclave

3 Factors:
1. Edema Factor – swelling of the skin
2. Protective Antigen
3. Lethal Factor – causes cell death

g. Toxic Shock toxin

- In S. aureus and streptococcus
- Seen in women using tampons
- Action: stimulates Class II MHC protein which activates CD4 cells which secretes IL-1 and
IL-2
- Effects: IL-1 is a vasodilator which causes fever hypotension or decrease pressure and
vasodilation or increase in diameter of blood vessels. Blood pressure falls as a result in
the dilation of blood vessels resulting to shock

h. Alpha toxin
- Potent ; Clostridium perfringens
- A phospholipase / lecithinase
- Also secretes collagenase, hyaluronidase, protease and DNAse
- Action: phospholipase destroys the cell membrane
- Effect: cell death

2. Endotoxin
- Found in the cell wall of gram – bacteria
- It is not secreted, the presence of the microbe causes the disease
- A lipopolysaccharide with low toxicity
- Symptoms are limited to shock and fever
- Poorly antigenic and heat stable
- No toxoids and no vaccines are derived
- Induces Tumor Necrotic Factor and IL-1

Effects:
1. Fever –Due to endogenous pyrogen which acts on the hypothalamus (thermoregulatory)
and an IL-1
2. Hypotension – Results to shock ; bradykinin causes vasodilation

3. Disseminated Intravascular Coagulation

- Action: activates the Hageman factor (F12) 🡪 activation of clotting, forming
microthrombi even in the absence of wound
- Result: consumptive coagulopathy because all factors will be used up and bleeding

Note: platelets are important for vascular integrity

 4. Complement Cascade – activates the alternative pathway
Macrophages Activation – for antibody production

Bacterial Genetics
Bacterial Chromosome
- Circular and double stranded attached to a bacterial cell membrane

Genetic Variations among Bacterial Cells

1. Mutation – due to chemical alteration of the DNA, usually heritable and irreversible
○ one genetic sequence has the problem
○ Mutations may be induced by mutagens (i.e., chemical or physical factors) in the
environment or by biologic factors, such as the introduction of foreign DNA into the cell.
○ The mutation may result in a noticeable alteration in the organism’s phenotype, and the
change may provide the organism with a survival advantage.
a. Substitution – a single base is changed
b. Deletion – a base is deleted
c. Insertion – addition of a base

2. Gene Transfer
a. Transformation
o Fragments of DNA is taken up by a recipient cell
o Can change the pathogenicity of the cell
o Can lead to a change in the antimicrobial susceptibility of a cell
o Certain bacteria are able to take up naked DNA from their surroundings; that is, they are
able to undergo transformation. Such bacteria are said to be competent. Among the
bacteria that cause human infections, competence is a characteristic commonly associated
with members of the genera Haemophilus, Streptococcus, and Neisseria.
o Competence- refers to the ability of the recipient cell to take up extracellular DNA from the
environment (only competent cells can be transformed)
b. Transduction
o A fragment of DNA is transferred from one organism to another by a bacteriophage
o Steps:
➢ Donor cell DNA packaged in bacteriophage
➢ Release of bacteriophage from donor cell
➢ Bacteriophage infects and releases donor DNA
○ The bacterial DNA may be randomly incorporated with viral DNA (generalized
transduction), or it may be incorporated along with adjacent viral DNA (specialized
transduction).
○ Method:
1. Lytic cycle (Generalized Transduction) - virulent phages; lyse or destroy the
chromosome of the donor cell; penetrate the recipient cells and replicate within and
lyse in order to be released.
2. Lysogenic cycle (Specialized transduction) - temperate phages; phages invade the host
but do not directly cause lysis; incorporate into the cell as prophages only specific gene
is transferred, thus a specific trait is expressed by the bacteria.
o Virulent phage - phages the are capable of causing infection and the destruction and death
of bacterial cell.
 o Temperate phage- phage DNA is incorporated into a bacteria’s DNA and is replicated with it.
c. Conjugation
o Plasmid DNA is transferred when a sex pilus from a donor bacterium comes in contact with a
recipient bacterium
o This process occurs between two living cells, involves cell-to-cell contact, and requires
mobilization of the donor bacterium’s chromosome.
o Two live bacterial cells come together and the donor cell directly transfer DNA to the
recipient cell
o Conjugation pilus (ex. E coli)
o Cell to cell contact
➢ F+ - donor cell
➢ F- - recipient cell
o F factor (f) - plasmid which contains about 100 genes which encodes for plasmid production.
o OriT - region where the plasmid is replicated
o High frequency of recombination (Hfr) strains - cell exhibiting the ability to donate
chromosomal genes; the F factor has attached to the chromosome.
d. Transposition
o Responsible for toxin production
o Process by which these genetic elements excise from one genomic location and insert into
another.
o Transposons carry genes that have products that help mediate the transposition process, in
addition to genes that encode for other accessory characteristics, such as antimicrobial
resistance.

Lecture 3:
Classificat
ion of
Bacteria
Classification of Bacteria
1. Morphology discovered by Antoine Val Leuwenhoek
1. Cocci – spherical microorganism
a. Diplococci – pairs of cocci
Eg.
Neisseria gonorrhoeae – catalase positive and gram-negative cocci ; an STI that
causes the clap or gonorrhea
o When an infected person has sexual intercourse with
another person, their genitalia will produce a clapping
sound
o N. gonorrhoeae is a fastidious bacteria therefore it requires
CAM + antibiotic to make it specific → Thayer-martin agar
Diplococcus pneumonia

b. Streptococci – chains of cocci

Eg.
Sreptococcus pyogenes
 Abiotrophiza defective

c. Staphylococci – a cluster or group of cocci

Eg.
Staphylococcus capitis
Staphylococcus lugdunensis
S. aureus – gram positive ; gold colony in loefler’s serum slant / LSS
o A chromogenic agar – a differential agar that help in identification of a certain
group of organisms according to the pigment they produce
o S. aureus is manifested after 16-18 hours of incubation only while 18-24 hours of
incubation the colony will appear white in MSA or Mannitol Salt Agar

d. Tetrads – tuft of four cocci arranged within the same plane

Eg.
Micrococcus luteus
Peptococcus niger (renamed as Peptostreptococcus)

e. Sarcina – tuft of cocci arranged in cube

Eg.
Sarcina lutea
Sarcina aurantiaca
Rhodococcus – acid fast cocci ; may change in morphology from cocci to bacilli after 24
hours of further incubation

2. Bacilli – elongated bacterial cell ; rod shaped

a. Diplobacilli – bacilli arranged side-by-side with each other
Eg.
Coxiella burnetti
Klebsiella rhinoscleromatis

b. Streptobacilli – bacilli arranged in chains

Eg.
Streptobacillus moniliformis
c. Coccobacillus – circular shaped bacilli
Eg.
Haemophilus influenzae
Chlamydia trachomatis
Gardnerella vaginalis – forms “clue cells” when it infects a squamous epithelia

d. Palisade – bacilli bent at the point of division

Eg.
Corynebacterium diphtheriae
Corynebacterium pseudodiphteriacum – normal flora of the throat

Lactobacillus – gram positive

- L. acidophilus – normal flora of female genitalia
o Boas-Opler: GIT
 o Doderlein’s Bacilli: female genitalia

- L. leishmania – for the diagnosis of megaloblastic anemia and estimation of

B12
- L. cassei – for estimation of folate acid

Escherichia coli – gram negative ; causative agent of UTI among children

Mycobacterium – acid fast bacilli
- M. tuberculosis – tubercle bacilli
- M. gordonae – tap water bacilli

3. Spiral
a. Vibrio – comma shaped organism
Eg.
Vibrio cholerae
Vibrio mimicus

b. Spirochete – helical in shape with flexible bodies that move with the use of axial filaments
(flagella of sphirochete) ; cannot be stained with gram and acid-fast stain instead use a metallic
stain such as Fontana Leva Dye Sliver Stain ; cannot be cultured
Eg.
Treponema pallidum
Borrelia recurrentis
Leptospira interogans

c. Spirillum – rigid spiral structure and does not contain any axial filament for locomotion
Eg.
Campylobacter jejuni
Helicobacter pylori
Spirillum minor

d. Filamentous – very thin with long filamentous bodies that may form a mycelium when grouped
together
Eg.
Candidatus liberibacter

e. Pleomorphic – do not have any characteristic shape

Eg.
Mycoplasma pneumoniae

f. Star-shaped
Eg.
Stella humosa
Stella

Note: Enterobacterales page 1144-1145 24th ed Henry’s Clinical Diagnosis and Management by
Laboratory Methods
Staining
1. Gram Staining (Hans Christian Gram, 1884)
- Develop by Hans Christian Grams
- Differential stain, a polychromatophilic stain
- A principal stain
- Based on the peptidoglycan layer
- Gram positive (thick) ; Gram negative (thin)
- most clinically significant bacteria are detected except:
● intracellular bacteria
● bacteria that lacks cell wall
● bacteria with insufficient dimensions to be resolved by light (i.e. spirochetes)
- provides preliminary diagnosis for initial treatment
- Principle:
o Gram + bacteria have thicker peptidoglycan layer (40) with numerous teichoic acid cross-
linkages than that of G (-) (1 or 2)
o Teichoic acid prevents decolorization
o Gram + bacteria may loose CW integrity by:
1. Antibiotic treatment
2. Old cells
3. Use of autolytic enzymes
- Once stained, the smear is examined using the low power or 40 objectives (400 magnification).
The microbiologist should scan the slide looking for white blood cells, epithelial cells, debris, and
larger organisms such as fungi or parasites.
- When clinical material is Gram stained (e.g., the direct smear), the slide is evaluated for the
presence of bacterial cells as well as the Gram reactions, morphologies (e.g., cocci or bacilli), and
arrangements (e.g., chains, pairs, clusters) of the cells seen
- Rule:
1. All cocci are Gram + except: Neisseria, Branhamella, Veilonella
2. All bacilli are Gram – except: Bacillus, Clostridium, Corynebacterium, Erysipelothrix,
Lactobacillus, Listeria, Mycobacterium

Gram’s stain
Reagent Function
Crystal violet Primary stain, stains all bacteria blue to purple
Gram’s Iodine Mordant, enhances reaction between cell wall and primary stain
Ethyl alcohol or Gram positive bacteria retain the primary stain because of the
acetone peptidoglycan and teichoic acid cross-links. Gram negative bacteria
lose the primary stain because of the large amount of
lipopolysaccharide in the cell wall
Safranin O or Counterstain, no effect on gram positive bacteria, stains gram
Carbolfuchsin negative bacteria pink to red

Accentuator – accelerate staining time

 Common Bacteria
Gram positive – bacterial cells that have a very thick peptidoglycan layer thus, they are very
difficult to decolorize ; blue or violet
Gram negative – have very thin peptidoglycan layer which makes them easy to decolorize with
ethanol ; red or pink

❖ Not all can be stained with gram stain specifically those with very high lipid content
(legionella) thus, they can be stained with Acid Fast Stain
Gram Positive cocci (aerobe) Micrococcus, Staphylococcus, Streptococcus
Gram Positive cocci (anaerobe) Peptococcus, Peptostreptococcus, Sarcina
Gram Negative cocci (aerobe) Branhamella, Neiserria
Gram Negative cocci (anaerobe) Veilonella
Gram Positive bacilli (aerobe) Bacillus, Corynebacterium, Erysipelothrix, Lactobacillus,
Listeria, Mycobacterium, Nocardia
Gram Positive bacilli (anaerobe) Actinomyces, Clostridium, Propionobacterim
Gram Negative bacilli (aerobe) Acinetobacter, Aeromonas, Alcaligenes, Bordetella,
Brucella, Enterobacteriaceae/Enterobacteriorales,
Fransicella, Legionella, Pasteurella, Pseudomonas, Vibrio
Gram Negative bacilli Fusobacterium, Bacteroides
(anaerobe)

● Mechanism of Gram Staining: Different theories are put forward regarding mechanism of Gram
reaction as exact mechanism is not known. They are:

❖ Acidic protoplasm theory:

➢ Gram positive bacteria have more acidic protoplasm as compared to Gram negative
bacteria.
➢ Gram positive bacteria tend to retain, the primary stain (basic in nature) more than Gram
negative bacteria.
➢ Additionally, because of iodine, the cytoplasm becomes more acidic and acts as mordant.
Hence it enhances the attraction of the primary stain to the cell cytoplasm. As a result, it
helps to fix the stain in bacterial cell.

❖ Cell wall permeability:

➢ Gram positive bacterial cell wall contains more mucopeptide. It makes the cell wall thicker
and stronger. As a result, due to iodine complex cannot come out of Gram-positive cell wall.
 ➢ On the contrary Gram-negative cell wall contains less mucopeptide and so its cell wall is
thin and less strong. It is the reason that dye iodine complex diffuses out of cell and color
of the counter stain is taken up.

2. Acid Fast Staining

- Performed if microorganism has a high lipid content
- Ability of the microorganism to resist dealcoholization
- Commonly used for organisms with a waxy material in their cell wall
- for mycobacterium species
- Required temperature: 60°C
- Fixation time: 2 hours
- Discovered by Paul Ehrlich but modified by ziehl-nilson
- Principle:
o Designed for bacteria whose cell wall contains long chain fatty acids (mycolic acid)
o Mycolic acid renders the cell resistant to decolorization
o Acid fast organisms may be gram +
o Acid-fast–stained smear is read with 1000 magnification, acid-fast–positive organisms stain
red
- Acid Fast Bacilli: red or pink Non-acid Fast Bacilli: blue or green
- 2 Classification:
o Partial Acid Fast: Nocardia, Legionella, Tsukemurella, Rhodococcus
o Complete Acid Fast: Mycobacterium, Isospora, Cryptosporidium

Acid Fast Staining

Ziehl-Neelsen Kinyuon Method / Pappenhei Baumgarten
Method / Hot Cold Method m Method Method
Method - For tissue - For color
biopsies blind
- For detection of
non-pulmonary
mycobacterium
Primary 3 grams of Carbol 4 grams of Carbol Carbol Carbol Fuschin
Stain fuschin + 5% fuschin + 9% phenol Fuschin
phenol
Mordant Heat Turgitol
- stain that
enhance but
not speed up
the staining
process
Decolorizer 3% Acid alcohol Rosolic Acid Dil. Alcohol
Fuschin
Secondary Methylene Blue Methylene Methylene
Stain Blue Blue

3. Special Stains:
- Utilize for highlighting a specific structure of a microorganism
 Organelle Stain
Cell Wall Dyar Stain
Capsule Welch Stain
Metachromatic Granule Albert Stain, Burke’s Modification of Gram Stain
Endospore Schaeffer-Fulton
Flagella Leifson Stain
DNA Feulgen

Cultural Characteristic
Bacterial Growth Phase
- Amount it takes for the bacteria to double in population
- Refers to the cell number pattern in cell numbers

Growth Cycle

1. Lag phase – no cell division, start of biosynthesis, period of adjustment, no utilization of nutrient ;
series of adjustment
◦ Period of adaptation
◦ Increase in the metabolic activity of the cell
◦ Synthesis of new enzymes, cofactor and metabolic intermediates
◦ No cell division take place
2. Log/ Exponential phase – exponential increase (2-4, 16-32…); most sensitive to antibiotics
– active division, used for physiological and biochemical testing, target of chemotherapy
a. Log increase – exponential phase
b. Log decrease – decline phase
◦ Influenced by:
❖ Temperature
❖ Carbon sources
❖ Nutrition
❖ Oxygen
3. Stationary/ plateau phase – balance between division and dying ; 1:1 ratio of microorganism and
nutrient
◦ There is exhaustion of nutrients
◦ Accumulation of metabolic waste products
4. Decline phase – increase amount of toxic waste
◦ Theories:
 ❖ Viable but Non - culturable (VBNC)
- Organism are in the stationary phase without morphological changes
- Genetic response to starving
- When introduced in a medium where physiologic requirements are met, cell division
resumes.
❖ Programmed Cell Death
- A fraction of the population is programmed to die in order for the nutrients to leak out
and to be used for continuous growth.

Methods of Quantitation of bacterial growth

1. Viable Plate Count
• Most common method of estimating bacterial growth
• Counting of colonies after 18-24 hours of incubation
• Significant colony count is between 30-300 only
• Formula: number of colonies x dilution factor / volume of sample = number of colonies

2. Direct Count
• Direct microscopic counting of bacteria using a counting chamber
• No incubation is needed
• Microbes in a measured volume of a bacterial suspension are counted with the use of a specially
designed slide:
❖ Breed Count Method
❖ Petroff - Hausser Cell Counter
❖ Eletronic Cell Counter
• Formula: Number of bacteria in a sample x number of squares x dilution factor = no. of colony

3. Turbidimetric Method
• Determination of bacterial growth in a liquid media
• Spectrophotometer or Photometric

Bacterial Growth Factors

- Essential for a bacterial cell to grow especially 1-4 which are already incorporated in a culture
media like in broths which require meat extract
- The presence of protein may already supply the carbon, hydrogen, oxygen and nitrogen which is
needed for the manufacture of different enzymes

Note: carbohydrate, hydrogen, oxygen and nitrogen are the basic nutritional growth factor needed
by a bacterial cell and without these four, the organism will not thrive and will eventually die. These
compounds makes up the protein which are used in the manufacture of flagella, toxin, formation of
enzymes and other important structures needed by bacterial cell
Note: all culture media contains protein using peptide or peptone

1. Carbon
− Organisms require a source of carbon for the synthesis of cell wall
o Cell wall provides the structure or act as a backbone of a bacterial cell
− Acquisition from different sources: sunlight, other carbon producing organism (human),
 inorganic sources (CO2)
− Used to classify several bacterial cell into several groups
− Only inorganic molecule that contains carbon
o Autotrophs – Free-living, non-parasitic bacteria which use carbon dioxide as carbon
source. ; seen in plants ; synonymous with heterotrophs
o Photoautotrophs – Requires sunlight to metabolize carbons
o Chemoautotrophs – Harvest the carbon component from inorganic compounds
o Heterotrophs – bacteria require more complex organic compounds as their source of
carbon and energy ; Human pathogenic bacteria are heterotrophs. ; requires carbon
from other organism (usually human)

Note: Human pathogenic bacteria are heterotroph

2. Hydrogen
− Important in the synthesis of proteins ; for the cytoskeleton of the cell
− Obtained from water
− Essential for the growth and maintenance of cell

3. Oxygen
- Needed for the production of protein component or structure
- Utilize in the categorization of bacterial cell
● Obligate aerobic bacteria – grow only when free oxygen is available to support their
respiratory metabolism
● Obligate anaerobic bacteria – grow in the absence of oxygen
● Facultative anaerobic bacteria – grow in the presence or absence of oxygen
● Microaerophilic bacteria – grow best at reduced oxygen tension ; utilizes semi-solid
media as the preferred culture media

4. Nitrogen
− Constitutes 10% of dry weight of bacterial cell.
− Obtained from organic molecules like proteins and inorganic molecules like ammonium salts
and nitrates
− Main source of nitrogen is ammonia, in the form of ammonium salt.

5. Salt
− For halophiles → Vibrio (except V. cholera and V. mimicus), Enterococcus and Staph
− Utilizes wagatsoma agar for vibrio species
− S. aureus requires a high salt content (MSA)
− Enterococcus → NaCl broth
a. Extreme Halophiles – 15% salt concentration
b. Moderate Halophiles – 3% salt concentration

6. Temperature
- Optimum temperature: best temperature needed for an organism for them to grow
 ● Psychrophilic bacteria – thrive at a very low temperature ; usually non-pathologic
because it requires a very cold storage however there are some that are able to grow
also in room temp (Yersinia ertolitica and C. bacter jejuni)
➔ C. bacter jejuni – obtained from poultry products0
➔ Y. enterocolitica – obtained from refrigerator of blood bag ; causes sepsis (but
mostly seen in Pseudomonas)
● Mesophilic bacteria – thrive at a body temperature
● Thermophilic bacteria – thrive in a very hot environment (e.g.: Pyrococcus & Bacillus
encrinus)

7. pH
- majority thrives at neutral environment
- Acidic: Tomato juice agar (Rogosa tomato juice agar /RTJA for lactobacillus acidophilus ;
MacConkey for E. coli)
- Alkali: Vibrio is an alkali bacterium which requires Alkali peptone water media

Classification of Culture Media

- In ostea, ceraccia was discovered
- Gelatin and potato agar were first used agar in the laboratory
A. According to consistency
Purpose of Agar: act as a solid
i. Liquid Medium
1. 0% agar ; broth
2. BHI, TSB

ii. Semi- Solid Medium

1. 0.5 to 1% agar
2. SIM
3. For microaerophiles and motility detection

iii. Solid Medium

1. 2-3% agar
2. TSI, MAC, BAP, CAP

B. According to composition
i. Synthetic (Research)
ii. Non-synthetic (isolation of bacteria)
iii. Tissue culture (Rickettsia and Chlamydia)

C. According to how it is dispensed

 i. Plated media
ii. Tube media
a. Butt – for organisms that does not require oxygen
b. Slant - for organisms that require oxygen
c. Butt-Slant – the but must be equal to the size of the slant ;
testing for a group of microorganism (e.g.: TSI)

D. According to type
o Common Ingredients of Culture Media
a. Peptone – product of animal and plant protein extracted
b. Meat extract – provides amino acids, vitamins and minerals
c. Mineral salts – sulfates, phosphates, salts
d. Carbohydrates – simple and complex sugars for energy source and carbon
e. Agar – not used by bacteria but instead used as a hardening agent
f. Water – for hydration

o Types of Media
i. Basic /Simple / All-purpose media
1. Primary culture ; It is a media that supports the all growth of micro-
organisms that do not require special nutrients
2. For culturing unknown bacteria

ii. Enriched media

1. Media that are enriched with whole blood, lysed blood, serum, special
extracts or vitamins to support the growth of pathogenic bacteria
2. E.g.: BAM & CAM

iii. Enrichment media

1. Fluid / Liquid media that increases the numbers of a pathogen by
containing enrichments and/or substances that discourage the
multiplication of unwanted bacteria
2. Similar with selective media but selective media is used for solid agar

iv. Selective media

1. Media which contain substances (Eg. Antibiotics) that prevent or slow
down the growth of bacteria other than pathogens for which the media
are intended
a. Inhibitors:
i. Gram Positive – Crystal violet, bile salt
ii. Gram Negative – PEA, Potassium tellurite

v. Transport media
1. Media containing ingredients to prevent the overgrowth of commensals
and ensure the survival of pathogenic bacteria when specimens cannot
be cultured soon after collection
 2. Amies transport media, Stuart media, Kelly-Blair media

vi. Differential media

1. Media to which indicator substances are added to differentiate bacteria
2. Utilize for categorizing a group of organism according to a certain characteristics
- MacConkey Media
o utilized for isolating suspected Enterobacteriaceae
o used for ruling out UTI caused by E. coli
o categorized Enterobacteriaceae into three
● Rapid Lactose Fermenters (24 hours)
o Escherichia
o Klebsiella
o Enterobacter

● Late Lactose Fermenter (48 hours)

o Arizona
o Serratia
o Hafnia
o Yersinia
o Citrobacter

Note: For Rapid and Late Lactose Fermenters produces a pink colony after 24-
48 hours of incubation due to the fermentation of lactose to lactic acid

● Non-Lactose Fermenter
- Have colorless colony due to non-fermentation of lactose to lactic acid
but there is the breaking down of protein into ammonia because these
organisms utilize the carbon portion of protein as a source of carbon
unlike for lactose and late lactose where the source of carbon is the
carbohydrate thus, producing an ammoniacal odor
o Salmonella
o Shigella
o Proteus
o Providencia
o Morganella
o Edwardsiella
o Erwinia

Plating Media for Routine Bacteriology

Medium Component/Comment Primary Purpose
Bile Esculin Agar (BEA) Nutrient agar based with ferric citrate. Differential isolation and
- Always for Group D Hydrolysis of esculin by Group D presumptive
Streptococci Streptococci imparts a brown color to identification of Group D
the medium; sodium desoxycholate Streptococci and
inhibits many bacteria Enterococci
Bile esculin azide Contains azide to inhibit Gram- Selective and differential
agar with negative bacteria, Vancomycin to for cultivation of
vancomycin select for resistant Gram- positive Vancomycin- resistant
bacteria, and bile esculin to enterococci from clinical
differentiate Enterococci from other and surveillance
Vancomycin-resistant bacteria that specimens
may grow
Blood Agar Trypticase soy agar, Brucella agar, Cultivation of non-
or beef heart infusion with 5% fastidious
sheep/horse/rabbit blood microorganisms,
determination of
Hemophilus – rabbit or horse blood hemolytic reactions
to prevent the in division of the
clotting factor
Bordet-Gengou agar Potato-glycerol-based medium Isolation of Bordetella
enriched with 15-20% defibrinated pertussis and
blood; contaminants inhibited by Bordetella parapertussis
methicillin (final concentration of 2.5
um/mL)
Brain Heart Infusion Dextrose, pork brain and heart Cultivation of fastidious
agar or broth dehydrated infusions organisms
Buffered Charcoal Yeast Yeast exract, agar, charcoal, salts Enrichment for Legionella
Extract agar (BCYE) supplemented with L- cystein HCl, spp., Francisella and
ferric pyrophosphate, ACES buffer, Nocardia
and a-ketoglutarate
Buffered Charcoal Yeast BCYE supplemented with polymyxin Enrichment and selection
Extract agar with B, vancomycin, and ansamycin, to for
antibiotics inhibit Gram- negative bacteria, Legionella spp.
Gram-
positive bacteria, and yeast
respectively
Burkholderia cepacia Bile salts, gentamycin, ticarcillin, For recovery of B.
selective agar polymixin B, Peptone, and yeast cepacia from cystic
extract fibrosis patients
Campy-blood agar Contains Vancomycin (10mg/L), Selective for
trimethoprim (5 mg/L), polymixin B Campylobacter
(2500 U/L), amphotericin B (2 mg/L), spp. requires cold
and cephalothin (15 mg/L) in Brucella incubation because they
agar base with sheep blood are cycrophiles which
may also cause harm
Campylobacter Thioglycollate broth supplemented Campylobacter spp.
thioglycolate broth with antibiotics incubated at 4oC for cold
enrichment
CDC anaerobe 5% sheep Tryptic soy broth, 5% sheep blood Improvement growth of
blood agar and added nutrients obligate, slow-growing
anaerobe

Cefoperazone, Blood-supplemented enrichment Selective medium for

Vancomycin, medium containing cefoperazone, isolation of
Amphotericin (CVA) vancomycin, and amphotericin to Campylobacter spp.
medium inhibit growth of most Gram-negative
bacteria, Gram-positive bacteria, and
yeast respectively
Cefsulodin-Irgasan- Peptone base with yeast extract, Selective for Yersinia
Novobiosin (CIN) agar or mannitol, and bile salts; spp.; may also be useful
broth supplememented with cefsulodin, in the isolation of
irgasan, and novobiocin; neutral red Aeromonas
and Yersinia will have a
crystal violet indicators target colony
Chocolate agar Peptone base, enriched with solution Cultivation of fastidious
of 2% hemoglobin or isoVitalex microorganisms such as
(BBL) Haemophilus spp.,
Contains lysed sheep RBC Brucella spp., and
pathogenic Neisseria spp.
Chromogenic media Organism-specific nutrient base, Chromogenic media are
selective supplements and designed to optimize
chromogenic substrate growth and differentiate
a specific type of
organism. Chromoagars
are routinely used in the
identification of yeasts,
methicillin resistant
Staphylococcus aureus
(MRSA), and a variety of
other
organisms

S. aureus – yellow
S. epidermidis – whitish
S. citrus – orange
Columbia colistin- Columbia based agar with 10mg Selective isolation of
nalidixic acid (CNA) colistin per liter, 15mg nalidixic acid Gram- positive cocci
agar per liter, and 5% sheep blood
Cysteine-tellurite blood Infusion agar base with 5% sheep Isolation of C. diptheriae
agar blood; reduction of potassium tellurite
by Corynebacterium diphtheriae
produces black colonies
Eosin Methylene Blue Levine: Peptone base containing Isolation and
(EMB) agar (Levine) lactose; eosin Y and methylene blue as differentiation of
Halt-Harris EMB indicator lactose-fermenting and
non- lactose-fermenting
Difference: Halt-Harris: lactose and sucrose enteric bacilli
carbohydrate
component Sucrose: is in coordination with
lactose because sucrose will detect
the presence of late lactose
fermenters
Gram-negative broth Peptone base broth with glucose and Selective enrichment
mannitol; sodium citrate and sodium liquid medium for enteric
desoxycholate act as inhibitory pathogens
agent
Hektoen Enteruc (HE) Peptone base agar with bile salts, Differential, selective
agar lactose, sucrose, salicin, and ferric medium for the
ammonium citrate; indicators include isolation and
bromthymol blue and acid fuschin differentiation of
Salmonella and
Shigella spp. from
other Gram-negative
enteric bacilli

Salmonella: black colony

; positive for sulfuric acid
Loeffler’s medium Animal tissue (heart muscle), dextrose, Isolation growth of
egg and beef serum, and sodium Corynebacterium
chloride
MacConkey agar Peptone base with lactose; Gram- Isolation and
positive organisms inhibited by crystal differentiation of lactose
violet and bile salts; neutral red as fermenting and non-
indicator lactose fermenting
enteric bacilli ; For
coliforms in the isolation
of urine sample
MacConkey sorbitol agar A modification of MacConkey agar in For the selection and
which lactose has been replaced by differentiation of E. coli
sorbitol as the primary carbohydrate O157:H7 in stool
specimens
Mannitol salt agar Peptone base, mannitol, and phenol Selective differentiation
red indicator; salt concentration of of
7.5% inhibits most bacteria Staphylococci
New York City agar Peptone agar base with cornstarch, Selective for Neisseria
supplemented with yeast dialysate, 3% gonorrhoeae, also
hemoglobin, and horse plasma; supports the growth of
antibiotic supplement includes Ureaplasma urealyticum
vancomycin (2ug/mL), colistin (5.5 and some Mycoplasma
ug/mL), amphotericin B (1.2 ug/mL),
 and trimethoprim (3 ug/mL)

Phenylethyl Alcohol (PEA) Nutrient agar base ; Phenyl-methanol Selective isolation of

agar inhibits growth of Gram-negative aerobic Gram-positive
organism cocci and bacilli and
anaerobic Gram-
positive
cocci and negative bacilli
Regan Lowe Charcoal agar supplemented with horse Enrichment and selective
blood, cephalexin and amphotericin B medium for isolation of
Bordetella pertussis
Salmonella-Shigella (SS) Peptone base with lactose, ferric Selective for Salmonella
agar citrate, and sodium citrate, neutral red and some Shigella spp.
as indicator; inhibition of coliforms by
brilliant green and bile salts
Schlaeder agar Peptone and soy protein base agar Nonselective medium
with yeast extract, dextrose, and for the recovery of
buffers; addition of hemin, L-cystein, anaerobes and
and 5% blood enriches for anaerobes aerobes

Selective for
Campylobacter
and Helicobacter spp.
Selenite broth Peptone base broth; sodium Enrichment of isolation of
selenite toxic for most Salmonella spp.
Enterobacteriaceae
Skirrow agar Peptone and soy protein base agar Selective for
with lysed horse blood; vancomycin Campylobacter
inhibits Gram- positive organsims;
polymixin B
and trimethoprim inhibits most Gram-
negative organsims
Streptococcal Contains crystal violet, colisitin, and Selective for
selective agar (SSA) trimethoprim sulfamethoxazole in 5% Streptococcus pyogenes
sheep blood agar base and Streptococcus
agalactiae
Tetrathionate broth Peptone base broth; iodin and Selective for Salmonella
potassium iodide, bile salts, and and Shigella spp. except
sodium thiosulfate inhibit ; Gram- Salmonella typhi
positive organisms and
Enterobacteriaceae
 Thayer-Martin agar Blood agar based enriched with Selective for N.
(modified Thayer- hemoglobin; contaminating organisms gonorrheae and N.
Martin agar) are inhibited by colistin, nystatin, meningitidis;
vancomycin, and trimethoprim lactate Supports the growth of
Francisella and Brucella
spp.
Thioglycollate broth Pancreatic digest of casein, soy broth, Supports the growth of
and glucose enrich growth of most anaerobes, aerobes,
microorganisms; includes reducing microaerophiles, and
agents thioglycolate, cystine, and fastidious organisms
sodium sulfite; agar is semi-solid
medium with a low concentration of
agar reducing oxygen diffusion in the
medium
Thiosulfate citrate- Peptone base agar with yeast extract, Selective and differential
bile salts (TCBS) agar bile salts, citrate, sucrose, ferric citrate, for
and sodium thiosulfate; bromothymol Vibrio spp.
blue acts as an
indicator
Todd-Hewitt broth Todd-Hewitt, an enrichment broth for Selection and enrichment
supplemented with Streptococci, supplemented with for Streptococcus
antibiotics (LIM) nalidixic acid and gentamicin or agalactiae in female
colistin for greater selectivity; genital specimens
thioglycolate and agar reduce redox
potential
Trypticase Soy broth All-purpose enrichment broth that Enrichment broth
(TSB) can support the growth of many used for subculturing
fastidious and non- fastidious bacteria various bacteria from
primary agar plates
Xylene lysine Yeast extract with lysine, xylose, Isolation and
desoxycholate (XLD) lactose, sucrose, and ferric differentiation of
agar ammonium citrate; sodium Salmonella and Shigella
desoxycholate inhibits Gram-positive spp. from other Gram-
organisms; negative enteric bacilli
phenol red as indicator

Lecture 4:
STERILIZA
TION AND
DISINFEC
TION
Sterilization and Disinfection
✔ Sterilization – Destruction of all forms of microbial life including spores for inanimate objects ;
inhibition of all life forms (pathologic or not)
✔ Disinfection – Destruction of microbes that cause disease ; inhibits growth of pathologic organisms
only and living tissues

Variables of Disinfection
1. Concentration – inversely proportional
2. Time – the longer the exposure the lesser is the survival rate
3. Temperature
4. pH

Note: increased concentration and time > decrease survivors

A. Classification of chemical methods of sterilization and disinfection

- Chemical sterilant or biocides (kill all life form)
- Antiseptics (kills only the pathologic organism ; form of disinfection)

Mechanism Example
Cell Damage QUATS (not utilize in P. aeruginosa because it is resistant to
QUATS), Iodophor, Phenols
Protein Denaturation Acid, Alkalis
Modification of Functional ETO, Glutaraldehyde, Heavy metals
Group

1. Antiseptic
- A chemical substance that prevents growth of bacteria by either inhibiting or destroying
microbes
- Used on the surface of skin or mucous membranes ; for tissues

2. Disinfectant
- Kills many but not all microbes
- Aims to kills disease-causing microbes but not spore formers
- Used on inanimate objects

3. Bacteriostatic – Agents that inhibit the growth of bacteria

4. Bactericide – Agents that kill bacteria

Chemical Agents
A. Disruption of Cell membrane
2. Alcohols
- Diuretic ; denatures protein
- Disorganizes the lipid structure in the membranes
- 70% isopropyl alcohol: requires water for maximal activity

3. Detergents / soaps
- Absorbs bacteria ; surfactants interact with the lipid in the cell membrane and with
the surrounding water
 - Increases the surface tension (e.g.: quaternary ammonium→ quats or zephiran)

4. Phenols
- Original disinfectant of Lister
- Denatures protein (carbolic acid / cresol / Lysol)

B. Modification of Proteins
1. Halogens
a. Chloride → (e.g.: hypochlorite in restaurants and swimming pools)
b. Iodine → most effective skin antiseptic (tincture of iodine 🡪 2% solution of KI in
ethanol)
Iodophore – used as an antiseptic

2. Heavy metals
- Inhibits enzymatic activity
- E.g.: Hg: merthiolate
Ag: AgNO3, silver sufadiazine 🡪 antiseptic for babies infected with gonorrhea
(eyes) via vertical transmission

3. H2O2 – For healing wounds and contact lenses ; oxidizes microbes

4. Formaldehyde
- Formalin → 37% in water ; concentrated form
- Denatures protein
- Bactericide and fungicide

5. Glutaraldehyde
- 10x more effective than formaldehyde and less toxic
- Used for respiratory therapy equipment

6. Ethylene oxide
- For sterilization of heat-sensitive equipment
- Most effective cold sterilization technique for syringe

7. Acids and Alkali’s

- Denatures protein
- Weak acids are used as food preservatives (bacteriostatic) (e.g.: benzoic, propionic
and citric acids)

Modification of Nucleic Acids

1. Crystal Violet (gentian violet)
- Skin antiseptic
 - Binding of (+) charged dye molecule to the (-) charged PO4 groups of nucleic acids
(e.g.: mumps)

2. Malachite Green
- Selective medium ; kills other bacteria except MTB (e.g.: in L-J medium for MTB)

B. Physical methods of sterilization and disinfection (killing of microorganism as a whole)

1. Heat – the most reliable and universally applicable method of sterilization ; coagulates
protein
Mechanism of action
a. Dry heat – denatures protein which involves the cell wall; kills by oxidation
✔ Direct Flame / Incineration – most effective ; It is an efficient method of
sterilization and disposal of contaminated needles / inoculating loops and
needles, syringes and cover slips at high temperature ; heating / burning
of material until ashes
✔ Red heat – Inoculating wires, loops and points of forceps are sterilized by
holding them in the flame of a Bunsen burner until they are red hot ; for
objects that cannot be burned into ashes
✔ Flaming – Scalpels and neck of flasks, bottles and tubes are exposed
for a few seconds, but it is of uncertain efficacy ; introduced by
Theodore Schwann ; prevents entry of air contaminants
✔ Hot Air Sterilizer (Oven) – It is done by applying 160-180°C for 1-2
hours
Use: metals and glassware → Sterilizes
glassware, oils, greases, lubricants and
powders

❖ Time depends on the penetration of heat

on objects to be sterilized

b. Moist heat – utilizes moisture ; denatures and coagulates protein ; water aids in the
disruption of covalent bonds
✔ Boiling – ; most common method but unreliable especially for lyophilize
objects (agar) ; 100°C for 15-30 mins. ; kills vegetative forms but not spores
and viruses
✔ Fractional Sterilization (fraction by fraction)
1. Inspissation – used in sterilizing protein rich media (LJ agar for M.
tuberculosis with the appearance of cauliflower white colony and
Ogawa media which are known to be high in protein component);
thickening through evaporation; 70-80 C for 2 hours for 3 successive
days ; performed using Arnorld’s sterilizer
➔ On the first day, old spore-forming organisms are being killed. On
the second day, the vegetative organism produces spore. On the
third day, all the remaining organism (spore-forming and
vegetative) are killed.
 ➔ Organism may thrived if heating only at 100 C for <30 minutes a
day
2. Tyndallization – Intermittent steaming
o Steaming of the material is done at 100°C for 30 minutes on 3
consecutive days. The principle is that spores which survived the
heating process would germinate before the next thermal
exposure and then would be killed. It is used for sterilizing heat
sensitive culture media containing materials such as
carbohydrates, egg or serum ; performed inside an Arnold’s
sterilizer
✔
Pasteurization – It is the process of application of heat at temperature of
62°C for 30 minutes (Holder method – most commonly used on dairy
products) or 72°C for 15 seconds (Flash method) followed by rapid cooling
(Flash Pasteurization) to discourage bacterial growth ; among dairy
products (milk)
✔
Autoclaving – Steam under pressure ; 15-30 minutes at 121°C at 15 psi

2. Desiccation – drying under the sun ; lack of water prevents multiplication (e.g.: Neisseria
vs. MTB)
3. Freezing / Freeze-Drying / Lyophilization – Inactivation of living bacteria by cold ;
preservation method by stopping the replication of bacteria
4. Filtration – Mechanical / physical separation/sieving through membrane filters (e.g.:
asbestos, Millipore, nitrocellulose); used in vaccine production
5. Osmotic Pressure – by plasmolysis (e.g.: immersion of meat in salt solution ; fruits and
vegetables in sugar solution)
6. Sonic vibration, Trituration, Agitation – mechanical method that disintegrate bacteria
a. Sonic vibration – sound waves (high decibels)
b. Trituration – grinding
c. Agitation - shaking

7. Radiation
a. UV light
- DNA destruction by inhibiting replication of DNA
- Forms thymine dimers ; may damage the cornea and skin
b. X-rays
- With higher energy and higher penetrating power than UV
- Produces hydroxyl radicals by the hydrolysis of water
- Breaks covalent bonds on the DNA
- Spores are resistant due to its low water content
c. Gamma rays
- Its high energy breaks chemical bonds in molecules that are vital for cell growth
and integrity
 d. Ionizing – DNA mutation

Biological Indicators
- determines success of sterilization
- sporulated organism because these organisms are most difficult to kill
2. Autoclave – Geobacillus stearothermophillus/ Bacillus stearothermophillus
3. Ionizing Radiation – Bacillus pumitis
4. Dry Heat Oven – Bacillus subtilis var Niger
5. Ethylene oxide (ETO) – Bacillus subtillis var Globujii

Clinical Specimens
1. Blood Culture
- Use 0.025% SPS as anticoagulant serving as an anti-complement activator and anti-phagocytic
agent
- Always require 2 extraction to determine if the bacterial growth is really causing an infection or
the bacteria is just a contaminant
- In conjunction with procalcitonin

2. Throat and Nasopharyngeal Cultures

- Indicates deep respiratory infection
- Collected with Endotracheal Aspirate / ETA tube since it can reach up to the lower respiratory
tract where the target organisms are located
- Used for detecting S. pyogenes infection
- Culture on Modified Tinsdale Media, Tellurite Blood Media or Todd-Hewitt broth for
fluorescence microscopy (for immediate observation of Streptococci)
- Nasopharyngeal swab is used for H. influenza, N. meningitidis, B. pertussis

3. Sputum
- Culture at BAP, CAP, LJ Media
- Bartlett’s Classification
● determines if it is a sputum or saliva
- <10 Epithelial cells & >25 PMNs

4. Urine culture (4°C)

- Boric Acid is used for preservation or simply refrigerate
- 105 indicates infection
- Formula: colony count = number of colonies x dilution factor

5. CSF (37°C)
- Collected by lumbar puncture using University of Illinois in L3 and L4 or L4 and L5
- 3 vials
- Opening pressure must be first measured before collection of CSF
- For diagnosing meningitis

Birth – 1 month Neonate Grp B. Agalactiae

1 month -5 years old Grade school H. influenza
 5- 29 years old Adolescent N. meningitidis
>29 years old Adult S. pneumoniae

Types of Meningitis
Bacterial Tubercular Viral Fungal
✔ Positive limulus ✔ Pellicle ✔ Normal glucose ✔ Lactate level
lysate test with formation and lactate >25 mg/dL
Gram negative ✔ Lactate level because they do ✔ Positive India
organism >25 mg/dL not utilizes ink with C.
✔ Lactate >35 carbohydrate thus neoformans
mg/dL – always no production of
affected lactate

Specimen Possible Pathogen Remarks

Sputum S. pneumoniae Best collected in the morning
S. aureus
S. pyogenes
H. influenzae
K. pneumoniae
Proteus
Yersinia
ETA (Endotrachial aspirate) S. pneumoniae
Throat samples C. diphtheriae
H. influenzae
N. meningitidis
B. pertussis
B. parapertussis
Klebsiella

Ear Discharge S. pneumoniae

S. pyogenes
H. influenzae
K. spenil
S. aureus
P. aeruginosa
Eye specimens Moraxella lacunata Loeffler’s serum slope
C. Trachomatis
N. gonorrhea
Note: Meningitis occurs inside the skull since it contains less oxygen thus the
organism that metabolizes glucoses produces lactate instead of pyruvate
Note: the gold standard in identification of microorganism is culture

Limulus Lysate Test – agglutination test ; uses horseshoe crab indicating blue
color due to increase copper ; detects endotoxin

Microbiology Test:
1. First Generation Test: Culture, Biochemical Test, Identification
2. Second Generation Test: API Test – color test
3. Third Generation Test: Vitek, BacteAlert
4. Fourth Generation Test: specific with high sensitivity ; MALDI-TOF

Lecture 5: ANTIMICROBIAL
SUSCEPTIBILITY TESTING AND
ANTIMICROBIAL AGENTS

Antimicrobial Susceptibility Testing

– Method used to measure the in vitro susceptibility of microorganisms to various microbial agents
based on the presence or absence of a zone of inhibition
Group of Antimicrobial Agents
▪ Group A – appropriate for routine and primary testing
▪ Group B – includes antimicrobial agents that supplement the Group A agents; or those that do
not respond to group A agents and can be used for infection prevention
▪ Group C – alternative drugs for organisms that produce an epidemic of resistant strains to primary
drugs; can be used to treat individuals allergic to primary drugs; used for treating unusual
organisms
▪ Group U – used for treating UTIs; should not be used for treating organisms from other sites
(except for Enterobacterales)
▪ Group O – agents not routinely reported
▪ Group inv. – agents that are investigational and have not yet been approved by FDA

Definition of Terms
1. Breakpoint – Minimum Inhibitory Concentration (MIC) or zone diameter used to categorize if
an organism is susceptible, susceptible dose dependent, intermediate or resistant ; reference
value
2. Interpretative Category – category derived from microbial characteristics
a. Susceptible – refers to organisms with a Minimum Inhibitory Concentration (MIC) at or below
the susceptible breakpoint
b. Susceptible-dose dependent – refers to a breakpoint based on the dosage of a patient
c. Intermediate – includes breakpoint within the intermediate range
 d. Resistant – MIC is above the zone diameter and is usually not inhibited by the usual
concentration of agent with a normal agent
e. Non-susceptible – category used for which a susceptible breakpoint is designated because of
the absence or rare occurrence of resistant strain
Categories of Antibmicrobial Testing (CLSI, M100S30 2020)
a. Routine test
b. Supplemental test – identify a specific resistance mechanism
c. Screening test – provides presumptive result and requires additional testing
d. Surrogate agent test – test performed with a different agent from the agent of interest
e. Equivalent agent test – a type of test that predicts the outcome of a closely related agents of the
same class
Standard Components of Antimicrobial Susceptibility Testing
1. Bacterial inoculum size
 − Affects indirectly or the directly the action of antibiotic thus, it is necessary to maintain the
standard number of a bacterial colony
− Should be pure and standardized
− Should be obtained from 4-5 colonies of the same morphology
− Should be compared with 0.5 McFarland Turbidity standard/Barium sulfate (1% H2SO4 + 1.175%
BaCl)
o 0.5 McFarland Turbidity Standard has an optical density of 1.5 x 108 CFU/mL
o Positioned in front of Wickerham card

2. Culture Media
Broth
Enterobacteriaceae Mueller-Hinton
Enterococci Mueller-Hinton
Haemophilus influenzae Haemophilus test medium
MRSA Mueller-Hinton + 2% NaCl
Neisseria meningitidis Mueller-Hinton + 2-5% horse blood
Pseudomonas aeruginosa Mueller-Hinton
Streptococcus pneumoniae Mueller-Hinton + 2-5% horse blood

Agar & Disk Dilution Susceptibility Testing

Enterobacteriaceae Mueller-Hinton
Enterococci Mueller-Hinton
Streptococcus 5% SRBA
MRSA Mueller-Hinton + 2% NaCl
Neisseria gonorrheae GC-Lect agar
Pseudomonas aeruginosa Mueller-Hinton
Streptococcus pneumoniae Not recommended
Penicillin resistant Staphylococcus Mueller-Hinton Agar + 6 microgram oxacillin + 4% NaCl
Vancomycin resistant Enterococcus BHIB + 6 microgram vancomycin/mL
Aminoglycoside resistant BHIB + 500 microgram gentamicin + 1000 microgram
Enterococcus streptomycin

3. Atmosphere of incubation
4. Incubation temperature (30-35C)
5. Time of incubation (16-24 hours)
6. Concentration of antimicrobial agent

❖ Susceptibility disk must be placed within 15 minutes of inoculation

Methods for Antimicrobial Susceptibility Testing

Component Comment
Agar MHA
pH of Agar 7-2-7.4
Depth of Agar 4mm
Size of Inoculum 1.5 x 108 CFU/L
Incubation Time 16-18 hrs. at 35C
Size of Filter paper 2.4-2.6 mm
1. Broth Dilution Method
a. Broth Macrodilution – tubes are viewed by the anaided eye to determine whether
growth is present; labor intensive
b. Broth Microdilution – most common dilution method; measures the in vitro
activity of an antimicrobial agent; uses cation-adjusted Mueller Hinton broth
(CAMHB)
2. Agar Dilution Method – allows multiple testing of multiple isolates on a single plate; used
for Bacteroides fragilis (anaerobic species)
3. Disk Diffusion Method (Kirby Bauer Method)
Affected by:
o pH
o Thymidine or thymine – can reverse the inhibitory effect of sulphonamides and
trimethoprim
o Divalent cations
o Mg and Ca – affects the results of aminoglycosides and tetracycline with P. aeruginosa
o Zn – reduces carbapenem zones
4. Antimicrobial gradient method or E-test (Epsilometer) – combination of dilution and diffusion
5. Automated AST – provides rapid, automatic and standardized validation; can detect resistance
pattern

Mechanism of action of anti-microbial drugs

Mechanism Drug
Cell Wall Synthesis Inhibitor Penicillin
- Bacteriostatic ; from Penicillium notatum
- By Alexander Fleming (1929)
- Susceptible: G+ bacteria, spirochetes

Disadvantages:
- Counterattack: destroyed by acids
- Production of Enzymes: destroyed by penicillinase / B –
lactamase (Enterobacteriaceae, staphylococcus)
- Mostly for gram (+), Neisseria and Treponema pallidum
only

Ampicillin
- Improved penicillin
- For gram (+) and (-)

Bacitracin
- From Bacillus licheniformis
- Against staphylococcus, streptococcus, Neisseria and
haemophilus

Cephalosporin, Vancomycin, Ristocetin, cycloserine

Cell Membrane Damage Polymixin B
- From Bacillus polymyxa
- For gram (-) 🡪 Brucella abortus, Klebsiella pneumoniae
 Amphotericin B, Tyrocidin, Garamicidin S, Imidazole and
Triazole
Protein Synthesis Inhibitor Aminoglycosides, Gentamycin, Lincomycin, Clindamycin

Tetracyclin – broad spectrum

Erythromycin – freezes the ribosome

Streptomycin
- From Streptomyces griseus
- For MTB and N. gonorrhea

Chloramphenicol
- From Streptomyces venezuelae
- For gram (+) and (-)
- bacteriostatic
Nucleic Acid Inhibitor Nalidixic Acid, Rifampin
- destroys genetic material of
a microorganism therefore,
antibiotic consumption
must be taken seriously
since mutation may occur if
there is an alteration in
antibiotic ingestion
Nucleotide Synthesis Inhibitor Sulfonamide, Trimethorpim, Rifampin, Quinolones

Antimicrobial Agents and Antibiotics

Antimicrobial Agents – Manufactured synthetic
Antibiotic Agents – Produce ; fungi-like bacterial cells

Chemotherapy
- treatment of disease with chemical compounds
- Paul Ehrlich: father of chemotherapy ; discovered salvarsan / Arsphenamine (arsenic) / 606 for
syphilis
- Sources: chemically prepared: antibacterial From fungi or bacteria: antibiotic / antimicrobial

Characteristics of Antimicrobials and Antibiotic:

1. Selective toxicity
2. Wide spectrum of activity
3. Host should not become allergic to it
4. The microorganism should not become readily resistant to it

Sources Antibiotics
1. Bacillus subtilis Bacitracin
2. Bacillus polymyxa Polymyxin
3. Streptomyces nodosus Amphotericin
4. Streptomyces venezuelae Chloramphenicol
5. Streptomyces erytheus Erythromycin (discovered by Pilipino scientist)
6. Streptomyces noursei Nystatin
7. Streptomyces fradiae Neomycin
 8. Micromonospora Gentamicin
purpurea
9. Cephalosporium Cephalosporin
10. Penicillium notatum Penicillin

Bacteria Treatment
Actinomycetes Clindamycin, Penicillin, Erythromycin
All Streptococci except Penicillin, Erythromycin
Enterococci
Bacillus anthracis Ciprofloxacin, Gentamicin, and Penicillin
Bacillus cereus Clindamycin and Aminoglycosides
Bordetella pertusis Erythromycin
Borellia recurrentis Penicillin, Tetracycline
Brucella abortus Doxycycline + Rifampin, Tetracycline + Streptomycin
Campylobacter jejuni Erythromycin
Chlamydia pneumoniae Tetracycline, Doxycycline, Erythromycin
Chlamydida trachomatis Tetracycline, Chloroamphenicol
Clostridium dificile Metronidazole and Vancomycin
Clostridium perfringens Penicillin
Clostridium tetani Penicillin
Clostrium botulinnum Intravenous Trivalent Antibody
Corynebacterium diphtheriae Penicillin and Erythromycin
Enterococci Penicillin, Gentamicin
Erysipelothrix rheusiopathiae Penicillin G
Escherichia coli Perform Kirby-Bauer
Francisella tularensis Streptomycin, Gentamycin, Tetracycline
Haemophilus ducreyii Erythromycin, Cotrimoxazole
Haemophilus influenzae Amopicillin, Chloramphenicol, Cotrimoxazole, Cephalosporin
Helicobacter pylori Bismuth, Amoxicillin, Metronidazole
Klebsiella pneumoniae Perform Kirby-Bauer
Legionella pneumophilia Erythromycin, Rifampin
Leptospira interrogans Doxycycline, Ampicillin
Listeria moncytogenes Ampicillin, Erythromycin, Cotrimaxazole
MRSA Vancomycin
Mycobacterium leprae Dapsone, Rifampicin, Clofazimine
Mycobaterium tuberculosis Rifampin, Ethambutol, Isoniazid
Mycoplasma pneumoniae Tetracycline, Erythromycin
Neisseria gonorrheae Ceftriaxone and Ciprofloxacin
Neisseria meningitidis Penicillin, Chloramphenicol
Nocardia asteroides Cotrimoxazole, Amikacin, Imipenem, Cefotaxime
 Pasteurella multocida Penicillin, Tetracycline
Penicillin Resistant Staphylococci Cloxacillin, Nafcillin
Penicillin Sensitive Staphylocci Penicillin, Ampicillin
Proteus perform Kirby-Bauer
Pseudomonas aeruginosa Ticarcillin, Piperacillin, Aztreonam, Imipenem, Ceftazidine,
Cefoperazone, Fluroquinolones
Rickettsiae Tetracycline, Chloroamphenicol
Salmonella Chloramphenicol, Ampicillin (for carriers)
Shigella Ciprofloxacin, Cotrimaxazole
Streptococcus pneumonia Amoxicillin, Chloramphenicol, Cephalosporin
Treponema pallidum Penicillin, Tetracycline, and Erythromycin
Vibrio cholerae Tetracycline, Chloroamphenicol
Yersinia pestis Streptomycin, Tetracyclin, Chloramphenicol

Lecture 6: BIOCHEMICAL TESTS FOR IDENTIFICATION OF ORGANISMS

1. Beta Lactamase Test

- Performed to indicate whether a microorganism is a
Beta Lactamase produce
- Becomes the basis of doctor in giving beta lactam
- The red color is due to the hydrolysis of beta lactam
ring
Principle: Cefinase disk is impregnated with the
chromogenic cephalosporin Nitro- cefin. A rapid color
change from yellow to red results when the amide bond in
the beta- lactam ring is hydrolyzed by beta lactamase
Result: Positive: color change from yellow to red
Negative: color change does not occur
Control: Positive: S. aureus
Negative: H. influenzae

2. Kirby- Bauer Diffusion Test

- Allows impregnated disk with antibiotic to diffuse in the agar
- Compares zone of inhibition to the breakpoint value
Principle: Standardized suspension of organisms is inoculated onto MH agar. Paper
disks impregnated with specific antibiotic concentrations are placed onto the agar.
After 18 to 24 hours of incubation the diameters of the zones of growth are
measured. The results are compared with established values to determine the
organism’s susceptibility or resistance to each antibiotic
3. Catalase Test
- An exotoxin ; major virulence factor of gram positive bacteria
- Causes CGD seen in sepsis and bacteremia
- Avoid contamination with RBC because hemoglobin contains
pseudoperoxidase activity
Principle: Catalase converts hydrogen peroxide into water and oxygen. A
positive reaction is indicated by rapid and continuous bubble formation
Result: Positive: rapid and continuous bubbles
Negative: lack of bubble formation 10 sec later
Control: Positive: S. epidermidis (bubble formation)
Negative: S. pyogenes (no bubbles)

4. Modified Oxidase Test

Principle: Those bacteria that possess Cutochrome C produce dark blue end
product when reacted with modified oxidase reagent (6%
tetramethylphenylenediamine hydrochloride in dimethyl sulfoxide)
Result: Positive: dark blue color
Negative: no color change
Control: Positive: Micrococcus luteus (dark-blue color)
Negative: Staphylococcus epidermidis (no color change)

5. Bacitracin Susceptibility
- Utilizes 4 units ; anti-susceptibility test
- Measures zone of inhibition
- Differentiates Group A Strep from Group B Strep and
Micrococcaceae from Staphylococcaceae
Principle: Bacitracin inhibits the growth of Micrococcus and Stomacoccus while having no effect on
Staphylococcus, which is resistant
Result: Zones greater than 10mm indicate susceptibility and are typical of Micrococcus. Staphylococcus
organisms typically produce no zone of inhibition and are considered resistant
Control: Micrococcus luteus: zone diameter greater than 10mm- susceptible
Staphylococcus epidermidis: no zone of inhibition- resistant

6. Coagulase Test
- Utilizes rabbit’s plasma
- Staphylococcus covers itself with fibrin strand thus, cannot be
detected by monocyte
Principle: In the presence of coagulase, fibrinogen is converted to fibrin.
There are two forms of coagulase: bound or clumping factor and free.
Bound coagulase is detected in the coagulase slide test, and free
coagulase is detected in the coagulase tube test
Result: Positive: white fibrin clots in plasma
Negative: smooth suspension, no clot formation
Control: Positive: S. aureus
Negative: S. epidermidis
7. Novobiocin Susceptibility Test
Principle: After incubation with 5 ug novobiocin, S. saprophyticus is not
inhibited by the antibiotic. Other cons, such as S. epidermidis, are
susceptible to novobiocin
Result: Susceptible: zone diameter greater than 16mm
Resistant: zone diameter less than or equal to 16mm
Control: Susceptible: S. epidermidis
Resistant: S. saprophyticus

8. DNAse Test
Principle: DNase medium using methyl green becomes colorless in the presence of
DNase due to hydrolysis
Result: Positive: hydrolysis of surrounding medium, resulting in clear zone
Negative: no clearing observed
Control: Positive: S. marcescens, S. aureus
Negative: E. cloacae, S. epidermidis

9. Mannitol Salt Agar (MSA)

- Two main ingredients: mannitol (carbohydrate) and salt
- Staphylococcus aureus is halophiles and the only one who can
ferment mannitol
Principle: MSA contains high level of sodium chloride (7.5%).
Staphylococcus spp are able to grow in this level of NaCl, wheras most
gram-negative organisms and other gram positive organisms cannot. MSA
contains beef ectract, NaCl, D-mannitol and phenol red indicator. S. aureus grows on MSA and ferments
mannitol, producing yellow colonies. Most coagulase- negative strains of Staphylococcus do not
ferment mannitol and grow as small red colonies surrounded by red or purple zones. Differential colors
are caused by reactivity of phenol red indicator, which is red at pH 8.4 and yellow at pH 6.8
Result: Growth without fermentation: plate remains pink to red, with no yellow halo surrounding
growth
Growth and fermentation: yellow halos surrounding growth
Control: Growth, yellow zone around colonies: S. aureus
Growth, red zone around colonies: S. epidermidis

10. Bacitracin and Sulfamethoxazole- Trimethoprim

Susceptibility tests
Result: Group A beta- hemolytic streptococci are susceptible to
0.04 U bacitracin but resistant to 1.25 ug sulfamethoxazole- trimethoprim (SXT). Group B beta-
hemolytic streptococci are resistant to both bacitracin and SXT
Result: Any zone of inhibition around either disk indicates that the organism is susceptible
Control: Bacitracin susceptible and SXT resistant: Grp. A Streptococcus
Bacitracin resistant and SXT resistant: Grp. B Streptococcus
Bacitracin resistant and SXT susceptible: Grp. C Streptococcus
11. CAMP Test (Christie, Atkins, Munich, and Paterson)
- Arrow head is due to synergistic hemolysis with S. aureus
meaning, hemolysis of S. aureus and S. agalaceae will
combine
Principle: The CAMP factor is a diffusible, protein-like compound
produced by Streptococcus agalactiae. A characteristic
“arrowhead” hemolytic pattern results when the organism is
streaked perpendicularly to beta hemolytic S. aureus
Result: Positive: a zone of enhanced hemolysis given by an arrowhead appearance at the junction of the
Staphylococcus and Streptococcus indicated the presence of group B Streptococcus. Report “Group B
Streptococcus, presumptive by CAMP reaction”
Negative: Absence of hemolysis
Control: Positive: S. agalactiae
Negative: S. bovis

12. Bile-Esculin Agar (BEA)

- Blackening of media indicates hydrolysis of esculin to esculitin
Principle: Esculin is a coumarin derivative of a glycoside that contains a
glucose moiety and a glycone moiety. Differentiation is based on the
organism’s ability to grow in 40% bile and to hydrolyze esculin to
produce esculetin. Esculetin reacts with ferric citrate to form a brown-
black precipitate
Result: Positive: growth indicates tolerance to 40% bile (4% oxgall).
Blackening indicates hydrolysis of esculin
Negative: Lack of growth indicates inability of organism to grow in 40% bile, and lack of color
change indicates inability of organism to hydrolyze esculin
Control: Growth with black color: E. faecalis
No growth: S. viridans

13. 6.5% Salt Broth

- Turbidity indicates presence of growth
- Indicator: bromcresol purple → absence indicates
growth
Principle: Medium without added salt is tested along with
the 6.5% salt broth to serve as a growth control. If
growth is equivalent in both media, the organism is
tolerant of salt and a positive result is noted. However,
if the growth on the salt- containing medium is very weak or absent but growth in the salt- free medium
is
good, a negative result is noted. An indicator, such as bromcresol purple, can be incorporate into the
medium to facilitate interpretation. A positive result is indicated by a change in the color of the indicator
from purple to yellow or by the appearance of obvious growth
Result: Positive: Growth of organism
Negative: No growth of organism
Control: Positive: E. faecalis
Negative: S. bovis

14. PYRase (Pyrrolidonylarylamidase) Test

Principle: PYR-impregnated disks serve as substrate for the detection
of the enzyme pyrrolidonylarylamidase. After inoculation of the disk with the test organism, hydrolysis
of the substrate occurs, forming beta- naphthylamide, which produces red color with the addition of the
color developer, pdimethyl-aminocinnamaldehyde
Result: Positive: pink to cherry- red color after addition of color developer
Negative: No color development
Control: Positive: Enterococcus
Negative: Non-enterococcus

15. Optochin (Taxo P)

Principle: In the presence of optochin (ethylhydrocupreine hydrochloride), colonies of
S. pneumonia are selectively lysed. Lysis is indicated by a zone of inhibition after
incubation under increased CO2. Other alpha- hemolytic streptococci are resistant to
optochin and give a negative test
Result: Positive: Zone of inhibition
Negative: No zone of inhibition
Control: Positive: S. pneumonia
Negative: Viridans Streptococcus

16. MacConkey Agar

Principle: Bile salts inhibit gram-positive bacteria, which allows
for the isolation of gram-negative bacteria. Neutral red and
crystal violet further inhibit the gram-positive bacteria. Lactose
is the only carbohydrate source. Neutral red indicator is brown
in pH 6.8 to 8.0 and pink-red at pH less than 6.8
Result:
Rapid Fermenters Late Lactose Fermenters Non-Lactose Fermenters
Escherichia Arizona Salmonella
Klebsiella Serratia Shigella
Enterobacter Hafnia Proteus
Yersinia Providencia
Citrobacter Morganella
Edwardsiella
Erwinia

17. Eosin Methylene Blue Agar

- Two Agars:
a. Halt-Harris Eosin Methylene Blue agar: lactose and sucrose
b. Levine Eosin Methylene Blue agar: Lactose
Principle: Eosin and Methylene blue are dyes that inhibit the gram-positive
bacteria. Lactose is the only carbohydrate source in most formulations
18. Triple Sugar Iron Agar (TSIA)
- Butt-slant media
- Contains lactose, sucrose and glucose which may require oxygen or not
- Ratio of lactose:glucose:sucrose: 10:10:1
- Slant: lactose and sucrose
- Butt: glucose
Principle:
Medium contains protein source that permit growth of most bacterial strains. Lactose, sucrose
and
glucose is present as well as phenol red indicator. Glucose is in a concentration one-tenth that of the
other carbohydrates. Ferrous sulfate is present as an indicator of hydrogen sulfide (H2S) production.
The TSI is a two-reaction chamber with an aerobic slant portion and an anaerobic deep portion.
The slant portion of the tube is exposed to atmospheric oxygen and will become alkaline due to
oxidative
decarboxylation of peptides and amino acids.
The slant tends to become and remain alkaline(red). Amino acid degradation is minimal in the
deep
(anaerobic) portion and thus a small quantity of acid produced can be detected because of a few amines
are being formed from amino acids.
Bacteria that ferment glucose but not lactose or sucrose, only produce small quantities of acid
and
can’t counteract the degradation of amino acids at the slant, which results in an alkaline pH due to
oxidative
decarboxylation. Such organisms characteristically produce an alkaline slant over an acid deep (K/A) →
ferments glucose
Organisms that ferment both glucose and lactose and/ or sucrose produce large quantities of
acid,
which overcome the alkaline reaction of the slant, yielding an acid slant over an acid deep (A/A) →
ferments glucose, lactose and sucrose
An organism incapable of fermenting glucose produces no change in the indicator and is
characterized by an alkaline slant over an alkaline deep (K/K)
A sulfur source, sodium thiosulfate, provides sulfur atoms to detect the production of H2S gas.
H2s
reacts with iron salts (ferrous sulfate or ferric ammonium citrate) to produce the black precipitate of
ferrous
sulfide.
The production of gas during fermentation is indicated by the presence of cracks in the medium
or
the “pulling away” of the medium from the walls of the test tube.

Results:
A/A/g Eshcerichia, Klebsiella, Enterobacter
K/A/g/+ Salmonella, Proteus, Citrobacter
K/A/g Morganella
K/A Shigella, Serratia, Providencia
K/K Pseudomonas, Aeromonas, Virbrio, Burkholderia, Stenotrophomonas
Control: None

19. Indole Broth

- Tryptophan to Indole
Principle: Tryptophan present in peptone is oxidized by certain bacteria to
indole, skatole, and indole- acetic acid. The intracellular enzymes that
metabolize tryptophan are known as tryptophanase. Indole is detected
in broth cultures of bacteria with an alcoholic p- dimethylaminobenzaldehyde reagent. Indole reacts
with
the aldehyde to form a red product. Two reagents may be used to detect indole, Kovac’s and Ehrlich’s.
Ehrlich’s reagent is believed to be more sensitive than Kovac’s and is recommended for indole detection
in
anaerobes and non-fermentative bacteria.
Result: Positive: red ring at the interface of reagent and broth
Negative: absence of ring
Control: Positive: E. coli
Negative: E. aerogenes

20. Methyl-Red-Voges-Proskauer (MR-VP)

- detects type of fermentation of bacterial cell
- Methyl red: mix acid reaction
- Voges-Proskauer: glucose to acetoin (substrate:
potassium hydroxide) to butanediol fermentation
- Positive MRVP: S. aureus
Principle:
In the first pathway, mixed acid products result,
leading to a decrease in the pH of the medium and
a positive MR test. The pH must drop to 4.4 or less for the MR indicator to take on its acidic red color.
In the second pathway, acetyl-methyl carbinol (acetoin) is an intermediate product to butylene
glycol. Acetoin is the neutral product detected in the VP reaction. The broth should be heavily
inoculated,
with a small volume of broth used for the VP test to obtain favorable results at 24 to 28 hours of
incubation.
In the presence of oxygen and 40% KOH, aceotin is converted to the diacetic form, which results
in a red color in the presence of alpha- naphthol
Result: Positive MR test: distinct red color at surface of the medium
Negative MR test: yellow color at surface of the medium
Positive VP test: pink- red color at the surface of the medium
Negative VP test: Yellow color at the surface of the medium
Control: MR+,VP-: E. coli
MR-,VP+: E. cloacae
21. Simmon Citrate Reaction
Principle: Simmons citrate agar contains sodium citrate, which
serves as the only carbon source. If the organism can utilize
citrate, the sodium citrate is converted to ammonia, which is then
converted to ammonium hydroxide. The alkalinity of the
compound formed raises the pH of the medium, and the
bromthymol blue indicator takes in its alkaline color, which is
blue
Result: Positive: blue color
Negative: green color
Control: Positive: K. pneumonia
Negative: E. coli

22. Motility
Principle: Medium contains a small amount of agar which allows
motile bacteria to move out from the line of inoculation.
Nonmotile organisms grow only along the line of inoculation; 1% triphenyltetrazolium chloride may be
added to the medium to aid visualization of the reaction. Bacteria incorporate this colorless dye and
reduce it to a red pigment. Thus, reddening of the medium can be used as an indication for the extent of
bacterial growth.
Result: Motile: diffuse growth extending laterally from line of inoculation
Nonmotile: growth only along the line of inoculation
Control: Motile: P. mirabilis
Nonmotile: K. pneumonia

23. Decarboxylase Test

- Detects ability of microorganism to remove the carboxyl group
from an amino acid
Principle: Decarboxylases are enzymes that attack the carboxyl group of
specific amino acids, forming amines and carbon dioxide. The amines
formed are alkaline, and they alter the color of the pH indicator. The amino
acid to be tested is added to the Moeller base medium in a 1% concentration. Each decarboxylase
reaction is specific for a particular amino acid. Tests for lysine decarboxylase, ornithine decarboxylase
and arginine dihydrolase are generally performed on the enteric bacteria. Lysine is decarboxylated to
cadaverine; ornithine is decarboxylated to putrescine and arginine undergoes a dihydrolase reaction to
form citrulline, which is then converted to ornithine in decarboxylation. A control tube of Moeller
decarboxylase medium, which contains glucose but no amino acid, should be set up with each culture.
The medium contains bromcresol purple indicator which is purple in alkaline conditions and yellow in
acidic. Conversion of the control tube to yellow indicates the organism is viable and has fermented
glucose. During the early part of decarboxylation reaction, glucose is fermented leading to a yellow
color. As the pH decreases, an optimal env’t for decarboxylation occurs and the amino acid is
decarboxylated, which leads to an increased pH and purple-blue color.
24. Deaminase Reaction
- Removal of amino group from amino acid
Principle: Phenylalanine → (Phenylalanine deaminase) → Phenylpyruvic acid + 10% FeCl3
(green)
: Tryptophan → Tryptophan deaminase → indole-pyruvic acid +10% FeCl3 (brown)
Result: Intense green color: + deamination for phenylalanine
Brown color: + deamination for tryptophan
Control: Positive: P. vulgaris
Negative: E. coli

25. Lysine Iron Agar (LIA)

- Combination of decarboxylation and deamination
Principle:
LIA contains a small amount of protein, glucose, lysine,
sulfur, H2S indicator, agar and pH indicator bromcresol purple.
Lysine decarboxylation produces alkaline cadaverine and leads
to reversion of the deep from yellow to purple.
Lysine deamination occurs in the presence of oxygen
and results in production of a red color. H2S production is
noted by a black precipitate in the deep as H2S gas reacts with
ferric ammonium citrate
Result: Lysine decarboxylase positive: purple/purple
Lysine decarboxylase negative: purple/ yellow
Deaminase positive: red/yellow
H2S positive: blackening

26. ONPG Reaction

- Detect and confirm presence of late lactose fermenters
Principle:
Lactose fermentation requires 2 enzymes: lactose permease,
which actively transfers lactose into the bacterial cell, and beta-
galactosidase, which degrades lactose into glucose and galactose.
Non-lactose fermenters lack both enzymes, and those known as slow
or late lactose fermenters possess the beta-galactosidase but lack
permease. Lactose fermenters possess both enzymes.
ONPG is useful in detecting late lactose fermenters because the ONPG molecule is structurally
similar to lactose. ONPG can enter the bacterial cell without a permease. In the presence of beta-
galactosidase, ONPG is converted into galactose and o-nitrophenyl, which is yellow chromogen and the
alkaline end product
Result: Positive: yellow color within 20 min to 24 hours
Negative: No coloration
Control: Positive: E. coli
Negative: S. typhimurium
27. Urease Reaction
Principle: Urease splits urea molecule into ammonia, carbon dioxide
and water. Ammonia reacts in solution to form an alkaline compound,
ammonium carbonate, which results in an increased pH of the medium
and a color change in the indicator to pink-red.
Result: Positive: Red coloration
Negative: Absence of coloration

28. Oxidation-Fermentation Test (OF)

- Tube 1: cover with mineral oil to prevent entry of oxygen
- Tube 2: no mineral oil
Principle:
OF medium contains a high con’s of carbohydrate (1%) and a
small con’c of peptone (0.2%), which facilitates the oxidative use of
carbohydrates by non-fermenting gram-negative bacilli.
Medium used to identify Enterobacteriaceae is not suitable for
the non-fermenters for several reasons. First, the amount of acid produced during the oxidative process
is not sufficient for the indicator to take on its acidic color. Second, the alkaline end products produced
during the reaction neutralize the acid produced.
Result: Positive: a yellow color, as bromthymol blue indicator becomes yellow in an acidic environment
Negative: green or blue- green coloration
Open Tube Sealed Tube Interpretation
Yellow Green Oxidative +
Yellow Yellow Fermentative +
Green or blue Green No carbohydrate fermentation

29. Oxidase Test or Cytochrome Oxidase

Principle:
Used in the identification of Pseudomonas, Neisseria, Vibrio and Pasteurella. An oxidase
producing
organism will oxidize Phenylenediamine producing a deep purple color. Acidity inhibits oxidase activity
and
should not be used in colonies fermenting carbohydrates. Nitrate containing medium can be unreliable.
Oxidase Reagent: 10g/l solution of tetramethyl-p-phenylenediamine dihydrochloride.
Result:
Blue-purple color production (within 10 sec) Positive
No blue-purple coloration (within 10 sec) Negative

Control: Positive: P. aeroginosa

Negative: E. coli

30. Niacin Accumulation Test

- Differentiates tubercular mycobacterium to non-tubercular mycobacterium
Principle: Acidified potassium thiocyanate reacts with chloramines-T release
cyanogens chloride. In the presence of para- aminosalicylic acid, cyanogens chloride
reacts with free niacin to produce a yellow color
Result: Positive: yellow color
 Negative: clear
Control: Positive: M. tuberculosis
Negative: M. avium intracellulare

31. Litmus Milk Reduction

Principle: Lactose, casein, and lactalbumin are
major milk substrates capable of milk
transformation. Litmus is added to distinguish the
metabolic changes in milk. It is an excellent
differential medium to differentiate organisms
according to their milk metabolism
Result:
Positive: Acid pH: Pink to red
Alkaline pH: Purplish-blue color
Acid curd: hard curd with clear
supernatant
Rennet curd: soft curd following peptonization
Negative: no color change

32. Bile Solubility Test

- For S. pneumoniae vs. E. faecalis
Principle: An inoculum is emulsified in saline, sodium
deoxycholate is added. The sodium deoxycholate
dissolves S. pneumoniae indicated by clearing of
turbidity after 15 min.
Control: Positive: S. pneumoniae
Negative: S. faecalis

33. Tween 80 Hydrolysis Test

Principle: Tween 80 (polyoxyethylene sorbitan mono-oleate) is converted
to oleic acid by Tween 80 lipase. Neutral red indicator takes on its red
acidic color on the release of oleic acid
Result: Positive: pink to red color
Negative: no color change
Control: Positive: M. kansasii
Negative: M. chelonei

34. Phenylalanine Deaminase Test or Phenylpyruvic Acid (PPA)

- Differentiates P. providencia from other enterobactereales
Principle: Based on the ability of Proteus and Providencia to breakdown
phenylalanine with the production of phenyl-pyruvic acid. The cultured
organism on a slope is incubated overnight, the deamination of
Phenylalanine to Phenyl-pyruvic acid is detected by adding iron III Chloride (FeCl) which will then
produce a green color on the surface of the culture
Result: Positive: Green color
Negative: no color change
Control: Positive: Proteus spp.
Negative: E. coli

35. Motility Indole Urea Medium

Commonly used for differentiating
Enterobacteriaceae. Proteus spp. Are strong
urease producers.
Result: Positive: Red-pink color
Negative: no color change
Control: Positive: Proteus vulgaris
Negative: E. coli

36. Sulfide Production

Commonly used in the detection of Enterobacteria and can be used to differentiate Bacteroides and
Brucella. Sulfide is produced when sulfur-containing amino acid is decomposed
Methods:
a. Kligler Iron Agar (KIA)
• Best used for Entrobacteria
b. Lead acetate paper test
• Sensitive detection of sulfur
Control: Positive: P. vilgaris
Negative: Shigella

37. Arylsulfatase Test

Principle: Arylsulfatase splits the sulfate group from tripotassium
phenolphthalein to form free
phenolphthalein, which is detected by adding a small amount of an alkaline
solution, sodium carbonate
Result:
Control: Positive: M. chelonei
Negative: M. phlei

Lecture 7: GRAM-POSITIVE
COCCI
Micrococcaceae & Streptococcaceae

A. Micrococcaceae
- Divided into two genera:
i. Staphylococcus
Staphylococcus epidermidis Staphylococcus caprae
Staphylococcus haemolyticus Staphylococcus warneri
Staphylococcus saprophyticus Staphylococcus hominis
Staphylococcus lugdunensis Staphylococcus cohnii
Staphylococcus schleiferi Staphylococcus xylosus
Staphylococcus aureus Staphylococcus simulans
Staphylococcus capitis
- Commonly colonizers of the skin and of mucosal surfaces
- Can be considered as part of the normal flora
- May cause inflammation in the mucous membrane
- Considered as the most virulent
- Carbohydrate fermenters (glucose) but some are non-fermenters
- Group in clusters or grid like configuration
- Causes: suppuration, septicemia, food poisoning
- With variable hemolytic reactions with >30 species
- Rosenbach – Identified two specie according to pigment produced
- S. aureus: yellow S. epidermidis / albus: white
- Morphology: Spherical about 1 um arranged in clusters, gram positive, non-motile and non-
spore formers
- Culture:
● Aerobic and microaerophilic
● 37°C, room temperature (pigment production) requires 9% NaCl
● Resistant to drying
- S. aureus: gray – deep golden yellow S. epidermidis: gray – white

Culture Media
Media Information Significance
BAM Enriched / differential S. aureus – Beta hemolysis
S. epidermidis – gamma
hemolysis
MSA Color of Agar: Red S. aureus – golden yellow
Type: selective agar
Requirement: 7.5% salt 🡪 prevents growth of
other bacteria
Fermentation = acid production
Indicator: phenol red
VJ Content: mannitol & tellurite S. aureus – black with
yellow halo
S. epidermidis – black
without yellow halo
Baird Parker Content: LiCl2, egg yolk emulsion for S. aureus – black with clear
cholesterol content and tellurite zone (lipolysis of egg
because it produces lipase)
S. epidermidis – black
without clear zone
Chapman Enhances pigmentation S. aureus – yellow halo
Stone Contains mannitol and gelatin S. epidermidis – no yellow
halo
DNAse Agar Clearing around colony due to destruction of Green colonies
DNA
 Antigenic Structures
- Elicits antibody production
a. peptidoglycan – provide a rigid exoskeleton of the cell wall
b. Teichoic Acid – reaction with Ab is used in the diagnosis of endocarditis
c. Protein A – antiphagocytic ; causes coagulation ; binds with IgG molecules in
a wrong orientation ; disrupts opsonization and phagocytosis
d. Slime Layer (Biofilm) – found in some only, similar with the capsule

1. Staphylococcus aureus
General Characteristics
- Most virulent
- Can cause localized infection
- Non-fastidious meaning it does not require any special requirement and forms a pigment
(golden colony) at 20-25C
• Basis: chromogenic agar → Loffler’s Serum Slant
➢ S. aureus: golden colony
➢ S. albus or S. epidermidis: white
➢ S. citreaus: orange
➢ MRSA: mauve
Note: Gold colony of S. aureus may be observed in BAP during 16-18 hours
- Catalase positive
- Beta hemolytic

Virulence Factors
Catalase Anaerobic Catalase Test 15% H2O2
- Converts H2O2 into Aerobic Catalase Test 3% H2O2
H2O and O2 How does Catalase work?
- Differentiates When a pathogen enters inside the body, phagocytosis occurs.
Staphylococcus from During phagocytosis, neutrophil undergoes degranulation.
Streptococcus Degranulation is the release of granules from the neutrophils to
- 1 drop H2O2 in S. the pathogen. The granules are rich in H2O2 which oxidizes the
aureus colony and cell membrane of bacterial cell but since S. aureus is rich in
observed rapid catalase enzyme, instead the granules oxidizing, the oxidation
bubbling or reaction will be simply neutralize and is called Chronic
effervescence Granulomatous Disease (CGD).

Chronic Granulomatous Disease

Note: Staphylococcus
- A disease wherein during the process of degranulation H2O2
makes H2O2 useless is being neutralize by catalase enzyme
since it contains
catalase
Coagulase Slide Coagulase bound to cell wall
- Clots oxalated or Tube Coagulase free from the cell wall
citrated blood Coagulase – for fibrin clot formation
- For S. aureus only
What happens inside the body?
Due to presence of coagulase enzyme produce by the bacterial
 cell, fibrin will cover the S. aureus so that phagocytic cell will not
detect the presence of S. aureus, thus no phagocytosis will occur
Hemolysin Responsible for lysing blood cells seen in BAP giving a beta
hemolytic pattern
Leukocidin/ Valentine Responsible for lysing leukocytes
Phantom Leukocidin With several components which acts together
Damages membranes
Necrotizing skin infections
Fibrinolysin Breaks down fibrin clot
Lipase Break down fat
- S. aureus causes SSS or Staphylococcal Scalded Skin Syndrome
where Staph specie migrates to the dermal layer by breaking
down the fat.
- Upon breaking down the fat, exfoliative toxin is released and
epidermolytic toxin is also release to cause lysis of the skin
causing a scaley appearance of the skin
Hyaluronidase Spreading factor
- An enzyme that
breaks down
hyaluronic acid in
the connective
tissues
B-lactamase Destroys Beta lactam (antibiotic)
Protein A Necessary for coagglutination test
➢ An agglutination test where there is a carrier particle
➢ Direct agglutination: antibody directly reacts on the
antigen
➢ Indirect Agglutination: a carrier particle is utilized to
carry either antigen or antibody depending on the type
of test
Carrier Particles:
• Latex particle
• RBC
• Human, alligator, sheep
• Bentonite clay
• S. aureus since it contains Protein A which adheres
on the antibody present in the reagent
Anticomplement
No complement = no phagocytosis
Enterotoxin Causes food poisoning and emesis and gastrointestinal enteritis
Chemotaxis inhibitory Inhibits migration of neutrophils
protein
Exotoxin e. A-toxin or a-hemolysin
- Most potent membrane damage toxin
- Platelets and monocytes are sensitive
- Destroys by osmotic lysis
- In S. aureus

f. B-toxin (sphingomyelinase)
- Damages cells rich in lipids
- Encoded by lysogenic bacteriophage
 g. Gamma-toxin
- Peptide toxin with no known role in pathogenesis
- Both in S. aureus and S. epidermidis
Exfoliation Toxin Causes Staphylococcus Scalded Skin Syndrome (SSS)
Staphylokinase, TSS – superantigen that causes formation of rashes and multiple
Proteinase, Lipase, TSS organ failure ; causes interleukin increase
Toxin

Colonial Appearance
- Medium to large; smooth slightly raised, translucent
- Creamy yellow
- Beta hemolytic

Laboratory Detection
a. Tube Coagulase: Positive
b. Vogues-Proskauer: Positive
c. PYR/Pyrolidonyl aminopeptidase: Negative
d. Methyl Red: positive

Mode of Transmission
- Introduction to a sterile site by trauma such as surgery
- Can be introduced thru air and fomites (inanimate object that may serve as a reservoir)

Clinical significance
- Staphylococcal food poisoning due to enterotoxin
- Staphylococcal scalded skin syndrome, Toxic shock syndrome
- Bacteremia, endocarditis, pneumonia
- Folliculitis (pimples), furuncles (boils), impetigo, osteomyelitis in open fractures

2. Staphylococcus epidermidis
General Characteristics
- non- hemolytic
- coagulase negative staphylococcus
- part of CONS (Coagulase Negative Staphylococcus)

Colonial Appearance
- Small to medium, translucent, gray-white colonies
- Some are sticky that adhere to the agar
- Non-hemolytic
- Natural habitat: skin

Laboratory Detection
a. Tube Coagulase: Negative
b. PYR/Pyrolidonyl aminopeptidase: Negative
c. Novobiocin Susceptibility Test: Susceptible
d. Alkaline Phosphatase: Positive
e. Polymyxin B: Resistant
 f. Vogues-Proskauer: Positive
* In practice TUBE COAGULASE and NOVOBIOCIN SUSCEPTIBILITY TEST is Primarily used

Mode of Transmission
- Use of contaminated medical equipment and implanted heart valve
- Contaminated prosthetic devices

Clinical Significance
- Endocarditis and bacteremia following infection of cannulae, indwelling catheters, shunts or
other appliances positioned in the body
- prosthetic heart valve endocarditis

3. Staphylococcus saprophyticus
General Charactristic
- Urinary tract infections (UTI) in sexually active women
- Non hemolytic
- Saprophytic → feeds on dead organic matters
- Habitat: rectal canal

• NOTE: ALWAYS REPORT GRAM POSITIVE ORGANISM & GRAM-NEGATIVE

BACILLI in URINE
• DO NOT REPORT GRAM POSITVE BACILLI (ACIDophilia) – Normal Flora of
Urinary Tract of FEMALES, but it is NOT normal for MALES because of the
Alkalinity of their genitalia

Colonial Appearance
- Large, entire, very glossy, smooth opaque, and convex
- Usually white

• BOARD EXAM: Urinary Tract Infection (UTI) = common are E. coli (younger) and S. saprophyticus
(older)
• * E. coli is common in younger ages because of contamination from coliform or normal flora of
feces

Laboratory Detection
a. Tube Coagulase: Negative
b. PYR/Pyrolidonyl aminopeptidase: Negative
c. Novobiocin Susceptibility Testing: Resistant
d. Urease Test: Positive
e. Oxidase Test: Negative
f. Alkaline Phosphatase: Negative
g. Kloos and Scheifer Test: Positive

4. Staphylococcus lugdunensis
- At first glance it looks like S. aureus because it is Catalase: POSITIVE, Slight Coagulase POSITIVE
- Perform TUBE COAGULASE for differentiation b/w S. aureus & S. lugdunensis

Laboratory Detection
 a. Tube Coagulase: Negative
b. PYR Broth Hydrolysis: Positive
c. Novobiocin Susceptibility Test: Susceptible
d. Ornithine Test: Positive

Organism Colony App Catalase Coagulase Novobiocin Hemolysin

S. aureus Golded Positive Positive Sensitive Positive
S. epidermidis White-Gray Positive Negative Resistant Negative
S. White-Gray Positive Negative Resistant Negative
saprophyticus
Differential Tests
Test S. aureus S. saprophyticus S. epidermidis
Coagulase + - -
DNAse + - +
Mannitol fermentation + + -
Trehalose fermentation + + -

Sucrose fermentation + + +
Novobiocin S R S

i. Micrococcus
General Characteristics
- Most are non-pathologic
Laboratory Detection
Test Staphylococcus Micrococcus
Catalase + +
Oxidase - +
0.4 U Bacitracin R S
100 ug Furazolidone S R
200 ug/mL Lysostaphin S R
B. Streptococcaceae
- Lodges on lower respiratory tract
- Pairs or chains
- Most Group A, B and C are encapsulated
- Gram + and non-motile cocci with different reactions to hemolysis
- Does not belong to micrococcaceae
Classification of Streptococci
1. According to Hemolysis – on 5% sheep blood agar
a. Alpha hemolysis – incomplete ; greening of the surrounding media
b. Beta hemolysis – complete destruction of RBC ; zone of hemolysis (clearing) around
colonies
c. Gamma hemolysis – no destruction of RBC ; CM remain red
d. Alpha hemolysis or wide zone hemolysis – combination of the partial and complete
hemolysis

2. Group Specific Substance

- Lancefield classification (Rebecca Lancefield)
- Precipitin reactions with specific antisera determines arrangement
- A-H and K-U
- A-D, F and G are pathogenic to man
 Bergey’s Classification Smith and Brown Classification Lancefield Classification
Basis: pathogenicity and Basis: Hemolytic Pattern Basis: C-carbohydrate on the cell
morphology wall which contains antigenic
property
1. Pyogenic 1. Alpha Hemolytic (green) - incomplete 1. Group A
- pus producers 2. Beta Hemolytic (clear) - complete 2. Group B
– Strep. pyogenes, agalactiae, 3. Gamma Hemolytic - none 3. Group C
pneumoniae, scarlateae 4. Group D
5. Group H
2. Viridans 6. Group K
- produce incomplete hemolysis
- Incomplete hemolyzers
- Produce green decolorization
- Strep. mutans, sanguinis, mitis
& Abiotrophia & Rothia
dentocariosa

3. Lactoccus
- Milk Fermentors

3. Capsular Polysaccharide – Classifies S. pneumoniae into 84 types and S. agalactiae into

different sub-species

4. Biochemical Reactions
- CHO fermentation
- Presence of enzymes
- Anti-microbial susceptibility test

MEMBERS:
Streptococcus pyogenes Streptococcus bovis
Streptococcus agalactiae Abiotrophia
Streptococcus pneumoniae Enterococcus
Streptococcus mutans
Streptococcus mitis

- Non-motile and non-sporulating

- Grow best in solid media supplemented with blood, sugar and serum
- Mucoid colonies is seen among capsular strained streptococcus

• ALL COCCI ARE NONE MOTILE due to LACK of FLAGELLA which are common in BACILLI

Tests for Confirming Streptococcus

1. Phadebact Test
- Confirmatory test to determine Strep in CSF sample
Principle: Antibodies, raised in rabbit and specific against Streptococcus
pneumoniae, Neisseria meningitidis, Haemophilus influenzae type b or
Streptococcus agalactiae are bound to Protein A on the surface of non-viable
staphylococci (6,7). When a CSF-sample containing microorganisms belonging to one of these groups is
mixed with the reagents, the specific antigens on the surface of the microorganisms bind to the
corresponding specific antibodies. A co-agglutination lattice is formed, which is visible to the naked eye.
2. Fluorescent Antibody Test – antigen-antibody reaction with
fluorescent dye
3. Lancefield Precipitation Test – ring test → formation of ring due to
presence of an antigen

Members
1. Streptococcus pyogenes (Group A, B-Hemolytic)
General Characteristics
- Most aggressive pathogen encountered in the laboratory because it
cannot be easily seen by the body
- Systematic or localized infections
- Sometimes referred to as the “Flesh-eating bacteria”
- Gram positive cocci, in short chains, but also in pairs and singly
- Long chains are formed in fluid cultures ; main human pathogen
- Catalase negative ; susceptible to bacitracin ; beta hemolytic
- PYR positive ; contains Group A Ag ; pyrrolidinyl phosphate positive

Virulence Factors
Streptolysin Streptolysin S Aerobic Non Antigenic
- responsible for lysis of RBC in BAP Streptolysin O Anaerobic Antigenic
- Streptolysin O: utilize for diagnosis (immunogenic)
Streptokinase Lyse fibrin
Hyaluronidase Antigenic, Spreading factor
Leukocidin Destroys WBC
Lipoteichoic Acid Adhesion to pharyngeal cells
M-Protein Hair-like projections; major virulence factor
Mimic the protein present in the heart → myocardial protein
→ results to undetection by the body → propagates in the
throat and tonsils → migrates to the circulation resulting to
mild bacteremia → sore throat with febrile and red rashes
due to erythrogenic toxin → M protein sheds off as it travels
the circulation and the moment it reaches the heart, the body
now detects the streptococcus → immune reaction occurs in
the heart causing irregular pumping of blood leading to
rheumatoid heart fever (RHF) → Strep specie will be pulled
out and be transported to the kidney → the antibody and
strep is irreversibly clump together and may not penetrate
the glomerular filtration → will deposit on the nephron
causing to damage in the nephrons resulting to chronic
kidney disease or CKD-5
Remedy: dialysis
Nicotinamide Adenine Dinucleotidase Kills WBC
(NADase)
DNA-ase Breaks down DNA
Pyrogenic / Erythrogenic Toxin Red rashes
Group Specific Cell Wall Ag basis of classification by Lancefield (A-H, K-U)
T substance obtained from proteolytic digestion of Streptococci
Nucleoproteins makes up most of the body of the bacteria
Streptodornase Depolymerizes DNA
Together with streptokinase, it is used for debridement for
better access of antibiotic
Colonial Appearance
- Grayish-white, transparent to translucent, matte, with a large zone of beta-hemolysis

Laboratory Tests
a. Catalase Test: Negative
b. PYR Test: Positive
c. VP Test: Negative
d. Hippurate Hydrolysis Test: Negative
e. CAMP Test: Negative
Morphology: Gram + cocci in chains
Culture: BAP, 10% CO2 (speeds up hemolysis)

Mode of Transmission
- Person-to-person direct contact
- Contact with contaminated droplets produced by cough or sneeze

Clinical significance
A. Due to Invasion
- Streptococcal throat and tonsillitis
- Scarlet fever (Strawberry tongue)
- Erysipelas – erythema and edema
- Acute rheumatic fever
- Acute glomerular nephritis with the presence of RBC cast
- Streptococcal toxic shock- like syndrome (TSLS)
- Cellulitis, necrotizing fasciitis, puerperal fever (childbed fever), sepsis, ostitis,
pyoderma
B. Sequelae (disease caused by preceding disease)
- Acute endocarditis
- Sub-acute endocarditis
- TSS and Scarlet Fever
C. Post Streptococcal Infections (non-suppurative)
- Acute glomerulonephritis
- Rheumatic fever

Treatment
- Penicillin, Vancomycin, Erythromycin

ADDITIONAL NOTES
Diagnostic Test
a. Bacitracin Susceptibility Test (TAXO-A)
- 0.02 – 0.04 units Bacitracin
- Group A (pyogenes): susceptible Group B (agalactiae): resistant

b. BAM
- Staphylococci are bigger with smaller zone of hemolysis
- Streptococci are pinpoint with a larger zone f hemolysis
 c. SXT
- Sulfamethoxazole trimethoprim
- Group A (pyogenes): resistant Group B (agalactiae): resistant

d. PYR
- Group A (pyogenes): cherry red Group D (enterococcus): negative

e. 6.5% NaCl
- Principle: it is used to determine the ability of an organism to grow in high concentration of
salt. It is used to differentiate enterococci (positive) from non-enterococci (negative). A
heart infusion broth containing 6.5% NaCl is used as the test medium. This broth also
contains a small amount of glucose and bromcresol purple as the indicator for acid
production
- Group A (pyogenes): purple 🡪 negative Group D (enterococci): yellow 🡪 positive

f. 40% Bile
- Differentiates S. pneumoniae and pyogenes
- Principle: differentiates S. pneumoniae form alpha-hemolytic Streptococci. Bile salt such as
sodium deoxycholate rapidly lyses pneumococcal colonies. Bile salts lower the surface
tension between the bacterial cell membrane and the medium, accelerating the organisms
autolytic process
o Group A (pyogenes): negative (intact colonies)
o Pneumococci: positive (colony disintegrates)

g. Dick’s Test
- Serologic test ; toxins are injected intradermally
o Positive: redness at the site of injection
o Negative: no rashes (immune)

h. Schultz-Charlton Test
- Blanching phenomenon
- Differentiates rashes of scarlet fever from rashes of German measles
- Principle: at the site of rashes, anti-erythrogenic toxin is administered, the fading or
disappearance of rashes indicates that the anti-toxin is neutralized by the toxic effects of
pyogenic toxin
Scarlet fever: rashes disappear Measles: no disappearance

2. Streptococcus agalactiae (Group B, B-hemolytic)

 General Characteristics
- CAMP test- arrow- shaped hemolysis
- Normal flora of the Vagina
- May show double zone of hemolysis when incubated at reduced oxygen
- GBS or Group B Strep – standard test for pregnancy abroad
➢ CARROT TEST: Carrot media → orange colony

Colonial Appearance
- Translucent, flat, glossy, with narrow zone of beta-hemolysis

Laboratory Detection
a. PYR Test: Negative
b. VP Test: Negative
c. Hydrolysis of Hippurate Test: Positive
d. CAMP Test: Positive

Mode of Transmission
- Mother in utero transmission
- Nosocomial
- Contamination of sterile site

Clinical significance
- Neonatal: sepsis, pneumonia, bacteremia, meningitis, puerperal sepsis, maternal septicemia
- Skin infections, bacteremia, UTI, endocarditis

Treatment
- Ceftriaxone, Cefotaxime, Vancomycin

ADDITIONAL NOTES
Laboratory Test:
1. Sodium Hippurate Hydrolysis
- Principle: Na Hippurate – (hippuricase) 🡪 Na benzoate + glycine

2. 7% FeCl3 Test (reaction with Na benzoate)

- Positive: persistence of a precipitate or turbidity for more than 10 minutes

3. 0.1% Ninhydrin Test (reaction with glycine)

- Positive: deep purple

4. Camp Test
- Performed by Cristie, Atkins, Mumch and Peterson
- CycloAdenosine MonoPhosphate
- BAP + inoculate vertically the S. aureus + inoculate horizontally the suspected
organism + incubate 🡪 after 24 hours = arrow head hemolysis indicating presence of
CAMP factor indicating S. agalactiae
- Camp Factor
o Protein like compound that is able to react with the Beta-toxin by S. aureus to
produce an even more potent hemolysis
 - Positive: arrow-head hemolysis

3. Streptococcus pneumonia / S. lanceolatum (No Lancfield Group)

General Characteristics
- Gram positive elongated (lancet – flat on one end and tapered on other end)
diplococcus
- Pneumococci are nonmotile and capsulated (non-capsulated following culture)
- Normal flora of the Respiratory Tract

Colonial Appearance
- Small, gray, glistening
- Colonies dip down in the center and resemble a doughnut as they age
- Some are mucoid

Specimen: Blood, sputum

Laboratory Detection
1. Optochin Test: Susceptible
2. Quellung Test: Positive
- Good for rapid identification
3. Catalase Test: Negative

Mode of Transmission
- Person-to-person spread by contact with contaminated respiratory secretions

Clinical significance
- Lobar pneumonia, bronchitis ,meningitis, bacteraemia, otitis media, sinusitis and
conjunctivitis.
- Childhood pneumonia and serious infections in patients with sickle cell disease.

Treatment
- Penicillin, Ceftriaxone, Telithromycin, Levofloxacin

ADDITIONAL NOTES
Laboratory Tests
Specimen: Blood, sputum
Test Reaction
Optochin / Taxo P - 5 mg/mL of ethylenehydroxycupreine HCl
- antibiotic - Disc used:
o 6 mm 🡪 14 mm zone of inhibition
o 10 mm 🡪 16 mm zone of inhibition
- Reaction: Sensitive (pneumoniae)
Catalase Negative
 Neufeld - Quellung
Test

- Serologic test or immunological test, capsular swelling

test
- Utilizes an antibody and antisera that are bound to the
capsule of microorganism 🡪 observe for swelling
- Sample is added with a specific anti-polysaccharide
- Positive: capsular swelling after 20-30 minutes
Bile Solubility Test - Uses 10% Na taurocholate and 10% Na Deoxycholate
o S. pneumoniae: susceptible → dies
o S. viridans: Resistant

i. Broth / Tube Test

- Todd-Hewitt Broth + organism
- pH: 7 Bile salt: 6.5 indicator: phenol red
o positive (pneumoniae): clearing
o negative (viridans): turbidity

j. Agar / Plate Test

- 5% sheeps blood
- Uses Na deoxycholate
- Positive: clearing or dissolution of colonies
Dreft - 4% duodenyl sulfate
o S. pneumoniae: susceptible
o S. viridans: resistant

Animal Inoculation Death of mouse

(obsolete)

Clinical significance
- lobar pneumonia, bronchitis, meningitis, bacteremia, otitis media, sinusitis and
conjunctivitis, peritonitis
- childhood pneumonia and serious infections in patients with sickle cell disease.

Culture: alpha hemolytic enhanced by 5-10% CO2 ; glucose fermenters

Antigenic Structures
1. M-Protein – anti-phagocytic
2. Polysaccharide – for capsule in quelling reaction
Virulence Factors
1. Capsule
 2. C Substance – teichoic acid ; detected in CRP test
3. Pneumolysin – O – toxin that lyses RBC ; autolytic function
4. Neuramidase – invades host tissues

ADDITIONAL NOTES
Group C – Beta hemolytic – S. dysagalactiae, zooepidemicus and equi

Clinical Significance: puerperal sepsis, endocarditis, cellulitis

Laboratory Test:
1. 10% Bile solubility test: positive
2. Hippurate Hydrolysis: negative

4. Viridans Streptococci
- Produces a green halo surrounding the colony indicating an incomplete hemolysis
Members
a. S. mitis
b. S. mutans
c. S. salivarius
d. S. sanguis
e. Abiotrophia / S. meteor – requires pyidoxal or cysteine for growth

ADDITIONAL NOTES
- S. mutans, S. sanguis, S. mitior
o S. mutans causes dental carries or tooth decay
o S. sanguins causes septicemia
o S. mitior causes endocarditis

- Optochin resistant, Non- bile soluble, “Exclusion”

- Most prevalent member of the normal flora of the upper respiratory tract
- Alpha hemolytic or non-hemolytic
Clinical significance
- Bacteremia, dental caries, endocarditis

5. Enterococcus (Group D) non- hemolytic

- Normal flora of gastrointestinal tract but some are pathologic
- usually, non- pathologic except for E. faecalis
- E. faecalis (most infections), E. faecium, E. durans, E. avium
- Ferment lactose ; gram + bacteria that are found in the intestines
- Hydrolyze esculin ; contains group D ag (Teichoic acid)
- Reduce litmus milk
- Catalase negative
- Culture media: Buffered Azide Glucose Glycerol (BAGG)
 a. Enterococcus species: E. faecalis, E. liquifasciens, E. zymogens
b. Non-enterococcus: S. bovis, S. equines
Laboratory Test
1. Bile Esculin Test
- Uses the bile esculin agar (BEA) containing 1-4% bile salt
- Reagents:
● Esculin: 6-beta-glucoside-7-hydroxy cumarin
● Sodium azide: inhibits gram – organisms (Enterobacteriaceae)
- Positive: black-brown complex

2. 6.5% NaCl: positive

3. CAMP: negative
4. Heat Resistance Test – tolerates up to 60°C

Clinical significance
- urinary tract, biliary tract, ulcers (e.g. bed sores), wounds (particularly abdominal)
- endocarditis or meningitis, diarrhea
ADDITIONAL NOTES

• Group F: Streptococcus anguinosus / constellatus

• Group G: S. canis
• Group H: S. sanguis
• Group K: S. lactis
• Group N: S. cremoris
• Anaerobic Streptococcus: peptostreptococcus, peptococcus
• Nutritionally Variant Strep (NVS) / Satellite Strep
- Requirement: Vit. B6 (pyridoxal) for growth, cross-streaking with S. aureus to
provide necessary growth factors
- S. defectivus & S. adjacens

Species Catalase Bacitracin Optochin Bile Litmus CAMP

solubility milk
reduction
S. pyogenes Negative Resistant Susceptible Negative Negative Negative
S. agalactiae Negative Susceptible Susceptible Negative Negative Positive
Enterococci negative Susceptible Susceptible Negative Positive Negative
Viridans Negative Susceptible Susceptible Negative Negative Negative
S. Negative Susceptible Resistant Positive Negative Negative
pneumoniae

Lesson 8: Gram Negative Cocci

- uncommon, COCCI are mostly POSITIVE
A. Neisseriaceae
- Gram negative, oxidase positive arrange in pairs
- Habitat: Normal flora of respiratory, alimentary and genito-urinary tracts
- Mostly opportunistic ; kidney-bean shaped with polymononuclears
- Consideration in detection: collection
o During collection a special type of swab is utilized because a cotton swab may cause the
inhibition of Neisseria
Type of Swab
✔ Dacron and Rayon Swab – for collection of samples
✔ Amies Media with charcoal – transport media

Culture Media:
1. Transport Media
- Doesn’t support the growth of bacteria
- Fineburg, Transgrow, Amie-Stuart, Jembek

2. Selective Media
a. Chocolate agar media
- Blood is added to the media while it is at around 65°C to allow growth of bacteria by the
lysis of RBC

Growth Environment:
- Requires 5-10% CO2
- With the use of candle jar, gaspak
- Easily killed by drying, exposure to sunlight, moist heat and with disinfectants

MEMBERS:
Moraxella catarrhalis
Neisseria gonorrhoeae
Neisseria cinerea
Neisseria lactamica
Neisseria subflava

Neisseria polysaccharea
Neisseria sicca
Neisseria mucosa
Neisseria flavescens
Neisseria meningitidis
 P a g e | 76

Culture: (all are chocolate agar plate but differ in the presence of antibiotic)
Media Antibiotic Function
Component
Vancomycin Gram + inhibitor
Thayer-Martin Agar
Colistin Gram – inhibitor
Nystatin Fungi inhibitor
Vancomycin Gram + inhibitor
Martin Lewis Colistin Gram – inhibitor
Anisomycin Fungi inhibitor
Vancomycin Gram + inhibitor
Colistin Gram – inhibitor
NYC Media
Trimethoprim Swarming inhibitor
Amphotericin B Fungi inhibitor
Vancomycin Gram + inhibitor
Modified Thayer- Colistin Gram – inhibitor
Martin Trimethoprim Swarming inhibitor
Nystatin Fungi inhibitor
Vancomycin Gram + inhibitor
Lincomycin Gram + inhibitor
GC-LECT Colistin Gram – inhibitor
(for antibiotic therapy) Trimethoprim Swarming inhibitor
- Not all Gram (+) are Amphotericin B Fungi inhibitor
Vancomycin Susceptible
like Vancomycin Resistant
Enterococcus

1. Neisseria gonorrheae / Gonococcus

- Rapidly killed by drying, sunlight, heat, and disinfectant
- Grows best at CO2 enriched aerobic media
- The Clap/ Clapping disease
o 1st theory: the scrotum of person infected hardens resulting to the clapping sound
produced during intercourse
o 2nd reason: “les clapiers” French word for Brothels/ house of prostitutes
- Intracellular parasite with coffee bean shape

Note: cotton swab are not recommended for Neisseria and Chlamydia because the fatty acid of cotton
swab destroys the cell wall of neisseria and the wood on swab also destroy chlamydia
• Ryon and Dacron Swab: utilize for PCR ; utilize for vaginal or penile discharge

Laboratory Diagnosis
a. Ferment carbohydrate producing acids but not gas
b. Oxidase test: Positive
c. Dacron and Rayon Swab – for collection of samples
d. Amies Media with charcoal – transport media

Virulence Factors
Por (Protein I) Pore formation on the surface of the cell for nutrient entry
Opa (Protein II) Important protein for the attachment of bacteria to host cell
 P a g e | 77

RMP (Protein III) Associated with por

ADDITIONAL NOTES
Antigenic Structures
1. Pili – for attachment and phagocytosis resistance ; for conjugation
2. Por – forms pores on the surface for nutrients to enter
3. Opa – opacity association protein ; for adhesion
4. Rmp – reduction of modifiable protein ; associated with Por
5. Los – lipooligosaccharide with long O Ag like LPS
6. Lip – heat modifiable like Opa
7. Fbp – iron binding protein
8. IgA Protease – inactivates IgA

Types of Neisseria According to Colony Morphology

T1 and T2: piliated / smaller colonies ; pinpoint virulent
T3, T4, T5: non-pathogenic

Laboratory Test:
Specimen: pus and secretion from urethra, cervix, rectum, conjunctiva, throat and
synovial fluid
Smear: gram negative intracellular diplococci

a. Oxidase Test
- Uses tetramethyl-p-phenylenediamine dihydrochloride
- Principle: It is used to determine the presence of bacterial cytochrome
oxidase using the oxidation of the substrate tetramethyl-p-phenylenediamine
dihydrochloride to indophenol
- Positive: deep purple color within 10 seconds

b. CTA Fermentation Test

- Cysteine Tryptic Digest Semi-solid Agar
- Indicator: phenol red
- Positive: change to yellow color

c. ONPG
- O-nitrophenyl galactopyranoside
- Principle: the test is used to determine the ability of an organism to produced
Beta-galactosidase, an enzyme that hydrolyzes the substrate ONPG to form
o-nitrophenol
- Positive: yellow color

d. Chromogenic Enzyme Substrate Procedure

- Detects enzymatic activity

B-galactosidase Y-glutamyl Prolyl

(blue) aminopeptidase aminopeptidase
(yellow) (red)
N. lactamica + - +
 P a g e | 78

N. meningitidis - + +/-
N. gonorrhoeae - - +
B. catarrhalis - - -

e. Gonocheck II
o Red: N. gonorrhoeae
o Yellow: N. meningitidis
o Blue: N. lactamica
o Canary Yellow: Presumptive for Branhamella catarrhalis

f. Fluorescent Antibody Technique (FAT)

- Positive: green fluorescence 🡪 due to cyan green

g. Coagglutination
- S. aureus coated with gonococcal Ab is mixed with gonococcal suspension
- Principle: It uses antibody bound to a particle (protein A of Staph) to enhance
visibility of the agglutination reaction between antigen and antibody
- Positive: coagglutination

h. ELISA
- Principle: the basic test consists of antibodies bonded to enzymes, the
enzymes remain able to catalyze a reaction while attached to antibodies

Test N. N. M.
gonrrheae meningitidis catarrhalis
Superoxol / Catalase + - -
MTM, ML, NYC + + V
growth
Growth on NA @ 25C - - V
Growth on NA @35C - - +
Acid production
Glu + + -
Mal - + -
Suc - - -
lac - - -
DNase - - +
Nitrate reduction - - +
Tributyrin hydrolysis - - +
B- galactosidase - - -

b. NVG
- Nutritionally variant gonococci ; AHU auxotype
- Requires arginine, hypoxanthine and uracil

c. CMRNG
- Chromosomally developed due to antigenic variety of N. gonorrhoeae
IgA and IgG produced are type specific
 P a g e | 79

2. Neisseria meningitidis (A, B, C, Y, and W135)

- Causes meningococcal meningitis
- Rifampicin is used as prohpylaxis
ADDITIONAL NOTES
- With several serogroups: A, B, C, Y and W-135 are associated with human
diseases
- Same antigenic structure as gonococci ; encapsulated and piliated
- Humans are the only natural host
- Portal of entry is thru the nasopharynx

Clinical Significance: Bacteremia, meningococcemia, meningitis

Laboratory Diagnosis
Specimen: blood, CSF, nasopharyngeal swab
Smear: shoes gram – diplococci that are intracellular or extracellular
Culture: CAP incubated at 5% CO2 tension at 37%
Tests: same with all other Neisseria

a. DNA Testing
- Genonotipic identification of microbial genes and no the gene products
1. Nucleic Acid Hybridization
2. Amplification
3. Sequencing and enzymatic digestion of nucleic acid

b. Particle Agglutination – serological testing ; latex agglutination

Lecture 9: Gram Positive Bacilli

- BACILLI are commonly GRAM NEGATIVE , Its not
common to have Gram Positive

a. Spore Forming
i. Bacillus
- Ubiquitous, large gram + rods in chains
- Spores are located centrally, subterminally or terminally
- Sterilized only by autoclaving
- Appears like an inverted fir tree on gelatin stab
- Appears as round “cut-glass” on culutre

Note: the spore of fungi is used for reproduction. In bacteria, spores will encapsulate the genetic
material of bacterial cell thus, preventing it from dying

1. Bacillus anthracis – “Anthrax Bacillus”

- Major gent in bioterrorism (Used as a biological weapon and for community infection)
- Bamboo fishing rod appearance or box car shaped bacilli
- Encapsulated and with a central spore

Virulence factors
1. Capsule – may be lyse or destroyed by Na desoxycholate creating a slimy colony
2. Protein Antigen (PA) – forms a channel that mediates entry of EF and LF
 P a g e | 80

3. Exotoxin
o Protective antigen(protection)
o Edema factor(edema) – adenylate cyclase forms the edema toxin
o Lethal factor(death) – LF + PA = lethal toxin 🡪 causes cell death

Laboratory Diagnosis
a. BAM: non-hemolytic medusa head colonies or frosted glass
b. Polymyxin- Lysozyme- EDTA- Thallous- Acetate: best selective medium
c. Gelatin liquefaction test: “inverted fir tree” on motile strain, non-motile
d. Pearl- string test: BAP + 5 units penicillin → mucoid colony → pearl string →
positive
- Reagent: Na desoxycholate

e. Ascoli test: positive

Clinical significance
a. Cutaneous anthrax (Black Eschar) – contact with the spore
b. Pulmonary anthrax (Woolsorter’s dse) – inhalation of the spore
c. Intestinal anthrax (Violent enteritis) – ingestion of the spore
* SPORE FORMING ORGANISM = SPORE is the VIRULENCE FACRTOR
ADDITIONAL NOTES
Culture Media
a. BAM – lion head, medusa head, chain of pearls, comet tail, barrister’s wig
appearance non-hemolytic
b. (PLET) Polymyxin- Lysozyme- EDTA- Thallous- Acetate – best selective
medium ; beaten egg appearance
c. NA with 5% NaHCO3
d. NB – heavy pellicle with little subsurface growth

Laboratory Diagnosis:
Specimen: body fluid, pus, blood, sputum
Smear:
- Gram + bacilli, encapsulated with spores
- Immunofluorescent stain may also be used

Tests
a) Animal Innoculation
b) Serological Test
1. Ascoli Test
- Thermo precipitation test for the diagnosis of anthrax using a tissue
extract and anthrax antiserum. Used for detection of anthrax bacilli
in animal hides and meat
- Positive: ring of precipitate
2. ELISA Test: pink color

2. Bacillus subtilis – common laboratory contaminant; Hay Bacillus

- may also caused food poisoning ; opportunistic
 P a g e | 81

3. Bacillus cereus – “fried rice bacillus”

- Swarming motility in semi-solid media
- Beta-lactamase producer

Laboratory Diagnosis
a. Lecithinase: positive
b. Gelatin hydrolysis: positive

Virulence factors
- Enterotoxin, lecithinase, proteinases, nucleases
- Dermonecrotic and lethal toxin, hemolysisns

Clinical significance
- Food poisoning (2 syndromes)
a. Emetic syndrome – 1-5 hours of incubation (fried- rice, pastries, noodles)
b. Diarrheal syndrome – 8-16 hours of incubation (vegetables, sauces, meat products)

ADDITIONAL NOTES:
- Non-encapsulated ; produces enterotoxin
- Present in normal stool ; acquired from eating contaminated fried rice
- Not the most cause of food poisoning
o Staphylococcus food poisoning is the most common cause of food
poisoning
5
- >10 bacteria per gram food is diagnostic
- Two Types of Infection:
a. Emetic Type
- Seen in fried rice and pasta
- Disease is self-limiting
b. Diarrheal Type
- In meat dishes and sauces
- Shows profuse diarrhea

- Lecithinase positive
- Motile
- Gelatin hydrolysis positive
- Grows in food
Virulence factors
- Enterotoxin, lecithinase, proteinases, nucleases
- Dermonecrotic and lethal toxin, hemolysisns

Clinical significance
- Food poisoning (2 syndromes)
a. Emetic syndrome – 1-5 hours of incubation (fried- rice, pastries,
noodles)
b. Diarrheal syndrome – 8-16 hours of incubation (vegetables, sauces,
meat products)

Difference between Bacillus and Clostridium:

Clostridium: anaerobic Bacillus: aerobic
 P a g e | 82

ii. Clostridium
- Motile ; found in soil and in GIT (of animals)
- Carbohydrate fermenters
- Culture: requires 10% CO2 ; aerobic incubator, candle jar, roll tube
- Clinical Significance:
Gas Gangrene, C. perfringens
Myonecrosis, Clostridial
Uterine Infection, Diarrhea
/ Food Poisoning
Tetanus C. Tetani
Food Poisoning C. botulinum, C. perfringens
Pseudomembranous colitis C. difficile
Miscellaneous C. ramosum, C. birementans, C. sporogenes

1. Clostridium botulinum
- Anaerobic
- Animal feces, “snow shoe” subterminal spore, swollen
- Most fatal type of food poisoning because in <24 hours a person may die
- Specimen: food remnants or stool
- Tests: animal inoculation & neutralization tests
-

Virulence factor
- Botulinum toxin (lethal dose – 1-2ug)

Laboratory Diagnosis
- Observance of toxin in food and serum
- Mice injection test – death of mice after injection of toxin
Clinical significance
- Botulism- food poisoning (incubation pd- 18-24 hrs)
o food poisoning (incubation pd: 18-24 hrs)
o intoxication ; also isolated from vacuum packed food
o s/s: visual disturbances, difficulty breathing or swallowing, paralysis leading to death

- Infant botulism
o Common cause of SIDS taken from improperly processed or fresh honey
o s/s: poor feeding, weakness, floppy baby (paralysis)

- Wound botulism
- Flaccid Paralysis / Floppy Infant syndrome
➢ In a normal muscle, muscle constrict when there is
acetylcholine and relaxes upon release of GABA
➢ In cases of C. botulinum there is blocker
(botulinum toxin) against acetylcholine thus, no
constriction occurs and only relaxation happens
 P a g e | 83

2. Clostridium tetani – “tetanus”

- Feces of horses and other animals, “lollipop, drumstick”, terminal spore, swollen
- Opposite of C. botulinum wherein, it blocks GABA resulting to spastic paralysis
- Toxin inhibits the inhibitory amino acid glycine and gamma aminobutyric acid (motor neurons)
are not inhibited resulting to continuous contraction

Virulence factor
• Tetanospasmin- neurotoxic; spastic paralysis (lethal dose- 2.5 ng/ kg) due to inhibition of GABA
• Tetanolysin – hemolytic property

Laboratory Diagnosis
a. Anaerobic culture

Clinical significance
- Tetanus (incubation pd – 4-5 days)
o Trismus (lock jaw)
o Risus sardonicus (Devil’s grin) – due to
inhibition of GABA which is responsible
in relaxation thus, there is continuous
constriction
o Opisthotonus (Arching of back) – due to
excess muscle constriction

Infection of Germination of the inhibition of

spore and
tissue with the development of
GABA secreting
spore vegetative organism neurons

- Neonatal tetanus

3. Clostridium perfringens or C. welchii - “Welch’s bacillus”

- Surgical incision, soft tissue infection, central spore, swollen, capsulated
- BAM – target hemolysis
- Process: spore germinates 🡪 cells multiply 🡪 ferments CHO 🡪
releases gas 🡪 distension of tissue, interference with blood,
release of toxins and hyaluronidase
● Results to spread of infection
● Necrosis extends with the increase in bacterial
growth
● Anemia, toxemia and death results

Virulence factors
- Alpha toxin - lecithinase
- Theta toxin - hemolytic and necrotizing
- Collagenase
- Hyaluronidase
- DNAse
- Enterotoxin – isolated from meat dishes ; diarrhea (same with B. cereus)
- Lambda - enzyme
 P a g e | 84

Laboratory Diagnosis
a. Lactose fermentation: reddening of the medium and colonies when exposed to air
b. Litmus Milk: stormy clot by fermenting litmus milk

Clinical significance
- Gas gangrene / myonecrosis
- Food poisoning

4. Clostridium difficile
- May be a normal flora in the colon in low concentration,
- community acquired diarrhea
- subterminal spore, non-swollen
- Remedy include: Ampicillin, clindamycin and cephalosporin but will inhibit normal flora
- Cycloserine Cefoxitin Fructose Agar

Virulence factors
- Toxin A – potent enterotoxin
- Toxin B – potent cytotoxin

Clinical significance
- Pseudomembrane colitis – pseudomembrane or
microabscesses

b. Non-Spore Forming
i. Corynebacterium (Aerobic)

1. Corynebacterium diphtheria – “Klebs-Loeffler” bacillus

- Possess irregular swelling at one end that give them a “club-shape
appearance”
- Forms metachromatic granules/volutin/babes-ernst granules near
the pole (beaded appearance)
- “Chinese letters or palisades” due to incomplete separation of
cytoplasm (V, Y, X, Z configuration)
o Schick’s Test – susceptibility test (injection with diphtheria toxin)
o Elek’s Test – virulence test (uses agar immunodiffusion)
- Loeffler’s methylene blue stain (Albert’s stain)
- Potassium Tellurite medium
- Burke’s Modification of Gram Stain
- Special Media: Philly Gorman Media
Biotypes
Biotype Appearance in Loeffler’s Agar Appearance in Potassium Tellurite

Gravis Club shape, few granulation Daisy Head colonies

Intermedius Short irregular rods without granules Frog Eggs
but in Chinese Letter configuration
Mitis Classic morphology with numerous Poached Egg appearance
granules and typical arrangement
Balfanti
 P a g e | 85

Virulence factors
▪ Pili
▪ Diptheria Toxin (Tox Gene)
- Action: causes ADP ribosylation of EF2 (for
protein synthesis)
- EF2 + NAD 🡪 EF2-ADP-ribose + nicotinamide
+ H- (inactivate substances) 🡪 stops protein
synthesis
- Result: causes cell death at the respiratory
tract

▪ Dermonecrotic Toxin – increases vascular

permeability
▪ Hemolysin A – responsible for hemolysis
Specimen: swabs from nose, throat or other lesions

Laboratory Diagnosis
a. Acid production
b. Serotyping by agglutination
c. Guinea pig inoculation test / Schick Test: injection of strain (anti-diphtheria) in one of the two
guinea pigs; death of guinea pig after 23 days
d. Gel-precipitation test or Elek’s Test
• Filter paper with toxin is incorporated in a
serum agar
• Suspected C. diphtheriae in streaked at the agar
in a right angle to the filter paper
• Observe precipitation line after 1-2 days
e. Schick Test: used to determine immunity due to
immunization or natural infection; observation of
erythema at the injection (0.2 ml of toxin) site at
36 and 120 hours
• Toxin reaction – slower and longer lasting
Clinical significance
- Diphtheria
- Myocarditis, polyneuritis
- Cutaneous Wound Infection
ADDITIONAL NOTES
Tests:
1. Animal inoculation
- Antitoxin is administered to one guinea pig while the other is not after the
antigen Is injected intracutaneously
- Unprotected: death Protected: survives

2. Elek’s Test / Forbisher’s Test

- A strip of filter paper is saturated with antitoxin is placed on agar with 20%
horse serum. A culture is streaked across the strip and incubated at 37°C for
24 hours
- Positive: presence of precipitin lines
 P a g e | 86

Tissue Culture Test

- Incorporation of bacteria on agar with cell culture monolayer
- Positive: cell death
3. FAT
4. Carbohydrate fermentation
5. PCR

Detection:
1. Titration of serum for Antitoxin
2. Schick’s Test
- Principle: diphtheria toxin is very irritating and results in a marked local
reaction when injected intradermally causing swelling and induration

Schick’s Dose
- Amount of standard toxin that when mixed with 0.01 U diphtheria anti-
toxin and injected intradermally into a guinea pig will induce a 10 mm
erythematous reaction
o Positive: induration <0.01 Lf U/mL, susceptible
o Negative: no reaction >0.02 Lf U/mL, immune to diphtheria

Biotypes
Biotype Appearance in Loeffler’s Appearance in Potassium
Agar Tellurite
Gravis Club shape, few granulation Daisy Head colonies
Non-hemolytic ; Medium, white,
opaque
Intermedius Short irregular rods without Frog Eggs
granules but in Chinese Letter
configuration
Hemolytic ; Medium, white,
opaque
Mitis Classic morphology with Poached Egg appearance
numerous granules and typical Coolie hat
arrangement
Non-hemolytic ; Small, gray,
translucent

2. Propionlibacterium acnes – casues ACNE

3. Corynebacterium ulcerans, Corynebacterium pseudotuberculosis – may carry diphtheria tox gene
4. Corynebacterium jeikium – immunocompromised patients
5. Listeria monocytogenes
- Distinct feature: Tumbling end- over- end motility (25C) due to the presence of flagella
- Acquired from Unpasteurized milk and raw vegetables
- Cold enrichment technique is recommended
- Zone of accentuated hemolysis in CAMP test

Virulence factors
- Internalin
- Listeriolysin O – hemolytic and cytotoxic (anti-phagocytic)
 P a g e | 87

- Act A surface antigen

- Monocytosis–producing agent

Laboratory Diagnosis
a. BAP: narrow zone of beta-hemolysis
b. Motility agar: umbrella pattern
c. Catalase Test: Positive
d. Oxidase Test: Negative

Clinical significance
- Listeriosis
a. Neonatal listeriosis (granulomatosis infantiseptica)
- Granulomatosis infantiseptica
- Intrauterine infection
- May also be disseminated causing menigitis

b. Adult listeriosis

6. Erysipelothrix rhusiopathiae – “seal finger, whale finger”

- Non-spore forming, non-motile, exhibits H2S production
- Clinical Significance: Erysipelas, Erysipeloid, Bacteremia
- Laboratory Diagnosis: skin biopsy

7. Gardnerella vaginalis
- Non spore-forming, non-branching
- Bacterial vaginosis
- Clue cells - Usually infect the CYTOPLASM of Epithelial Cell
ADDITIONAL NOTES:
8. Lactobacillus acidophilus / Doderlein’s Bacillus
- Pleomorphic rods
- Grows on Rogosa’s Selective Tomato Juice Agar
- Normal flora of the vagina, GIT and oral cavity
- Maintains the acidity of the vagina

Clinical Significance: endocarditis, meningitis

9. Eubacterium alactolyticum – seagull-wing shaped on smear

10. Bifidobacterium – gram positive rods which bifurcate at its ends resembling “Dog
Bones”

ii. Actinomyces (Anaerobe)

- Bacteria-like fungi
- Common soil saprophytes
- Facultative anaerobe substrate
- A heavy infection, often mistaken as a tumor

Group Actinomycetes (aerobic) Genus Actinomyces (anaerobic)

 P a g e | 88

Nocardia spp, Stretomyces spp, Actinomadura Actinomyces israelii

spp, Mycobacterium spp

1. Streptomyces
- Source of antibiotics
Antibiotic Source
Streptomycin S. griseus
Erythromycin S. erytheus
Aureomycin S. aerofaciens
Oleandomycin S. antibioticus
Spiramycin S. ambofacious

2. Actinomyces israelli
- Spider colonies – Molar Tooth colonies, sulfur granules, Spider Web colony
- Actinomycosis (cervicofacial, thoracic, abdominal)- also caused by Actinomyces Naeslundii

Lesson 10: Gram Negative Bacilli

a. Fermenting Bacilli
i. Enterobacterales

ii. Enterobacterales

Organism TSI Gas H2S Ind MR VP Cit PAD Ure ONPG

E. coli A/A + - + + - - - - +
Shigella A, K/A - - -/+ + - - - - -
B, C
S. sonnei K/A - - - + - - - - +
E. tarda K/A + + + + - - - - -
Salmonella K/A + + - + - + - - -
C. freundii A/A + + - + - + - +/- +
K/A
C. diversus K/A + + + + - + - +/- +
K. A/A ++ - - - + + - + +
pneumonia
K. oxytica A/A ++ - + - + + - + +
E. A/A ++ - - - + + - - +
aerogenes
E. cloacae A/A ++ - - - + + - +/- +
H. alvei K/A + - - -/+ + - - - +
S. K/A + - - -/+ + + - - +
marcescens
 P a g e | 89

P. vulgaris K/A +/- + + + - -/+ + ++ -

P. mirabilis K/A + + - + +/- +/- + ++ -
P. stuartii K/A - - + + - + + -/+ -
M. morganii K/A + - + + - - + ++ -
Y. K/A - - +/- + - - - +/- +
enterocolitica

TSI Reaction
A/Ag - Escherichia, Klebsiella,
Enterobacter
K/Ag + Salmonella, Proteus
K/A - Shigella
K/K - Pseudomonas

Triple Sugar Iron Agar – slant butt media

o Purpose of Slant: increases oxygen intake and entry for fermentation
o Purpose of Butt: decrease oxygen
o 3 Sugars Present: Glucose, Lactose, Sucrose
Gas Formation: may be seen in TSI ; indicated by a crack or gas
H2S: may be seen in TSI ; indicated by blackening of the butt
IMVIC Test contains: TSI, Gas, H2S, Indole, MR, VP, Citrate

LIA
− Ability to deaminate lysine (slant) and decarboxylate lysine (butt)
K/K + (purple/purple with black) Salmonella
K/A – (purple/yellow) Shigella
R/A – (red/yellow) Proteus, Providencia, Morganella

Families Genus
Enterobacteriaceae Escherichia, Citrobacter, Enterobacter, Klebsiella, Salmonella, Shigella
Erwiniaceae Erwinia, Pantoea
Pectobacteriaceae No human pathogen
 P a g e | 90

Yersiniaceae Yersinia, Ewingella, Serratia

Hafniaceae Hafnia, Edwardsiella
Morganellaceae Morganella, Proteus, Providencia
Budviaceae Leminorella

General Characteristics
- All are glucose fermenting with acid production
- All causes UTI and gastroenteritis

Antigens of Enterobacteriaceae:
- H Antigen – flagellar antigen and may interfere with O antisera
- K Antigen – Heat labile and associated with capsular polysaccharide
- O Antigen – found in the cell wall of Enterobacteriaceae; has affinity to IgM
• HEMOLYTIC UREMIC ORGANISM: E. coli O157: H7

Laboratory Diagnosis
Lactose Fermenter Non-Lactose Fermenter
Oxidase Negative Eshcerichia Salmonella
Klebsiella Shigella Proteis
Enterobacter
Citrobacter

1. Klebsiella pneumoniae, K. rhinoscleromatis, K.ozanae

Laboratory Diagnosis
a. MacConkey Agar: Large, mucoid, lactose fermenting
b. Capsular Serotyping (for Pneumonia causing pathogen): Positive
ADDITIONAL NOTES
1. Klebsiella
- Type specie: Klebsiella pneumoniae
- Non-motile, lactose fermenter with large sticky colonies on culture media

Virulence Factor
- Heat stable enterotoxin (ST)
- R-Plasmids
• Resistance plasmids confers resistance to antibiotics like ampicillin
• May be transferred to other bacteria of the same specie

Clinical Significance: Pneumonia, UTI (second to E. coli)

Laboratory Indications: non-motile

Lysine: + Citrate: + Indole: - Ornithine: - TSI reaction: +/+ w/
gas (A/A)

2. Enterobacter aerogenes
 P a g e | 91

ADDITIONAL NOTES
2. Enterobacter
- Highly motile, lactose fermenter and nosocomial
- Similar to Klebsiella in terms of biochemical activity except that it is ornithine
positive
- Specie: Enterobacter aerogenes, Enterobacter cloace

Laboratory Indications: motile

Lysine: + (except E. cloacae) Citrate: + Indole: -
TSI reaction: +/+ with gas Ornithine: +

3. Citrobacter freundii
ADDITIONAL NOTES
1. Citrobacter
- Normal flora ; late lactose fermenter and nosocomial
- Citrobacter fruendii → causes diarrhea and extraintestinal infection

Laboratory Indications:
Lysine: - Hydrogen Sulfide: + (C. frundii) Citrate: +
TSI reaction: +/+ w/ gas (A/A)

4. Salmonella typhi, S. paratyphi, S. enteritidis

- Non-sucrose fermenter, but ferment glucose and mannose

Laboratory Diagnosis
a. Bacteriology Test
- Generally, gas and sulfur producing

Differential media EMB, MacConkey, Deoxycholate agars

Selective media SS agar, Hektoen Enteric agar, XLD agar, Deoxycholate-Citrate agar
Enrichment media Selenite broth, Tetrathionate broth

b. Serologic Test
1. Tube dilution agglutination test (Widal Test)
- Obsolete, replaced by TyphiDot, TUBEX (iron granules where
salmonella toxin attaches to) etc.
o Result interpretation
• High titer of O antigen – active infection
• High titer of H antigen – past infection or immunization
• High titer of Vi antigen – present only in some cases

2. Slide agglutination test (Kauffman-White Test) – used to identify unknown cultures

Clinical Significance: Different TYPES of TYPHI

Characteristic Enteric Fever Septicemia Enterocolitis
Incubation 7-20 days variable 8-48 hours
 P a g e | 92

Onset Insidious Abrupt Abrupt

Duration Several weeks variable 2-5 days
GIT symptoms constipation None Diarrhea
Blood culture (+) in the 1st and 2nd (+) in high fever Negative
week
Stool culture (+) in 2nd week (+) (+)
Organism S. typhi and S. S. choleraesuis S. enteritidis and S.
paratyphi typhimurium

ADDITIONAL NOTES:
2. Salmonella
- Causes typhoid-like typhus (rashes & diarrhea)
- In 19th Century, all enteric fevers were characterized as typhoid
- Observed by Ebeth ; serodiagnosis was made in1896
- Major reservoir: chicken
- With 2200 specie

a. Salmonella typhi
- Carried by man ; some strain may produce a thermolabile toxin (LT)
similar to choleragen
- Non-lactose fermenter

Antigenic Structures:
1. O Ag
- OHNE: body ; somatic or cell wall Ag
- Heat stable and alcohol resistant

2. Surface Ag – envelope Ag ; also found in E. coli & Klebsiella

3. Vi Ag – found in typhi, paratyphi C & Dublin serovars
4. H Ag – flagellar Ag ; heat labile

Clinical Significance: Enteric or typhoid fever, Bacteremia, Diarrhea

Laboratory Diagnosis:
Specimen: blood, serum, stool
Culture:
1. Enrichment: Selenite-F broth
2. Differential: MacConkey, EMB, XLD, HEA, BSA
3. Selective: SSA

Serologic Tests:
1. Widal Test
- detects the presence of febrile agglutinins panel (OHAB –
somatic, flagellar, paratyphi A & B ; an antibody)
- Positive: agglutination with different concentration
- Indication of Infection: 1:8 ratio
 P a g e | 93

2. ELISA – typhirapid (detects salmonella accurately), typhidot

Laboratory Indications:
Lysine: + Hydrogen Sulfide: + Indole: + Citrate: +
ONPG: - TSI reaction: -/+ w/ gas Malonate: -

5. Yersinia pestis
- Short and pleomorphic aerophiles and exhibit bipolar staining
- Mouse Lice (Xenopsylla cheopis/ Xenopsylla Lice) – FOOT
Laboratory Diagnosis
a. Stain: Wright’s, Methylene Blue stain, Wayson’s stain (for bipolar
staining seen in Pasteurella, Yersinia and Morganella)
b. Catalase Test: positive
c. Oxidase Test: negative

Clinical Significance
a. Bubonic Plague – Inflammation in Lungs and Lymph nodes( Bubos)
b. Pneumonic Plague
c. Septicemic Plague

ADDITIONAL NOTE
3. Yersinia
- Invasive, penetrates the gut lining entering the lymphatics then the blood
stream of man
- Acquired by ingesting contaminated food ; non lactose fermenter
a) Yersinia enterocolitica
- Able to grow at cold temperature and motile at room temperature
- Releases enterotoxins causing severe intestinal pain
- Clinical significance: enterocolitis – severe intestinal pain with
diarrhea

Laboratory Indications:
Urease: + Ornithine: + TSI reaction: +/+ w/out gas Motility:
motile (at RT)

b) Yersinia pestis
- Vector: flea
- Releases toxins which inhibits the electron transport protein
- Clinical Significance: Bubonic Plague, Pneumonic Plague

Laboratory Indications:
Urease: - Ornithine: - Motility: non-motile (at room temperature)
 P a g e | 94

6. Proteus spp.
- Highly motile with peritrichous flagellation
- Shows swarming colonies on CM
- Resistant to some antibiotics
- P. mirabilis (indole: -)
- P. vulgaris (indole: +)
- Clinical Significance: wound infection and UTI
Laboratory Indications:
- Lysine: - Hydrogen Sulfide: + Urease: + Motility: motile

Laboratory Diagnosis
a. Culture: swarming motility
b. MacConkey: Non-lactose fermenting
c. Indole test: P. vulgaris (positive) and P. mirabilis (negative)

7. Shigella
- Producer of endotoxin (dysenteri) and exotoxin (neurotoxin)
- Discovered by Shiga ; colorless
- Anaerobic and non-lactose fermenters
- Diarrhea is accompanied with fever ; invasive

Virulence Factors:
1. Endotoxin
- released upon autolysis causing irritation to the bowel
- LPS component of gram – bacteria
- Upon death of shigella specie, it releases endotoxin causing diarrhea

2. Shiga Toxin / Verotoxin

- Produced by S. dysenteriae
- Inactivated by steam treatment, bleach, glutaraldehyde
- <20 ug/kg injected peritoneally to mouse is fatal

Clinical Significance: Dysentery, Reiter’s Syndrome, Hemolytic Uremic Syndrome

Laboratory Indication: Lysine negative, non-motile, +/- TSI reaction with no gas production
(K/A), NLF
-
Shigella dysenteriae Group A Shiga’s bacillus Japanese diarrhea Bacillary
dysentery
Shigella flexneri Group B Flexner’s/ Strong’s Philippines diarrhea
bacillus
Shigella boydii Group C Boyd’s bacillus British diarrhea
Shigella sonnei Group D Sonnei- Duval’s US diarrhea
bacillus
• NOTE: Salmonella is POSITIVE in ALL TEST while Shigella is NEGATIVE in ALL TEST

8. Escherichia coli
- (Theodor Escherich)
- Most common bacteria encountered in the laboratory (genetics)
- No. 1 cause of UTI in children
- Produces vitamin K from undigested material in the large intestines
 P a g e | 95

- With >700 sero-types based on O, H and K antigens

- Lactose fermenters

Laboratory Diagnosis
a. Indole Test: Positive
b. Nitrate Test: Positive
c. MacConkey Agar: Lactose fermenting with mucoid colonies

Disease Virulence factors

Adherence Toxins
Enterotoxigenic E. coli ( ETEC) Colonization factors of adherence, Endotoxin; Heat labile
– Has adhesion to the Type 1 pili enterotoxin (LT), Heat stable
epithelium of the small enterotoxin (ST)
inrestines
Enteropathogenic E. coli (EPEC) Bundle- forming pili (BFP), Type 1 Endotoxin
– Self-limiting type of pili, Intimin
diarrhea
Enteroaggregative E. coli (EAEC) Mucus- associated Endotoxin, cytotoxin (
– Produces Shigella toxin- autoagglutination, Type 1 pili Enteroaggregative ST- like toxin
like EAST)
Enteroinvasive E. coli (EIEC) Shigellosis Afimbrial adhesions, Type 1 pili Endotoxin
like diarrhea
Enterohemorrhagic E. coli (EHEC) Afimbrial adhesions, Type 1 pili Shiga toxin, verotoxin, Endotoxin
Hemorrhagic colitis, hemolytic uremic
syndrome
– Cytotoxic verotoxin
producing E.coli O157:H7

ADDITIONAL NOTES:
Virulence Determinants of Pathogenic E. coli
Adhesins
- Proteins that attaches E. coli to the walls of intestines
● CFAI/CFAII
● Type 1 Fimbriae
● P fimbriae – binds specifically to P blood group
● S fimbriae
● Intimin (non-fimbrial adhesin)

Invasin
- Hemolysin
● Alpha hemolysin – lyses lymphocytes
● Beta hemolysin – inhibits phagocytosis and chemotaxis

- Siderophores and siderophore uptake system – for iron acquisition for bacterial
growth
- Shigella-like “invasins” for intracellular invasion and spread

Motility/chemotaxis – flagella
 P a g e | 96

Toxins
- Labile Toxin
- Stable Toxin
- Shiga-like toxin
- Cytotoxins
- Endotoxin LPS

Defense Against Serum Bactericidal Reactions: LPS and K Antigens

Defense Against Immune Response: capsules, K antigens, LPS, antigenic variation

Genetic Attributes
- Genetic exchange by transduction and conjugation
- Transmissible plasmids
- R factors and drug resistance plasmids
- Toxin and other virulence plasmids

Clinical Significance: UTI, neonatal meningitis

Intestinal Diseases (differs in toxin)

Disease Virulence factors Siderophor Invasion Other
Adherence Toxins es Information
Enterotoxig Colonization Endotoxin; Enterocheli Non- Traveler’s
enic E. coli factors of Heat labile n invasive Diarrhea due
(ETEC) adherence, enterotoxin to poor
Type 1 pili (LT): cyclic- sanitation
K-88: piglets /AMP (underdevelo
K-99: calves & ped regions)
lamb Heat stable
CFA I & II: enterotoxin
humans (ST): cyclic-
GMP
Enteropathog Bundle- Endotoxin Enterocheli Poorly Infant
enic E. coli forming pili n invasive / Diarrhea
(EPEC) (BFP), Type 1 moderatel
pili, Intimin
y invasive Similar to
ETEC but
without the
LT and ST
toxins

With an
outer
membrane
protein
 P a g e | 97

Enteroaggregat Mucus- Endotoxin, Enterocheli Non- Persistent

ive E. coli associated cytotoxin n invasive & diarrhea in
(EAEC / autoagglutina (Enteroaggreg non- young
tion, Type 1 ative ST- like
EAggEC) pili toxin EAST) inflammat children but
ory without
bloody
diarrhea

Attaches to
tissue culture
cells in an
aggregative
manner

Resembles
ETEC
Enteroinva Afimbrial Endotoxin Enterocheli Type III, Diarrhea
sive E. coli adhesions, n secretion with fever
(EIEC) Type 1 pili system
Shigellosis very Resembles
like invasive shigellosis
diarrhea
Penetrates
and
multiplies
within
epithelial
cells of the
intestine
Enterohemorr Afimbrial Shiga toxin, Enteroche Probably O157 : H7
hagic adhesions, verotoxin, lin, Hema poorly
E. coli (EHEC) Type 1 pili Endotoxin uptake invasive / Cytotoxic
Hemorrhagic system moderat effect on
colitis, ely the vero cell
hemolytic
uremic invasive (kidney) of
syndrome green
monkeys

Hemolytic
Uremic
Syndrome

Common to
undercooke
d
hamburgers

With
 P a g e | 98

shigella-like
symptoms

Laboratory Indications: Lysine: + Citrate: - Indole: + Lactose: +

9. Edwardsiella
- Edwardsiella tarda (most important specie)
- Biochemically similar to E. coli
- H2S producer & non-lactose fermenter ; usually in aquatic animals and reptiles
- May cause gastroenteritis and wound infection
- Nosocomial
Laboratory Indication
Lysine: + Hydrogen Sulfide: + Indole: + TSI reaction: +/- with gas
Citrate: - Methyl Red: +/- Vogues Proskauer: -/-

10. Serratia
- Type specie: Serratia marcescens
- Produces a characteristic red pigment
- Produces DNAse, lipase, gelatinase therefore, pathogenic
- Clinical significance: UTI, wound infection, pneumonia

Laboratory Indications:
Lysine: + Citrate: + Indole: -
TSI reaction: +/+ w/out gas DNAse: + (green clearing spots on culture media)

11. Morganella
- Type specie: Morganella morganii
- Non-lactose fermenter
- Clinical Significance: diarrhea, wound infection and UTI

Laboratory Indications:
Indole: + Ornithine: + Citrate: +

Decarboxylation Test / Moeller’s Test: ornithine is hydrolyze to form putrescene with red
color formation

12. Providencia
- Rarely isolated as a pathogen (nosocomial) ; non-lactose fermenter

Laboratory Indication:
Indole: + Hydrogen Sulfide: - Citrate: + Lysine: - Lactose: -
 P a g e | 99

b. Non-Fermenting Bacilli
i. Campylobacteriaceae
1. Campylobacter jejuni
- Common cause of bacterial gastroenteritis
- Non-fermentative
- “sea gull wing shape” with darting motility
- Campy thio, skirrows media, microaerophilic
- Amphitrichous, cruved rods/ seagull-winged, nonspore forming

Virulence factors
- Enterotoxin, endotoxin
- Adhesions, intracellular survival, ability to penetrate cells

Laboratory Diagnosis
a. Skirrow’s Media and Butzler’s Media: selective media
b. Oxidase Test: positive
c. Catalase test: positive
d. Hippurate Hydrolysis: C. jejuni (positive) and C. coli (negative)

Clinical significance
- Gastroenteritis : C. jejuni/ C. coli (incubation pd- 1-10days)
- Systemic infections : C. fetus ssp. Fetus

• Laboratory Indications:
Motility: motile Hippurate hydrolysis: + Catalase: + Oxidase: +

ii. Helicobacter
- Similar in appearance with Campylobacter and Vibrio
- Causes stomach ulcers: Peptic ulcer and chronic gastritis
- May be treated with antacids
- Urase and catalase positive
1. Helicobacter pylori (Campylobacter pylori)
- Spiral shaped, lophotrichous
- Oxidase positive, catalase positive
- habitat: human gastric mucosal - mucous layer of the antrum and fundus
- Stronger urease positive test than Camplylobacter
o Urea breath Test
o Wartin- Strarry Stain
- Binds to Lewis antigen present in secretions

Virulence factors
- Flagella, protease, urease, collagenase/ mucinase, Endotoxin
- Adhesions, Vacuolating cytotoxin (VacA), Neutrophil- Activating protein (NAP), CagA

Laboratory Diagnosis
a. Skirrow’s Media: translucent colonies after 7 days
b. Catalase test: positive
c. Urease test: positive
d. Urea breath test: positive
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Clinical significance
- Type B gastritis
- Chronic, active gastritis and peptic ulcers, carcinoma to stomach

Specimens: tissue biopsy material (stuarts's medium) and urine (ammonia testing)

iii. Vibrio
- Halophilic / salt-loving except Vibrio minicus and cholerae
- Latin: to vibrate
- Isolated by Robert Koch
- Motile, obligate aerobes with characteristic curved shape with a polar flagellum / monotrichous
flagellation
- Non-invasive, water-borne which may also survive brackish water or estuary
-
1. Vibrio cholera (Fresh and salty water) “comma bacillus”
- Shooting star motility (best seen in dark-field microscopy)
- Shooting star motility
- Causes cholera
o Severe diarrhea with rice-water stool (white color) and consistency
o 60% of death is due to dehydration
o Sensitive to acids wherein it requires 1011 in acidic environment and 104 in alkaline
to cause infection
o Life threatening ; obtained from eating fresh seafood
o Two types: base on region / location of infection
a. Classic
b. El Tor – with less toxin but colonizes better and more resistant to environmental
factors
o Serotypes / Antigenic variations:
1. Ogawa
2. Inaba
3. Hikojima

Virulence factor
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- H Ag, O LPS
- Choleragen : LT enterotoxin of ETEC
o Induces activation of adenylate cyclase which triggers production of cAMP to hyper-
secrete Na, Cl which will hyper-secrete water
o May secrete 20 liters of fluid per day
o 108 vibrio per mL in intestines
- Endotoxin
- Protease/ mucinase, motility

Laboratory Diagnosis
a. TCBS Media (Thiosulphate citrate bile salt sucrose): Yellow sucrose fermenting colonies
b. APW (Alkaline Peptone Water)
c. Oxidase test: Positive
d. Sucrose and Maltose fermentation: Positive

Clinical significance
- Cholera
a. Serogroup O1 and O139 – epidemic & pandemic
b. serogroup non-O1 &O139 – cholera-like
- “Rice-water” stool

Classical El Tor
Vogues Proskauer - +
Chicken Erythrocyte - +
Polymixin B (50ug) susceptible resistant
Culture Media
1) Alkaline Peptone Water – enrichment broth
2) TCBS – forms small yellow colonies
ADDITIONAL NOTES
History of Cholera Epidemic:
1563: described by Gracia del Huerto
1849: MOT decribed by John Snow
1883: Koch isolated the bacteria
1817: start of the epidemic (6 waves until the early 20th century)
1961: the 7th Global pandemic occurred in the Philippines (El Tor)

El Tor
- More carriers (1:30 – 100) vs. (1:2 – 4)
- Longer duration
- Longer extraintestinal survival

1969 – 1974: El Tor replaced the Classic strain as the cause of pandemic
1991: Peru (after an absence of 100 years)
1992: Classic re-emerged in Bangladesh (O139 “Bengal)

Antigenic Variation
i. Flagellar Ag – H antigen ; seen in water vibrios
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ii. O Ag – distinguishes strains to 139 known serotypes ; avirulent except O1

Ogawa, Inaba, Hikojima – all are either El Tor or Classical phenotype

Serotype O Antigens
Ogawa A, B
Inaba A, C
Hikojima A, B, C
iii. Endotoxin
Laboratory Indications:
Oxidase: + (purple) Catalase: + (intense bubbling) Indole: +
(red/pink)
Lysine decarboxylase: + Ornithine deaminase: +

2. Vibrio vulnificus (lactose positive vibrio)

- From contaminated seafood
- Invasive and is able to enter the blood streams
- Causes bioluminescence on aquatic animals

3. Vibrio alginolyticus (halophilic vibrio)

4. Vibrio parahaemolyticus (halophilic marine org)

- Requires high NaCl content
- V. parahaemolyticus serotype O3:K6 (pandemic strain)
- Kanagawa phenomenon (mild form of hemolysis), “summer diarrhea”
o Wagatsuma agar
➢ BAP with increase 7% NaCl

Virulence factor
Cholera-like enterotoxins, endotoxin, cytotoxin

5. Aeromonadaceae (infections in cold- blooded animals)

- “bulls eye” colony, Apron-like colony-CIN (cefsulodine Iragasin Novobiocin)
- Rice watery stool and mucous flakes
- Carry Blair medium, Buffered glycerol saline
Virulence factors
Heat labile enterotoxins, cytotoxins

i. Pseudomonas
- Motile gram negative rods
- Utilizes glucose oxidatively
- Important due to its antimicrobial resistance
- Antibiotic resistance plasmids due to the permeability barrier of the outer membrane
- Resistant to high salt
- Some produce pigments that are fluorescent

Virulence Factor:
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1) Exoenzymes: hemolysins, leukocidins, proteases

2) Toxin A: causes ADP-ribosylation (similar to diptheria toxin in action)
3) Exoenzyme S: also an ADP-ribosyl transferase
4) Slime layer: antiphagocytic
5) Protease: for invasion

Clinical Significance: Endocarditis, RTI, Bacteremia, Septicemia, CNS infection, Ear infection, Eye
infection, Bone and Joint infection, UTI (3rd), GIT infection, skin infection

1. Pseudomonas aeruginosa
- Gram negative bacilli, obligate aerobes
- See in moist environment
- Agent of "blue pus"
- has the ability to invade the vascular walls of blood vessels
- Able to grow at 42C, optimum 35C, grape like odor, tortilla like odor
- Oxidase positive, nitrate reductase negative, utilize citrate

Virulence factors
- Pili, endotoxin, elastase, alkaline protease, soluble invasion proteins
- Pyocyanin, pyoverdin, ADP- ribosylates vimentin, ADP- ribosyl transferase activity
Pyocyanin (blue)
Pyoverdin (green)
1) Exoenzymes: hemolysins, leukocidins, proteases
2) Toxin A: causes ADP-ribosylation (similar to diptheria toxin in action)
3) Exoenzyme S: also an ADP-ribosyl transferase
4) Slime layer: antiphagocytic
5) Protease: for invasion

Laboratory Diagnosis
a. Culture: Bluish-green pigment with fruity odor
b. Oxidase Test: positive
c. Catalase Test: positive
d. Citrate Test: positive
e. Indole Test: Negative

Clinical Significance: Endocarditis, RTI, Bacteremia, Septicemia, CNS infection, Ear infection, Eye
infection, Bone and Joint infection, UTI (3rd), GIT infection, skin infection

PIgment:
- pyoverdin: yellow- green or yellow - brown pigment
- Pyocyanin: blue (only produced bu P.aeruginosa)
- Pyorubin: red
- Pyomelanin: brown or black

Distinguishing Characteristics:
- (+) Gluconate production
- (+) arginine dihydrolase (ADH)
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- Grow at 42C
- (+) acetamide and citrate utilization
1. Pseudomonas aeruginosa
- Gram negative bacili, obligate aerobes
- Moist environment
- Able to grow at 42C, optimum 35C, grape like odor, tortilla like odor
- Oxidase positive, nitrate reductase negative, utilize citrate
- Produces a chromoprotein:
a. Pyocyanin – blue ; from pyocyaneus
b. Pyoverdine - green

Virulence factors
- Pili, endotoxin, elastase, alkaline protease, soluble invasion proteins
- Pyocyanin, pyoverdin, ADP- ribosylates vimentin, ADP- ribosyl transferase activity

Laboratory Diagnosis:
Fermentation: non-fermentative Oxidase: + Odor: fruity
Hemolysis: Beta-hemolysis
Culture: Bluish green pigment with fruity odor
Citrate: Positive
Indole: Negative
Catalase: positive

2. Pseudomonas mallei – causes glanders ; respiratory lesion ; disease on horses

3. Psedomonas pseudomallei – meliodosis (whitmore disease), same with glanders ; rat-flea
borne
4. P. fluorescens and P. putids
- isolated from contaminated blood products, cosmetic, hosptial equipment and
respiratory specimens
- they can produce acid afrom xylose like P.aeruginosa
- grape-like or taco odor

ii. Burkholderia
-Do not usually causes infection in human (zoonotic infection only) but among horses

- bioterorism

1. Burkholderia pseudomallei
- Soil and water
- Melioidosis – among donkeys

2. Burkholderia mallei
- Glanders in equines / glander's / farcy
-only non motile member of teh genus
- zoonotic

3. Burkholderia cepacia
- Soil, env’t, unpasteurized dairy products, medications, mouthwash
- Bacteremia, UTI, respiratory infections
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- Lysine + ; causes death of baby

- Notorious and nosocomial

vi. Stenotrophomonas
1. Stenotrophomonas maltophilia
- DNAse positive, lysine decarboxylase positive
- maltophilia = maltose loving
- Nosocomial infections- ambulatory peritoneal- dialysis related peritonitis, bacteremia,
soft tissue infections
- Trimethoprim- sulfamethoxazole
Culture: lavander - green with ammonia smell (BAP)
brown pigment (BH infusionagar with tyrosine)

vii. Acinetobacter
- Soil and water, skin NF
- Nonmotile, oxidase negative
- Nosocomial infections
Culture: purple colonies (MacConkey Agar)
gummy colonies (CAP)
Acinetobacter Acinetobacter lwoffi Acinetobacter
baumanii hemolyticus
Glucose-oxidation + - -
Hemolysis - - +
Other Information Causes nosocomial Do not grow on
infections MacConkey

I. Alcaligenes faecalis – has a fruity odor, opportunistic pathogen

Other species:
Flavobacterium
- Common infection to premature infants and the immunocompromised
- Resistant to penicillin
Alcaligenes
- Oxidase positive, motile

viii. Legionella
- Discovered in 1976 from an outbreak of pneumonia at an Americal Legion Convention led to
29 deaths
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- Intracellular parasites that grows on BYCE with on off-white

color and circular
- acquired through inhalation
- it will not grow on BAP
- Culture: colonies appear iridiscent with sticky consistency
- Primary medium: BYCE supplemented with L-cysteine
buffered to pH 6.9

Laboratory Indications:
Motility: motile Urease: - Catalse: + Nitrate: - Gelatinase: +

1. Leigonella pneumophilia
- Facultative intracellular pathogen( alveolar macrophages)
- Isolate in air conditioning ducts
- Legionnaire’s dse (pneumonia, Pontiac fever, wound abscesses, encephalitis)
- produces black pigment in Felly-Gorman media
- Legionnaire’s disease (pneumonia, Pontiac fever, wound abscesses, encephalitis)
o Indirect Fluorescent Antibody (IFA) – common test for Legionnaire’s Disease
o Signs and symptoms: gradual flu-like symptoms
o Aerosol-borne

Laboratory Diagnosis:
a. Catalase test: positive
b. Oxidase test: positive
c. Hydrolase Hippurate test: positive

Colony morphology: grayish- white or blue-green, glistening convex colonies central portion has
"ground glass" appearance

1. Legionella micdadei
- May cause same flu-like symptoms

Laboratory Indications: Beta-lactamase: - Hippurate hydrolysis: -

Oxidase Test: test to determine enteric from non-enteric bacteria ; positive for non-enteric

ix. Haemophilus
- Has the ability to use the hemin and NAD factor by doing “Staph Streak”
● Process: done by inoculating S. aureus on BAM and cross streaking it with Hemophilus
● Positive: formation of growth between the inoculated samples
● Purpose of S. aureus: for hemolysis of RBC in BAM to release X and V factors
● Requirement: 5-10% CO2

- Large group of gram negative rods ; anaerobic

- Forms a satellite around the S. aureus
- Coccobacilli or pleomorphic ; encapsulated (groups a-f) thus pathogenic
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● Group B: most significant

X factor/Hemin V factor/NAD
(heat stable) (heat labile)
H. aegypticus + +
H. ducreyi School of fish + -
H. haemolyticus B- hemolytic + +
(Requires blood agar
with horse or human
RBC)
H. influenza Mousey/bleach odor + +
H. parainfluenzae Mannose fermentation - +
H. paraphrophilus Lactose and mannose - +
fermentation

General Characteristics
• Non-sporeforming pleomorphic bacteria
• Growth is enhanced with CO2
• Fastidous (requires growth factors)
Inoculated on chocolate blood agar plate because the RBC membrane contains Nadase enzyme which
inhibit the utilization of the NAD factor
Note: In H.haemolyticus do not use sheep red blood cell because this organism uses the NAD factor; use
horse or human RBC

1. Haemophilus influenza – “Pfeiffer’s bacillus”

- -By Richard Pfeiffer
- Normal flora of the nose and nasopharynx
- In immunosuppressed patients with pre-existing respiratory ailments may suffer
- Common to unvaccinated children
- Vaccine: HIB
- Pale pink, coccobacilli
- Capsulated coccobacilli
- Has 6 serotypes (A-F); Type B being the most common

Virulence factors
- Capsule, adhesions, IgA proteas, endotoxins

Laboratory Diagnosis
a. CAP with X and V Factor
b. Quellung Reaction
c. Satellitism Test
◆ Mix Haemophilus b in 2ml of sterile saline
◆ Inoculate the bacteria suspension on a plate
◆ Streak pure culture of S, aureus across the inoculated
plate which provides V. factor for H. influenzar
◆ Incubate plate upright in CO2 environment at 35C
◆ Look for satellite growth of colonies the following day
(-) Porphyrin test- this test detects the presence of enzymes
that converts beta aminoleuvuliniv acid into porphyrins

Culture: transluscent, convex, tan mucoid colonies with "mousy or bleach - like odor"
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2. Haemophilus aegypticus – “ Koch- Weeks bacillus”

- Pale pink long, slender rods
- Conjunctivitis (pink eye); Brazilian purpuric fever

3. Haemophilus ducreyi – “Ducrey’s bacillus”

- Pale pink slender/ coccobacilli (“school of fish”)
- Chancroid/ soft chancre (confused with syphilis), Genital ulcers
- Cultured at 20-30% rabbit blood agarChancroid/ soft chancre (confused with syphilis), Genital ulcers
- Biochemically similar to H. influenza
- Causes pink eye in children (conjunctivitis)
Distinguishing characteristics X factor (Hemin) V factor (NAD)
heat stable heat labile
H. aegypticus + +
H. ducreyi School of fish + -
H. haemolyticus B- hemolytic + +
H. influenza Mousey/bleach odor + +
H. parainfluenzae Mannose fermentation - +
H. paraphrophilus Lactose and mannose fermentation - +

x. Pasteurella (zoonotic bacteria)

- Non-motile anaerobic coccobacilli
- Not an intracellular parasite
- Produces an extremely foul odor on CAP
- shipping agent for "shipping fever in cattle"

1. Pasteurella multocida
- “safety-pin” appearance, no growth on MacConkey, “musty” odor
- grows only in BAP; susceptible in penicilin
- many “multi” peelings “cida”
- used for terrorism

Virulence factors
- Capsule, endotoxin

Laboratory Diagnosis
a. Catalase Test: Positive
b. Oxidase Test: Positive

xi. Bordetellae
- Strict aerobe and non-motile
- Borget- Gengou media, Regan – Lowe medium, modified Jones- Kendrick charcoal
- Charcoal Ceplea Lecin Blood Agar (CCBA)
- Culture: smooth, glistening, silber in color becoming whitish-gray with age
- By Jules Bordet (also discover complement pathway)
- Media: Borget-Gengou media, Regan – Lowe medium, modified Jones- Kendrick charcoal
- Small coccobacilli, strict aerobes
- Clinical Significance: whooping cough
Nitrate reduction Growth on Oxidase Urease Catalase
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Blood Free
peptone
B. pertussis - - + - +
B. parapertussis - + - + -
B. bronchoseptica + + + + +

1. Bordetella pertussis
- etiologic agent of whooping cough
- only causes disease in humans
- contains protective antigen when combined with antobody, it abolishes its
infectivity
- Culture: small, shiny colonies resembling "mercury drops"
Virulence factors
o Pili, pertactin
o filamentous hemagglutinin – cell surface protein that binds to the
host cell surface
o pertussis toxin – enters target cells and activates cAMP (important
for protein synthesis)
o tracheal cytotoxin – destruction of ciliated epithelial cells which functions to sweep oof
mucous that traps microorganisms
o Dermonecrotic toxin, lipooligosaccharide

Laboratory Diagnosis
a. Bordet-Gengou Media
b. Blood Agar: no growth
c. Oxidase Test: Positive
d. Catalase Test: Positive

Clinical significance
- Whooping cough (common among infants)
a. Catarrhal stage (2 weeks) – patient is highly infectious but not very sick
b. Paroxysmal stage (4-6 weeks) – explosive repetitive cough with a “whooping sound”
c. Convalescent stage (months) – prolonged coughing

2. Bordetella parapertussis- mild form of whooping cough

- mild form of whooping cough and pharyngitis
- similar to B pertussis but lacks some of the toxin and is differentiated by biochemical
tests

3. Bordetella bronchoseptica – respiratory and wound infections

Nitrate Growth on Oxidase Urease Catalase Motility
reduction Blood Free
Peptone
B. pertussis - - + - + -
B. parapertussis - + - + - -
B. bronchoseptica + + + + + +
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xii. Brucella
- David Bruce
- Strict aerobe ; carried by animals causing incidental infection to man
- In tissues are found intracellularly
- Human infection occurs via skin penetration, ingestion, respiratory,
and digestive tract entry
- Enters the blood and lymphatics and multiplies inside phagocytes
- Once used as a biological weapon

Virulence factors
- Intracellular existence, endotoxin

Laboratory Diagnosis
a. Castaneda’s media
b. Oxidase Test: positive
c. Catalase Test: positive
d. Urease Test: Positive
e. Dye inhibition: Positive

Clinical Significance
a. Acute Stage
b. Chronic Stage
Clinical Significance: Brucellosis (human), bangs’ disease, Gibraltar fever, malta fever, Maltese fever,
Mediterranean fever, rock fever, undulant fever, abortion in animals
Signs and Symptoms: inconstant / undulant fever, sweating, weakness, anemia headaches, depression and
muscular and bodily pain
Culture media: Castaneda’s media

xiii. Francisella
- Edward Francis
- Non-motile and strict aerobes
- Requires cysteine for growth
- Culture media: CTBA

1. Francisella tularensis
-From tick bites and may also be transferred via lesions and inhalation
-Vector: rodents, rats, mice
-Signs and symptoms: Flu-like, causes rabbit fever
-Requires cysteine/ cystine for growth
-Tularemia – rabbits and rodents
-Streptomycin or tetracycline
- Biosafety cabinet 4, “safety pin appearance”
- Culture: round, smooth, blue-gray to white, slightly mucoid colonies
- Growth factors: cysteine and thiosulfate
- Serological test: agglutination titer of 1:40- diagnostic value
- Virulence factor: capsule
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Clinical Significance
a. Tularemia : a zoonotic dx which iscan be acquired through ingestion, inhalation, arthropod bite or
contact with infected tissues
• Ulceroglandural Tularemia – ulceration of arms with lymphadenitis after tick bite
• Oculoglandular Tularemia – Accidental contamination of the conjunctiva
• Pneumonic Tularemia – Contracted through contaminated aerosols
• Typhoidal Tularemia – ingestion of improperly cooked food

xiv. HACEK group

Causative agent for bacterial endocarditis
gram negative non-fermenting bacilli
ü Haemophilus aphrophilus – no X or V factors; most commonly encountered
ü Actinobacillus actinomycetemcomitans- human oral cavity, star- like appearing colonies
ü Cardiobacterium hominis- teardrop appearance
ü Eikenella corodens- “corroding bacilli”, sharp odor of bleach
ü Kingella kingae- capnophilic

- they have slow growth on BAP and CAP (7 to 14 days)

- utilize b-aminolevulinic acid

Catalase Oxidase Glu Mal Suc Lac Others

Haemophillus - V + + + + V factor (-
)
A. + V + + - - X and V
actinomycetemcomitans factor (-)
C. hominis - + + + + - Indole (+)
E. corodens - + - - - - Ornithine
(+)
K. kingae - + + + - - Nitrate (-)

Lesson 10: Spirochetes

- cannot be stained with gram stain or acid fast stain only metallic stain (Fontana Leva Dye Silver Stain)
i. Treponema
- Highly invasive, motile (endoflagella / axial filament), microaerophilic and cannot be grown in-
vitro
- best observed in dark-field and phase contrast
1. Treponema pallidum
- Causes venereal syphilis
- Capable of infecting all body tissues but cannot survive at 4°C for 24
hours

Virulence factors
- Molecular mimicry
- Hyaluronidase 🡪 because it requires its pathway
- generation time: 30 hours
o Wasserman antigen
- very first test ; flocculation test
- detection of wasserman antigen
- diphosphatidyl glycerol (aka cardiolipin antigen)
o Reiter strain – Non-virulent variant of T. pallidum
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o Nichol's strain – Virulent

Stage Indication Treatment
Primary Hard Chancre Arsenic
Duration: weeks to months
Lesion heals spontaneously
Highly contagious
Symptoms: none
Secondary Condylomata lata – warts like lesion in abdomen, Penicillin
anogenital region, axilla and palms
Skin rash spreads from the palms and soles towards the
trunk
Duration: 2-6 weeks
A period of latency follows lasting for several years
Symptoms: fever, soar throat, weight loss, headache and
rash
Latent Vertical transmission ; occurs in utero
Duration: 10-15 weeks of gestation
Effect: 50% are stillborn, saddle nose, Hutchinson’s triad,
interstitial keratitis, notched incisors, deafness
Tertiary Gummas, Tabes dorsalis, Neurosyphilis Jerisch-Herxhimer

Laboratory Diagnosis:
Smear: Darkfield

Serological tests for syphilis

-uses VDRL
-detects the antibodies produce after syphilis infection
Lysis of host cell releases cardiolipin antigen resulting to reagin production (IgE)
a. Non Treponemal tests
- non specific
- detects reagin
o an IgE produced by infected cells

Antigen Component Function

Lecithin Anti-complement
Cholesterol Enhances binding
Cardiolipin Main anitgen

VDRL RPR
Specimen CSF/Serum Serum
Principle Microflocculation Flocculation
Ceramic Ring 16/14 mm 18 mm
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Rotation of 4 min at 180 RPM 8 min at 180

Slide RPM
Gauge of Qlty: 18g 20 g
needle Qty: 19g/23g
Drops of Qlty: 60 drops 60 drops
antigen Qty: 75 drops/100 drops

b. Treponemal tests
- detects actual treponemal pallidum
- detects antibodies produced by the body ; confirmatory test

Test Other Remarks

Treponema pallidum Immobilization Test Reference Method
FTA-ABS Primary Stage of Syphillis
Hemagglutination Test Erythrocyte coated with Nichol’s Strain

Treponema Pallidum Hemagglutination Glutaraldehyde Turkey Erythrocyte

MHA-TP Tanned Sheep Erythrocyte

Jarisch-Herxheimer Reaction – hypersensitivity reaction as a result of a rapid release of treponemal antigens

during lysis

Clinical significance
- Venereal Syphilis (Great Immitator)
2. Treponema pallidum subspp pertenue – yaws or tropical syphilis
- Common in tropical regions characterized by painless papule called Mother of Yaw
3. Treponema pallidum subspp endemicum – Bejel syphilis
4. Treponema carateum – pinta
- Common in tropical America
- Also characterized by painless papule on the hand, scalp and feet
- Lesions heal slowly unlike syphilis and yaws

2. T. carateum
- causative agent for pinta/ carate/ mal de pinto / ozul
- pinta: infection of the skin; the primary lesion is a slowly enlarging
papule with regional lymph node enlargement, followed in 1-12 month by
generalized red to slate blue macular rash

xv. Borrelia (blood spirochetes)

- Disease is characterized by remittent fever
- Transmitted to humans by ticks and lice
- composed of 3 to 10 loose coils and are actively motile
- has 15-20 axial filaments and 2 insertion disks
- multiply by binary fission

1. Borrelia duttoni – Endemic relapsing fever

2. Borrelia recurrentis – Epidemic relapsing fever
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Virulence factor (Vector: Humanus pediculus)

- Antigenic variation

Clinical significance
- Relapsing fever / on-off fever

3. Borrelia burgdorferi – Lyme disease or Erythema chronicum migrans

Virulence factor (Vector: Ixodes)
- Antigenic variation

Clinical significance
- Lyme disease (erythema chronicum migrans)
4.Borrelia hermsii – endemic relapsing fever (ticks)
Clinical Significance:
1. Relapsing fever
a. Epidemic
- Incubation period: 1 week
- Generalized infection follows
- Sings and symptoms: fever, headache, malaise lasting 4-10 days, afebrile period
lasting 4-6 days, recurrence of the symptoms

b. Endemic
- Less severe that the epidemic form
- Transmitted by ticks
- May have several relapses due to its cyclic antigenic variation

2. Lyme Disease
- Production of ulcerative lesions which may lead to arthritis and neurologic involvement
- Can be cultivated in vitro
o Media: Barbour-Stoenner-Kelly
o Incubated at 32°C
o Microaerophilic
o Usually transmitted by ticks of deers (Ixodes)

1) Stage on (early infection)

- Indistinct red rash (erythema migrans) which appears 3 days to a month after a
bite with a bull’s eye appearance

2) Stage two (dissemination stage)

- Signs and symptoms: fatigue, chills, fever, headache. Malaise, secondary annular
lesions, swollen lymph nodes

3) Stage three (persistent infection)

- Signs and symptoms: Intermittent episodes of joint pains, meningitis, bell’s palsy,
arthritis
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Laboratory Diagnosis: darkfield microscopy, ELISA

3. Spirilium minor / Sodoku – causes rat-bite fever

xvi. Leptospira
1. Leptospira interrogans
-an obligate aerobic spirochete and cen be grown in artificial
media
-they live in the lumen of the renal tubules
-animal of choice for cultivation: hamsters and guinie pigs
- Microscopy: tightly coiles, thin, flexible organism with 2
long axial filaments "question mark like" shape - both ends
of the organism have hooks
- Virulence factor: hemolysin
- Generation time: 6 to 16 hrs
-Weil’s disease, Leptospirosis (L. interrogans)
a. Leptospiremic Phase – generalized infection with bacteremia
b. Leptospiuric Phase – multiplies in the kidney leading to shedding inurine which persist
for weeks to years

- Thin, tightly coiled obligate aerobes, motile ; >180 serotypes

Laboratory Diagnosis
a. Fletcher’s Media semi-solid media

Lesson 11: Other Bacteria

i. Rickettsiaceae
General Characteristics
– Obligate intracellular pleomorphic gram negative coccobacilli
- Small pleomorphic gram negative coccobacilli which replicates in the cytoplasm and nucleus of
host cells except Coxiella which is in the phagolysosome

Pathogenicity:
- Bacteria enters the host cell, O fever by inhalation
- Replicates inside the cell and causes lysis
- Disseminated via the vascular system causing vasculitis and rash

Laboratory Diagnosis
a. Macchiavello’s Stain: red
b. Giemsa stain: blue
c. Special ctain : Gimenez, Macchiavelo

Other Lab Diagnosis:

Immunofluorescent Antibody – for examining biopsy
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Tissue Culture
Weil-Felix Test – not specific ; detects serum antibody against Proteus OX19, OX2 or OXK antigens

Organism Disease Vector Mammalian reservoir

Typhus group
Rickettsia prowazekii Epidemic typhus Louse Humans
Brill-Zinser Disease
Rickettsia typhi Endemic typhus Flea Rodents
Orienta tsutsugamushi Scrub typhus Mite Rodents
Spotted fever group
Rickettsia rickettsii Rocky mountain Tick Rodents, dogs
spotted fever
Rickettsia akari Rickettsial pox Mite Mice
Rickettsia australis Queensland tick typhus Tick Rodent, marsupials
Rickettsia conorii Fievre boutenneuse Tick Rodents, dogs
Rickettsia sibirica Siberian tick typhus Tick Rodents
Q fever
Coxiella burnetii Q-fever Airborne formites, tick Sheep, cattle, goats,
others
Ehrlichie
Ehrlichia chaffeensis Human monocyte Tick Deer
ehrlichiosis
Neorickettsia sennetsu Human monocyte Trematode- infected Mammals
ehrlichiosis fish
Anaplasma Human granulocyte Tick Mice, other mammals
phagocytophilium anaplasmosis
Ehrlichia ewingii Human granulocyte Tick dogs
ehrlichiosis
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ii. Chlamydia
- Obligate intracellular parasites
- Cell walls are similar to cell walls of gram negative bacilli, but lack muramic acid
A. Elementary Bodies – infectious stage
B. Reticulate Bodies
- Replicative form found inside of the host cell
- Returns back to EB after 24 hours
48-72 hours later, the cell is lysed releasing EB
- C. trachoma – leading cause of blindness

1. Chlamydia trachomatis
- Causes oculorogenital infections
- Infection occur via swimming in unchlorinated pools, sharing towels and passage thru
infected birth canal

Clinical Significance: Trachoma, Inclusion conjunctivitis, non-gonococcal urethritis,

lymphgranuloma venereum

2. Chlamydia psittaci
- Causes systemic infections

Clinical Significance: Ornithosis, Psittacosis

Laboratory Diagnosis:
Smear: respiratory disease in humans ranging from flu-like to pneumonia-like symptoms
acquired from the droppings of birds

iii. Mycoplasma/ Pleuropneumonia-like organism

- No distinctive cell wall of bacteria
- Plasma membrane ( high content of sterols)
- Inhabits the mucus membrane of the respiratory and
urogenital tracts
- Produces high concentration of toxic metabolites (H2O2)
- May also produce ciliostasis
- Culture media requires penicillin
-FAT is used to confirm diagnosis

1. Mycoplasma pneumonia (Eaton agent)

- Resembles fried eggs, stained by Dienes stain
- Atypical pneumonia (walking pneumonia)
- Clinical Significance: Primary Atypical pneumonia (walking pneumonia)
- Tetracycline or erythromycin

2. Mycoplasma hominis
- Genital tract infections

3. Ureoplasma urealyticum
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- Genital tract infections

- Requires urea to produce electrochemical gradient
- Smallest prokaryote
- Clinical Significance: NGU

Lesson 12: Miscellaneous Bacteria

Organism Gram stain reaction Distinguishing characteristics
Actinomyces spp Gram (+), branching, beaded or Resembles “molar tooth”)
banded, filamentous rods
Bacteroides fragilis Gram (-) pale staining pleomorphic Gray white, circular, smooth, non
rods, resembles safety pin hemolytic
Bifidobacterium spp Gram (+) diphtheroid, coccoid or Small, white, shine, convex colonies
pointed shape, bifurcated ends,
resembles dog bones
Eubacterium spp Gram (+) pleomorphic, has seagull wing Chartreuse color fluorescence
shape
Fusobacterium nucleatum Gram (-) spindle shaped rod, resembles Green colored medium upon air
subsp nucleatum Capnocytophaga exposure, “breadcrumb-like”
colonies
Lactobacilli spp Gram variable rods, short
coccobacilli, resembles streptococci

Leptotrichia spp Gram (-) large fusiform rods “raspberry-like” colonies

Peptostreptococcus anaerobius Gram (+) large coccobacillus in chain

Porphyromonas spp Gram (-) coccobacilli Brick red fluorescence

Propionibacterium spp Gram (+) diphtheroid- like rod, palisade
“anaerobic diphtheroids” arrangement

Lesson 13: Acid Fast Bacteria

Runyon’s classification Color produced Growth rate Species
Basis: grow in the
presence of light and
Photochromogens (Group Not pigmented unless exposed 10-21 days M. kansasii
I) to light M. asiaticum
A. Grows in the − Cream M. marinum
dark or buff M. simiae
B. Grows in the − Orange
Light or
yellow
Scotochromagens (Group Pigmented both in the dark and 10-21 days M. szulgai
II) light M. gordonae
− Yellow M. scrofulaceum
to M. flavescens
orange M. xenopi
colonies
Nonphotochromagens Non- pigmented both in the 10-21 days M. avium cmplex
(Group III) dark and light M. ulcerans
M. terrae complex
M. gastri
M. haemophilum
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Rapid Growers Pigment variation 3-7 days M. fortuitum

M. chelonae
M. smegmatis
M. phlei
M. abscessus
M. mucogenicum

A. Mycobacterium
General Characteristics
• Non-spore forming and non-motile
• Acid fast reaction is based on the lipid envelope-mycolic acid of the cell wall
• Resistant to decolorization
● Rod-shaped aerobic gram – non-spore forming
● Resist decolorization once stained (Acid Fast) ; stained acid fast cell due to cell wall content N-
acetylmuramic acid (mycolic acid)
● Contains much’s granules (metachromatic)
● Pathogenic to man: M. tuberculosis and M. leprae
● Other species are referred as: atypical mycobacteria
● MOTT: mycobacterium other than tuberculosis
● NTM: non-tuberculous mycobacteria

Laboratory Diagnosis
a. Morphologic Index – indicates the percentage if living bacteria in a smear; used for judging
body’s response to drugs
a. Bacterial Index – indicates the number of organisms present in a smear

Runyon Classification - based on their ability to grow on light and pigments

a. Photochromatogen – produces yellow to orange pigment after exposure to light (M. asiaticum, M.
marinum, M. kansaii, M. siame)
b. Scotochromatogen – produces pigment in the dark or even when exposed to light (M. flavescence, M.
scrofolaceum, M. gordonae, M. szulgai)
c. Non-photochromatogen – no pigment production (M. ulcers, M. gastri)
d. Fast Growers / Rapid Growers – colonies appear in <7 days (M. smegmatis, M. fortuitum)

Runyon’s Classification Color Produced Growth Rate Species

Photochromogens (Group I) Not pigmented varies 10-21 days M. Asiaticum
A. Grows in the dark exposed to light M. Marinum
B. B. Grows in the -Cream or tuff M. Kansaii
light -Orange or yellow M. siame
Scotochromatogen (Group Pigmented both in the dark 10-21 days M. Flavescence
II) and light M. Scrofolaceum
- yellow to orange colonies M. Gordonae
M. szulgai
Non-photochromatogen Non-pigmented both in the 10-21 days M. Ulcers
(Group III) dark and light M. Gastri
M. terrae complex
Fast Growers / Rapid Pigment variation 3 - 7 days M. Smegmatis
Growers M. fortuitum
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AFB smear- 5000-10000 org/mL- positive result

0 No AFB seen
1-2/300 fields Doubtful; request another specimen
1-9/100 fields 1+
1-9/10 fields 2+
1-9/field 3+
>9/field 4+

1. Mycobacterium tuberculosis – “Koch bacillus”, Cauliflower colonies

- Thin straight rods
- Non sporeforming and non motile
- AFB, exhibits yellow-orange fluorescence with
fluorochrome stains
- May be arranged in palisade
- Resistant to discoloration
- Obligate intracellular growth
- Cord Factor
● Virulence factor ; composed of trehalose-6, 6-
dimycolate, a glycolipid that attacks the
mitochondria

- Resistant to chemical agents due to its hydrophobic

nature and its clumped growth and also resistant to
drying

Laboratory Diagnosis
a. Semisynthetic agar media (Middle brook 7H10 and 7H11)
b. Inspissated egg media/ Lowenstein-Jensen Medium
c. Broth Media- Middlebrook 7H9 and 7H12
d. Morphologic index- indicates the percentage of living bacteria in a smear; used for judging body’s
response to drug s
e. Bacterial index- indicates the number of organisms present in a smear

Clinical Significance:
i. Tuberculosis
Stages
a) Droplet Nuclei Inhalation – air born and can be spread to people up to 10 feet away
b) Multiplication in Macrophages – begins 7-21 days after infection and doesn’t kill bacteria and
a s result macrophages lyses

c) Lymphocyte Infiltration
- Macrophages are activated and cytokines are released
- Patient becomes tuberculin positive due to delayed hypersensitivity reaction
- IL-1 and TNF are released by activated macrophages

d) Tubercle Formation
- Lump made up of bacteria called granuloma
- Granulomatous lesion due to accumulation of macrophages and granulocytes and
hypersensitivity to MTB proteins
- Caseation necrosis of the lung tissue 🡪 cheese-like
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- Miliary distribution: Tubercle may invade other organs

e) Liquefaction of Caseous Centers

- Bacterial cell multiply extracellularly
- Walls of the bronchi become necrotic and rupture
- Cavity formation

Gohn Complex
- Fibrous and calcified primary lesions that healed
- Readily seen in chest X-ray

Simon Foci
- Small metastatic foci containing few bacteria
- Also seen in in X-ray, indicates reactivation of disease

ii. Primary Infection

- Usually a past infection during childhood which can be reactivated
- S/S: fatigue, weakness, weight loss, fever, chronic cough with blood

Tuberculin Test
Principle: persons infected, past or present, with tubercle bacilli are allergic to tubercle bacilli and its
products

Preparation:
a. Old Tuberculin – culture + 5% glycerin then filtered
b. Purified Protein Derivative – precipitate of broth cultures

Positive Result of Both: induration of >10 mm in diameter or more read after 48 and 72 hours

Tests
✔ Von-Pirquet Test: Scratch + tuberculin = hypersensitivity
✔ Mantoux Test: injected intracutaneously = redness / swelling
✔ Volmer’s Patch Test: filter paper + tuberculin then patch for 48 hrs = redness / swelling
✔ Tuberculin Tine Test: metal disk with prongs + tuberculin then press to arm = redness / swelling

Laboratory Diagnosis
Specimen: sputum, bronchial brushing, bronchial washing, urine, bipsy
Other Tests: PCR, AFS, EIA, X-ray

2. Mycobacterium bovis – Bacille- Calmette- Guerin vaccine

3. Mycobacterium africanum – spacer oligotyping

● Tuberculosis
- “consumption”
- Ghon complexes – lesions
- Pott’s disease
- Granuloma
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2. Mycobacterium kansasii - “yellow bacillus”, “shepherd’s crook”

3. Mycobacterium marinum – “swimming pool granuloma”
4. Mycobacterium ulcerans – Bairnsdale and Buruli ulcers
5. Mycobacterium gordonae – “Tap water bacillus”

6. Mycobacterium avium complex

- M. avium (“Battey bacillus”), M. intracellulare, M. paratuberculosis (Johne’s disease), M.
lepraemurium, M. avium subsp silvaticum (“wood pigeon bacillus”)

7. Mycobacterium xenopi – “bird’s nest” colonies, hot and cold water taps
8. Mycobacterium terrae – complex ( M. terrae- “radish bacillus”)

9. Mycobacterium leprae - “Hansen’s bacillus”

- Cigar pocket/ pocket fence arrangement
- Decontamination and digestion
- Non-cultivatable in-vitro ; obligate intracellular
- Regularly found in scrapings from skin and mucous membrane

Clinical Significance:
a) Leprosy / Hansens Disease – lesion involve cooler body tissues like the nerves, pharynx, larynx,
eyes, testicles and the skin

Signs and Symptoms: nerve infiltration and thickening which results to anesthesia, neuritis,
paresthesia, tropic ulcers and bone resorption

1. Lepromatous
- Progressive and malign with nodular skin lesions
- Negative with lepromin test
- Without cell-mediated immunity
- Skin is with suppressor T cells
- Disseminated form of leprosy

2. Tuberculoid
- Benign and non-progressive
- With macular skin lesions
- Positive lepromin test
- Skin is with CD4 T cells
- With cell-mediated immunity

Laboratory Diagnosis
Specimen: skin scrapings, biopsy, earlobe skin
Smear: AFB
Culture Media: no culture media ; inoculated on footpads of armadillo or mice

Tests:
a. Lepromin Test – Similar principle with tuberculin test
b. 2-4% NaOH method – most common decontaminating agent (2mL sputum + 2mL NaOH)
c. 6% Oxalic acid method – for sputum with gram-negative rods like Pseudomonas and Proteus
d. Zephiran-trisodium PO4 method – for specimens containing large numbers of bacteria
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e. 1% Cetyl-Pyridium Chloride – prolonged shelf life of sputum for 8 days

f. N-Acetyl-L-Cysteine (NALC)- NaOH method-
g. dithioreitol – digestion agent

(sputolysin) AFB smear – 5000 – 10000 org/mL – positive result

0 No AFB seen
1-2/300 fields Doubtful; request another specimen
1-9/100 fields 1+
1-9/10 fields 2+
1-9/field 3+
>9/field 4+

Test for the Identification of Mycobacteria

1. Temperature Test – exposure to 30°C
2. Pigment Production - obsolete
3. Niacin Accumulation Test
- Niacin is a precursor of the biosynthetic pathways of coenzyme production in mycobacteria
- Positive: MTB, M. simmae, M. marinum Negative: M. intracellulare, M. chelonae

4. Nitrate Reduction Test

- Positive: Red diazonium dye for 30-60 seconds (MTB, M. szulgai, M. kansasii, M. flavescence, M.
fortuitum)

5. Catalase Test
a. Spot Test
b. Semi-quantitative Test
- Colonies from L-J is added with twin peroxide
- Positive: >50 mm height of bubble (M. kansaii) Negative: MTB

c. Heat Stable Catalase Test

- With addition of heat
- Sample is exposed to 60°C for 20 mins.
- With bubbles: heat stable Without bubbles: heat labile

6. Tween 80 Hydrolysis
- Detergent ; test for non-pathogenic scotochromatogens and non-photochromatogens
- Positive: pink color (M. kansasii, M. szulgai, M. flavescence, M. terrae, M. gordonae)
- Indicator: phenolphthalein

7. 5% NaCl Tolerance Test: L-J + 5% NaCl = turidity (M. triviale, M. flavescence)

8. Iron Uptake Test: 20% Ferric Ammonium Citrate = rusty brown (M. fortuitum)
9. TCH Susceptibility Test
- Thiophene-2-carboxylic acid hydrazide
- Determines M. bovis from MTB
- Positive (M. bovis): zone of inhibition

10. Urease Test

- Test for scotochromatogens and non-photochromatogens
- Indicator: phenol red
- Positive: red
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11. Arylsulfatase: pink color (3 days: M. fortuitum & M. chelonei ; 14 days: M. marinum & M. szulgai)

12. Tellurite Production

- Reduction of potassium tellurite in 3 days
- Positive: black color (M. avium complex)

13. Pyrazinamide: pink band in agar (M. marinum)

14. Growth on MacConkey without Crystal Violet: for M. fortuitum-chelonei complex
15. Nucleic Acid Testing
Chromatographic Analysis

Treatment
1. Mycobacterium bovis – Bacille- Calmette- Guerin vaccine
2. Mycobacterium africanum- spacer oligotyping
3. Mycobacterium kansasii- “yellow bacillus”, “shepherd’s crook”
4. Mycobacterium marinum – “swimming pool granuloma”
5. Mycobacterium ulcerans- Bairnsdale and Buruli ulcers
6. Mycobacterium gordonae – “Tap water bacillus”
7. Mycobacterium avium complex
8. M. avium (“Battey bacillus”), M. intracellulare, M. paratuberculosis (Johne’s disease),
9. M. lepraemurium, M. avium subsp silvaticum (“wood pigeon bacillus”)
10. Mycobacterium xenopi – “bird’s nest” colonies, hot and cold water taps
11. Mycobacterium terrae- complex ( M. terrae- “radish bacillus”)
12. Mycobacterium leprae- “Hansen’s bacillus”
• Cigar pocket/ pocket fence arrangement

Decontamination
1. 2-4% NaOH method- most common decontaminating agent (2mL sputum + 2mL NaOH)
2. 6% Oxalic acid method- for sputum with G(-) rods like Pseudomonas and Proteus
3. Zephiran- trisodium PO4 method- for specimens containing large numbers of bacteria
4. 1% Cetyl- Pyridium Chloride- prolonged shelf life of sputum for 8 days
5. N-Acetyl-L-Cysteine (NALC)- similar with NaOH method
6. Dithioreitol- digestion agent (sputolysin)