MICROPARA LAB REVIEWER -contains lenses that contribute to total

MIDTERMS magnification
-power of 10x (magnifies 10 times)
MICROSCOPE
- The first compound of the microscope MICROSCOPE PARTS AND FUNCTION
was developed in 1590. Two Dutch Body tube- hollow tube that keeps the
spectacle-makers and father-and-son lenses of the ocular and objectives at a set
team, Hans and Zacharias Janssen, distance
create the first microscope with 2 Nosepiece- holds objectives
lenses.
Objectives- contain lenses that contribute to
HISTORY OF THE MICROSCOPE: total magnification
1670 – Robert Hooke used a compound Arm- supports body tube
microscope to observe pores in cork. Hooke
saw tiny boxlike cavities, which he Base- supports entire microscope (when
illustrated and described as cells. he had carrying, keep hand back on base because
discovered plant cells. lamp will be hot!)

1675: Anton van Leeuwenhoek, used Stage- tray-like structure that supports
microscope with one lens to observe insects specimen/slide over stage opening
and other specimen. Leeuwenhoek was the Stage Clips- keep specimen/slide tight
first to observe bacteria. against stage
WHAT IS A MICROSCOPE? Diaphragm- controls amount of light that
• A microscope is laboratory equipment reaches your eye
that is used to magnify objects that cannot Light source- provides light to create the
normally be seen by the unaided eye. it image that you see
magnifies objects, producing an image in
which the object appears larger, Coarse adjustment- larger knob, that
moves 1 of 3 structures (body tube, stage, or
• A valuable tool that allows you to nosepiece) and allows for rough focus
examine specimens in more detail, whether
the specimen is a biological sample or a Magnification formula- calculate total
crystal structure. magnification (Total mag. = ocular power x
objective power)
MAJOR TYPES OF MICROSCOPE
Fine Adjustment- smaller knob, moves the
• Compound microscope objectives slightly and allows for fine
• Dissecting microscope focusing

• Electron microscope TO CALCULATE MAGNIFICATION

MICROSCOPE VOCUBOLARY Eyepiece at 10x

• Magnification: increase of an object’s Objective at 4x

apparent size Eyepiece + objective lens= magnification
• Resolution: power to show details 10x x 4x=40x
clearly
• Both are needed to see a clear image
COMPOUND LIGHT MICROSCOPE
PARTS AND FUNCTION:
Ocular (eyepiece)
- part you look through
 hereditary material and can make copies of
themselves.
TOTAL MAGNIFICATION
TYPES OF CELLS
Ocular objective
Prokaryotic cell
10x red 4= 40x
• cells were the first, the only form of life.
10x yellow 10 = 100x
• Do not have membrane- bound nucleus or
10x blue 40 = 400x
organelles.
microscopes we use are parfocal- can switch
Eukaryotic cell
b/t lenses without much adjusting (only use
lens paper to clean objectives) • cells are more complex.
General procedures on how to use a • contains nucleus and organelles bound by
microscope plasma membrane.
 Make sure all backpacks and
materials are out of the aisles and off
Plant cell
the tops of desks.
 Plug your microscope in to the outlet. • Large and has a fixed rectangular shape.
 Store with cord wrapped around • Cell wall is present.
microscope and the scanning
objective clicked into place. • Nucleus lies on one side of the cell.
 Carry by the base and arm with both • Mitochondria are present in fewer
hands. numbers.
5 TIPS TO PROPERLY CARE FOR • Plasmids are present.
YOUR MICROSCOPE
• Centrosomes are absent.
 Always use two hands when moving
• Large vacuole is present.
the microscope.
 Make sure the microscope is not
plugged in before moving it. Animal cell
 If using immersion oil, be sure to
clean the immersion oil lens • Small and irregular or round shape
thoroughly after use. • Cell wall is absent.
 Keeping your microscope clean
(especially the optics) will also ensure • Nucleus lies in the center.
that it lasts longer. • Mitochondria present in large numbers.
 When the microscope is not in use
• Plasmids are absent.
cover it with a dust cover or store it in
a microscope case. • Centrosomes are present.
PROKARYOTIC AND EUKARYOTIC •Small vacuoles are present.
CELLS

Cell Anatomy The cell is the structural and Animal cells under microscope divided in
functional unit of all living things and is a 3 parts.
complex entity. Cells are the basic building  Nucleus
blocks of all living things. The human body  Cytoplasm
is composed of trillions of cells. They  Plasma membrane
provide structure for the body, take in
nutrients from food, convert those nutrients PLANT CELLS UNDER MICROSCOPE
into energy, and carry out specialized • Onion
functions. Cells also contain the body’s
• Leaf
ANIMAL CELLS UNDER • Gram + and Gram – bacteria have
MICROSCOPE differences in Cell Wall and outer envelope
(the theory of why the gram’s stain works is
• BLOOD
based on this difference) Gram – are less
• SKIN sensitive to penicillin as a result of thinner
CW and outer membrane
MICROORGANISMS
• ALGAE • Cell shape is determined by the genetic
character of the organism. Its genes code for
• Euglena the synthesis of the CW material and the cell
• Bacteria division mechanism that results in a “round”
or “rod” or “spiral” shape
• One of the criteria used in ID of bacteria
MICROBIOLOGY – CHAPTER 4,
BACTERIA • Coupled with gram reaction = helpful (Ex.
Gram + cocci in clusters is
• Bacterial appendages: Pilli (fimbriae) and “Staphylococcus” Gram (-) bacilli, motile,
Flagella green sheen on EMB is E. coli)
• Pilli are short, hair-like, protein: function • Bacterial cell membrane: regulates what
“adherence” – stick to each other, stick to moves in and out of the cytoplasm
surfaces, harder to wash away
• Diffusion –concentration gradients > high
• Specialized “sex” pilus – conjugation to low
• Flagella: complex organ of motility, a • Osmosis – diffusion of water across a
“motor” VERY COOL semipermeable membrane (Isotonic,
• A = monotrichous B = amphitrichous Hypertonic, Hypotonic)

C = lophotrichous D = peritrichous • Nucleoid – area containing the bacterial

chromosome (DNA)
• Bacterial “Envelope” – Structures on the
outside of bacteria • Plasmid – Extrachromosomal DNA, not
part of “genome”, different genes
• Glycocalyx – sugar coat, if tightly bound =
capsule (Protects and prevents from drying, • Can transfer in a process – conjugation
also protects from phagocytes. Slimy, and across sex pilus, change the genetic
often a significant component of “biofilms”) character of the recipient Tool of “genetic
“engineer
• Outer membrane (outside of cell wall)
found in Gram (-) bacteria • Ribosomes: structure made of RNA, site of
protein synthesis
• Bacterial Cell Wall – macromolecule,
polysaccharide, repeating sugars, NAG and • Some antibiotics work by messing with the
NAM, cross-linked with shot chains of “ribosome”
Amino Acids • Slightly smaller than our eukaryote
• “Peptidoglycan, aka: murein” ribosome, so antibiotic can work on bacteria
but not affect us (selective toxicity)
• Tough outer coat, prevents rupture,
protects cell, gives it its distinct shape
• Certain antibiotics work by inhibiting cell EQUIPMENTS
wall synthesis (penicillins)
• Gram + and Gram – bacteria have Autoclave
differences in Cell Wall and outer envelope • Is a pressurized chamber used for the
(the theory of why the gram’s stain works is process of sterilization and disinfection by
based on this difference) combining three factors: time, pressure, and
steam.
• Autoclaves use steam as their sterilization • Specific shapes are the consequence of
agent. adaptive pressures optimizing bacterial
fitness. Shape affects critical biological
• The basic principle of an autoclave is that
functions, including nutrient acquisition,
all the items within the autoclave come in
motility, dispersion, stress resistance and
direct contact with the steam for a particular
interactions with other organisms.
period irrespective of the nature of the
material- whether it is liquid, plastic ware,
or glassware. GRAM STAINING
• The amount of time and the temperature
depends on the type of material being
sterilized and the increase in temperature of
the cycle allows for shorter periods.
• For: 121 degree Celsius, 15 min, 15 psi
Colony counter
• Specify the number of microbial colonies
present on sample plates for maximized
working efficiency in the lab.
Incubator
• In microbiology, is an insulated and
enclosed device that provides an optimal
condition of temperature, humidity, and
other environmental conditions required for
the growth of organisms.
Laminar flow
• Is defined as airflow in which the
Gram-positive bacteria have a thick mesh-
entire body of air within a designated space
like cell wall made of peptidoglycan (50–
is uniform in both velocity and direction.
90% of cell envelope), and as a result are
Biomedical refrigerators stained purple by crystal violet, whereas
gram-negative bacteria have a thinner layer
• Are medical equipment's that are used to
(10% of cell envelope), so do not retain the
store a variety of samples of biological
purple stain and are counter-stained pink by
origin such as blood, derivatives of blood,
safranin.
biological reagents, vaccines, medicines,
flammable chemicals, ribonucleic acid THERE ARE FOUR BASIC STEPS OF
(RBA), and deoxyribonucleic acid (DNA). THE GRAM STAIN:
Bacterial Morphology  Applying a primary stain (crystal
violet) to a heat-fixed smear of a
• Microorganisms are a heterogeneous group
bacterial culture. Heat fixation kills
of several distinct classes of living beings.
some bacteria but is mostly used to
Based on the difference in cellular
affix the bacteria to the slide so that
organization and biochemistry, the kingdom
they don't rinse out during the staining
Protista has been divided into two groups
procedure.
namely prokaryotes and eukaryotes.
Bacteria and blue-green algae are  The addition of iodide, which binds to
prokaryotes, while fungi, other algae, slime crystal violet and traps it in the cell
moulds and protozoa are eukaryotes.  Rapid decolorization with ethanol or
Bacteria are prokaryotic microorganisms acetone
that do not contain chlorophyll.  Counterstaining with safranin. Carbol
fuchsin is sometimes substituted for
safranin since it more intensely stains
 anaerobic bacteria, but it is less ACID FAST STAINING
commonly used as a counterstain.

4 PRIMARY THINGS TO USE IN GRAM

STAINING
 Crystal violet
 Iodine
 Decolorizer
 Safranin

PROCESS OF GRAM STAINING:

 Step 1- crystal violet, primary stain
added to specimen smear.
Gram (+): purple
Gram (-): purple

 Step 2- iodine, mordant males dye

less soluble so it adheres to cell walls.
Gram (+): purple
Gram (-): purple
 Step 3- Alcohol or decolorizer,
washes away the stain from gram (-)
cell walls.
Gram (+): purple
Gram (-): colorless
 Step 4- safranin, counterstain allows
dye adherence to gram (-) cell walls.
Gram (+): purple
Gram (-): red

GRAM STAINED BACTERIA

 Staphylococcus aureus
 Streptococcus pyogenes
 Clostridium tetani
 Neisseria gonorrhea
 Bacillus anthracis
 Escherichia coli
 Vibrio cholera
 Treponema pallidum  It is the differential staining
 Leptospira interrogans techniques which was first developed
by ziehl and later on modified by
 Spirillum minus
neelsen. So this method is also called
ziehl-neelsen staining techniques.

 Neelsen in 1883 used ziehl’s carbol-

fuchsin and heat then decolorized
with an acid alcohol, and counter
stained with methylene blue. Thus
 ziehl-neelsen staining techniques was STEPS IN ACID FAST STAINING:
developed.
 STEP 1- primary stain, application of
carbolfuchsin (basic fuchsin with
phenol)
 The main aim of this staining is to
 Step 2-mordant, application of heat
differentiate bacteria into acid fast
(mordant)
group and non-acid fast groups.
 Step 3- application of acid alcohol or
 This method is used for those
decolorizer (sulfuric acid, alcohol,
microorganisms which are not
acid alcohol)
staining by simple or Gram staining
method, particularly the member of  Step 4- counter stain, application of
genus Mycobacterium, are resistant methylene blue (methylene blue,
and can only be visualized by acid- malachite green)
fast staining. ACID-FAST STAINING OF
BACTERIAL CELL

PRINCIPLE OF ACID-FAST STAIN  Mycobacterium tuberculosis

 When the smear is stained with carbol ACID-FAST STAINING: KINYOUN

fuchsin, it solubilizes the lipoidal METHOD
material present in the Mycobacterial  Acid-Fast staining positive- RED
cell wall but by the application of  Acid-Fast negative- BLUE
heat, carbol fuchsin further penetrates
through lipoidal wall and enters into
cytoplasm. HANGING DROP TECHNIQUE
 Then after all cell appears red. Then
the smear is decolorized with THE HANGING DROP METHOD
decolorizing agent (3% HCL in 95%
 was discovered by Robert Koch in
alcohol) but the acid fast cells are
1877. It is a technique used to culture
resistant due to the presence of large
amount of lipoidal material in their microorganisms, in which a small
drop of liquid culture is suspended
cell wall which prevents the
from the underside of a coverslip.
penetration of decolorizing solution.
This method is particularly useful for
The non-acid fast organism lack the
culturing bacteria and other
lipoidal material in their cell wall due
microorganisms that require a small
to which they are easily decolorized,
volume of liquid to grow in, and it
leaving the cells colorless.
allows for the observation of
APPARATUS REQUIRED: microbial growth under a microscope.
1. Acid fast kit (includes above mentioned
reagents)
 has been the most common method to
2. Bunsen Burner study the cell’s movement and
3. Slides morphology, by collecting live
microorganisms and removing them
4. Glass rods from the liquid medium. Hanging
5. Inoculating loop drop technique is based by utilizing
the principles of preparation using wet
6. Mycobacteria culture mounts, since it involves subjection of
7. Microscope (oil immersion objective) living microorganisms to drops of
fluid.
RESULT AND INTERPRETATIONS OF 2. SEMI-SOLID MEDIA
HANGING DROP METHOD INOCULATION
 Directional intent motility is a  The most commonly used test for
favourable indicator. Positions of motility in microbiology lab.
mobile organisms fluctuate relative to  It depends on the ability of motile
one another. Brownian movement bacteria to move through semi-solid
(random jiggling or shaking owing to media.
molecular bombardment) in which  Ordinary solid media contain 1.5-
organisms remain in the same relative 2.0% agar
position to one another should not be  Semi solid media contain about 0.4%
confused with genuine motility. agar

 Campylobacter and vibrio cholerae

exhibit extremely vigorous movement
(darting motility) that appears as
small spots darting across the field.

SITE OF FLAGELLA
 Peritrichous (E. coli)
 Monotrichous (vibrio cholera)
 Lophotichous (pseudomonas)
 Amphitrichous (spirillum volutans)

TYPES OF MOTILITY
1- True movement (depends on flagella
2- False movement
A- Brownian movement
- Vibratory movement
B- Drafting movement MEDIA PREP & ASEPTIC
- organisms streaming along a tide TECHNIQUE

Culture Medium
THREE WAYS FOR DETECTING
MOBILITY A microbial culture medium is a mixture of
substances that promotes and supports the
1- flagella staining. growth and differentiation of
2- motility test in semi-solid media. microorganisms. Culture media contain
nutrients, energy sources, growth-promoting
3- hanging drop technique. factors, minerals, metals, buffer salts, and
gelling agents (for solid media).

1- FLAGELLA STAINING
Rosanalin dye Solid Medium
Silver nitrite + ferric tannate  Solid culture media contain agar at a
concentration of 1.5-2.0% or some
other primarily inert solidifying agent.
Solid medium has a physical structure
and allows bacteria to grow in
 physically informative or useful ways THESE ARE CLASSIFIED INTO SIX
(e.g., as colonies or in streaks). TYPES:
MacConkey agar, chocolate agar,
 Basal media,
nutrient agar, blood agar, etc., are
 Enriched media,
some examples of solid culture media.
 Selective
Uses of solid culture media  Indicator media,
 For isolating bacteria from various  Transport media, and
types of specimen  Storage media
 For determining the colony BASAL MEDIA
characteristics of the isolate (such as
colony morphology, hemolysis,  Basal media are those that may be
pigment production, etc. used for growth (culture) of bacteria
 For performing antimicrobial that do not need enrichment of the
susceptibility testing using the Kirby media. Basal media, also called
Bauer disc diffusion method general-purpose media, are simple
media that support the growth of most
Semisolid Medium non-fastidious bacteria. Peptone
 Semisolid culture media are prepared Water, nutrient broth, and nutrient
with agar at 0.5% or less agar (NA) are basal media. These
concentrations. Semisolid medium media are generally used for the
has a soft custard-like consistency and primary isolation of microorganisms.
is helpful for the cultivation of Examples: Nutrient broth, nutrient agar and
microaerophilic bacteria or for peptone water. Staphylococcus and
determining bacterial motility. Enterobacteriaceae grow in these media.
Motility test medium, Stuart’s and
Amies transport media, etc., are
semisolid media. ENRICHED MEDIA
Liquid (Broth) Medium  The media are enriched usually by
 These media contain specific amounts adding blood, serum or egg.
of nutrients but don’t have a trace of Examples: Enriched media are blood agar
gelling agents such as gelatin or agar. and Lowenstein-Jensen media. Streptococci
Commonly used liquid media in the grow in blood agar media.
lab are; nutrient broth, glucose broth,
brain-heart infusion (BHI) broth,
alkaline peptone water (APW), tryptic SELECTIVE MEDIA
soy broth (TSB), and selenite F broth.
 These media favor the growth of a
Broth medium serves various
particular bacterium by inhibiting the
purposes such as propagation of many
growth of undesired bacteria and
organisms, fermentation studies, and
allowing growth of desirable bacteria.
various other tests.
Examples: MacConkey agar, Lowenstein-
DIFFERENT MEDIA PREPARATION
Jensen media, tellurite media (Tellurite
 Agar plate inhibits the growth of most of the throat
 Agar deep tube organisms except diphtheria bacilli).
 Broth Antibiotic may be added 4. to a medium for
 Agar slant inhibition.
INDICATOR (DIFFERENTIAL) Aseptic technique means using practices
MEDIA. and procedures to prevent contamination
from pathogens. It involves applying the
An indicator is included in the medium. A
strictest rules to minimize the risk of
particular organism causes change in the
infection. Healthcare workers use aseptic
indicator, e.g. blood, neutral red, tellurite.
technique in surgery rooms, clinics,
Examples: Blood agar and MacConkey agar outpatient care centers, and other health care
are indicator media. settings.
 Mannitol salts agar (mannitol INOCULATION OF
fermentation = yellow) MICROORGANISM USING ASEPTIC
 Blood agar (various kinds of TECHNIQUE
hemolysis i.e., α, β and γ hemolysis)
 Flame loop
 MacConkey agar (lactose fermenters,
 Flame tube
pink colonies whereas, non-lactose
 Transfer from broth plate/broth
fermenter produces pale or colorless
 Flame tube
colonies.
 Cap & put back in rack
 TCBS (Vibrio cholerae produces
yellow colonies due to fermentation  Flame loop
of sucrose)
METHODS FOR THE ISOLATION OF
TRANSPORT MEDIA. BACTERIA

 These media are used when specie-  Pouring method

men cannot be cultured soon after  Spreading method
collection. Such media prevent drying  Streaking method
(desiccation) of a specimen, maintain  Serial dilution method
the pathogen to commensal ratio, and
inhibit the overgrowth of unwanted
bacteria.  Bacterial isolation, purification and
identification are the first steps to
Examples: Cary-Blair medium, Amies bacteriological studies. Isolation is
medium, Stuart medium. done to obtain pure bacterial cultures.
To obtain a pure bacterial culture is
the first step to bacterial
STORAGE MEDIA. Media used for storing
identification.
the bacteria for a long period of time.
 Pure culture is essential in the study
Examples: Egg saline
of the morphology, physiology,
 medium, chalk cooked meat broth biochemical characteristics, and
susceptibility to antimicrobial agents
of a particular bacterial strain. Pure
WHAT TO PREPARE cultures are best obtained by using
solid media, by streak plate or pour
 NUTRIENT AGAR
plate method
 NUTRIENT BROTH
OMNIPRESENT OF MICROOGANISM
Microbes are omnipresent, which means
COMPUTAION
they are found everywhere. They can be
g/1000 ml= g/100 ml found in soil, water, air, ice, inside bodies of
human beings, animals, and plants, and even
28g/1000 ml= 2.8 g/100ml
in thermal vents.
If we only need 200 ml of medium
WHAT IS ASEPTIC TECHNIQUE?
DAMAGE TO HOST CELLS
 Siderophores
 Direct damge
 Toxins
 Exotoxins
 Endotoxins
 Lysogenic conversion
 Cystopathic effects

PORTALS OF EXIT
 Generally, the same as the portals of
entry for a given microbe.

PORTALS OF EXIT AND ENTRY

 Respiratory tract
 Coughing, sneezing
 Gastrointestinal tract
 Feces, saliva
MICROBIAL MECHANISMS OF
PATHOGENICITY  Genitourinary tract
 Urine, vaginal secretions
 Skin
Virulence is the degree of pathogenicity of
 Blood
an organism.
 Biting arthropods, needles/syringes
Pathogenicity, Ability to cause disease.

Microbes gain access to systemic system

PORTALS OF ENTRY through:
 Mucous membrane  Bites
 Respiratory tract  Injections
 Gastrointestinal tract  Wounds
 Genitourinary tract  Called parenteral rout
 Conjunctiva
 Skin
 Parenteral route Preferred Portal of entry
Most cause infection only when they use a
specific portal of entry
PENETRATION OR EVASION OF
HOST DEFENSES Example:

Capsules  Yersinia pestis by a number of portals

 Streptococcus pneumoniae respiratory
Cell wall components
tract
Enzymes  Vibrio cholerae gut
Antigenic variation  Neisseria gonorrhoeae genitals
 Clostridium perfringes parenteral
Invasins
Intracellular growth
Adherence HOW BACTERIAL PATHOGENS
DAMAGE THE HOST CELLS.
 In order to get into a host the bacteria
must stick to it. Only some of the cell damage is caused by
 Surface projections (ligands) adhere bacteria themselves
to receptors on host cells. • Some bacteria enter the host cell and
 Mostly on structures called fimbriae damage as they leave
• Some bacteria harm the cell as they
Adhesions/ligands bind to receptors on host enter
cells
 Glycocalyx- Streptococcus mutans BACTERIA TOXINS
 Fimbriae- Escherichia coli
 M protein- Streptococcus pyogenes  Cause the most damage
 Opa protein- Neisseria gonorrhoeae  Toxins: poisonous substances
produced by microbes
 Tapered end- Treponema pallidum
 Toxigenicity: capacity of a microbe
to produce toxin
HOW BACTERIA ESCAPE  Toxemia: presence of toxins in the
PROGRAMMED HOST DEFENSES blood
 Toxoid: Inactivated toxin used in a
 Capsules are extracellular glycocalyx vaccine
material that surrounds the cell.  Antitoxin:Antibodies against a
Prevent recognition of the bacterial specific toxin
cell. Streptococcus pneumoniae
 Cell wall components may also resist
recognition and phagocytosis EXOTOXINS
 Produced by bacteria and released
ENZYMES THAT PROTECT into surrounding area. Causes
Disease
 Leukocidins- destroy neutrophils and  Cytotoxin or diphtheria toxin inhibits
macrophages protein synthesis
 Hemolysisn- break up red blood cells  Tetanus toxin prevents nerve
 Coagulase- prevents or breaks up transmission
blood clots designed to localize  Enterotoxins promote electrolyte and
infection fluid loss from cells.
 IgA proteases- Destroy IgA  Neurotoxins (Botulinum toxin)
antibodies prevent nerve transmission
 Antibodies produced in response are
antitoxins
BACTERIA MOVING INTO TISSUES
Kinases- destroy blood clots
Hyaluronidase- works on
mucopolysaccharide that holds cells together
Collagenase- hydrolyses connective tissue
collagen
Invasins- destroy cytoskeleton of individual
cells.
Exotoxins EXTRA GENETIC MATERIAL
A-B toxins or type III toxins • Some bacteria may carry extra genes
that help pathenogenisity
• Plasmids (extra chromosomal) can
carry genes for antibiotic resistance, toxins,
capsules and fimbriae
• Coagulase produced by
Staphylococcus aureus
• Fimbria in specific straines of E. coli

SHAPE, SIZE AND ARRANGEMENT

OF BACTERIA

BASIC SHAPES
Coccus (pl. cocci)- round or spherical
Bacillus (pl. bacilli)- rod or cylindrical

SHAPES OF BACTERIA
Cocci – spherical or ovoid cells, e.G.
Staphylococcus.
Bacilli
 Straight and cylindrical rods, e.G.
Bacillus spp.
Exotoxins  Long, thin filamentous form, e.G.
Actinomycetes
• Superantigens or type I toxins
Spirillum –
• Cause an intense immune response
due to release of cytokines from host cells  Comma-shaped (vibrio), e.G. Vibrio,
campylobacter
• Fever, nausea, vomiting, diarrhea,
shock, death  Spiral-shaped, loosely coiled
(spirochete), e.G. Spirochetes,
 Elongated, tightly coiled (spirillum),
A TEST FOR ENDOTOXINS. e.G. Azospirillum spp)

 Limulus amoebocyte lysate (LAL)- Pleomorphic – variable shape

used to detect endotoxins in drugs and
on medical devices.
OTHER COMMON SHAPES
 The amoebocyte will lyse in the
presence of endotoxins causing a Coccobacilli- cells in between round and rod
thickening of the media. shape
Vibrio- curved cell
Spirillum- spirilla, plural, rigid, wave-like
shaped cell
Spirochete- corkscrew shaped sell
SIZE OF BACTERIA of cells.
 Staphylococci - Cells divide in three
 Bacteria are very small in size
planes, in an irregular pattern
• cocci are approx. 0.5 to 1.0 μm in producing bunches of cocci, e.g.
diameter. Staphylococcus aureus
• rods range from 2 to 5 μm in length by 0.5  Spherical is called- coccus.
to 1.0 μm in width  Division along the same plane forms
chains; 2 cocci together –
• Spirochetes are longer (up to 20 μm) and Diplococcus
narrower (0.1 to 1.0 μm)  4 - 20 in chains - Streptococcus.
 varies with the medium and growth  Division along 2 different planes –
phase Tetrads
 usually smallest in the logarithmic  Division along 3 planes regularly –
phase of growth. Sarcinae
 Bacteria are very small compared to  Division along 3 planes irregularly -
cells with nuclei. Staphylococci
 Bacteria compared to a white blood
cell that is going to eat it.
BACILLI
Single
SURFACE AREA/ VOLUME RATIO
 Diplobacilli - in pairs
The surface area/volume ratio of a spherical  Streptobacilli – in chains, e.G.
bacteria of 1 µm in diameter is high (6:1) as Bacillus subtilis
compared to a spherical eukaryotic cell
 Trichomes - rod-shaped bacteria
having a diameter of 20 µm (0.3:1).
arranged in chains with a larger area
Consequently: of contact between
 Adjacent cells, e.G. Beggiatoa spp.
• The intake of nutrients and removal of
waste products is quick - the bacteria have  Palisade – the cells are lined side by
high rate of growth and metabolism. side as match sticks, e.G.
Mycobacterium tuberculosis.
• No circulatory mechanism for  Chinese letter like – e.G.,
nutrients is needed - the cytoplasmic corynebacterium spp.
streaming is absent.  Filamentous – long, mycelium like
branching, mono-nuclear, e.G.
Actinomycetes
ARRANGEMENT OF BACTERIAL
 Hyphae – long, branched,
CELL
multinucleate filaments, e.G.
Cocci Streptomyces.
 Rod shape is called- Bacillus.
 Diplococci - Cells divide in one plane
 Two bacilli together- Diplobacilli
and remain attached predominately in
pairs, e.g. pneumococci.  Chains of bacilli are called-
Streptobacilli
 Streptococci - Cells divide in one
plain and remain attached to form  Palisades - Rods side by side or in X,
chains, e.g Streptococcus V or Y figures
 Tetracocci - Cells divide in two
planes and forms groups of four cells.
(also called as
 ‘tetrads’), e.g. Aerococcus.
 Sarcinae - Cells divide in three planes,
in a regular pattern producing a
cubodial arrangement
BASIC BACTERIAL STRUCTURE MYCOPLASMAS (PPLPO)
Being small offers bacteria unique  Naturally lack cell walls
opportunities for survival and reproduction  Gram-negative
 Size ranges from 50-60 to 100-250
nm
BACTERIAL STRUCTURE: Cell  Highly pleomorphic eubacteria
Envelope  Five genera require sterols and three
Components of the bacterial cell envelope: do not.
 No free-living mycoplasma; strictly
 Cytoplasmic
parasitic
 Cell wall
 Parasitize a wide range of organism
 Capsule including humans, plants, animals,
 Slime and insects.
 Flagella  Facultative anaerobes and obligate
 Fimbriae/ pilli anaerobes.
 Growth on artificial media is slow
with a generation time ranging up to
Bacterial structure: intracellular nine hours in some species.
structure
 Supplementation with other factors,
Intracellular componets: such as serum, may be required
 Utilize glucose or arginine
 Nucleoid
as the major source of energy.
 Ribosomes
 ‘fried egg’ or ‘nipple shaped’
 Inclusion granules
colonies, which can be stained by
 Endospores
dienes’ stain.

STRUTURES OF BACTERIA
RICKETTSIA AND CHLAMYDIA
 Cell wall- protects and given shape
 Coccoid to rods in shape, with a
 cell membrane- regulates movement diameter Of 0.3-0.7 μm.
of materials, contains enzymes
 Gram-negative type cell walls
important to cellular respiration
 Except one rickettsia (rochalimaea),
 cytoplasm- contains DNA, ribosomes,
all are Obligate intracellular parasites.
essentials compound.
 Contain both dna and rna.
 Chromosome- Carries genetic
information
 Plasmid- Contains some genes ANTIMICROBIAL SUSCEPTIBILITY
obtained through recomb. TESTING
 Capsule & Slime Layer- Protects the
cell and assist in attaching cell to
other surfaces ANTIMICROBIAL SUSCEPTIBILITY
TESTING
 Endospore- Protects cell agains harsh
enviornments Dilution method
 Pilus- Assists the cell in attaching to
• vary amount of antimicrobial
other surfaces
substances incorporated into liquid or solid
 Flagellum- Moves the cell media
• followed by inoculation of test
bacteria
Streak Plating • Temperature of incubation Larger
zones are seen with temperatures < 35 oC
• streaking is a technique used to isolate
a pure strain from a single species of • Incubation time Ideal 16-18 hours;
microorganism, often bacteria. Samples can less time does not give reliable results
then be taken from the resulting colonies
• Potency of antibiotic discs
and a microbiological culture can be grown
Deterioration in contents leads to reduced
on a new plate so that the organism can be
size
identified, studied, or tested. The streaking
is done using a sterile tool, such as a cotton • Composition of medium Affects rate
swab or commonly an inoculation loop of growth, diffusion of antibiotics and
activity of antibiotics
• Acidic ph of medium tetracycline,
Susceptibility test, main purposes:
novobiocin, methicillin zones are larger
As a guide for treatment
• Alkaline ph of medium
• Sensitivity of a given m.o. to known aminoglycosides, erythromycin zones are
conc. of drugs larger
• Its concentration in body fluids or • Reading of zones subjective errors in
tissues determining the clear edge
As an epidemiological tool • Size of the plate smaller plates
accommodate less number of discs
• The emergence of resistant strains of
major pathogens • Depth of the agar medium (4 mm)
thin media yield excessively large inhibition
• Continued surveillance of the
zones and vice versa
susceptibility pattern of the prevalent strains.
• Proper spacing of the discs (2.5 cm)
avoids overlapping of zones
Stokes method
• Tests of organism of unknown
sensitivity and control organism of known
sensitivity are set up at the same time and in
identical conditions on the same plate
• The control inoculum is spread in two
bands on either side of the plate, and the test
organism is inoculated onto the central area
of the plate
• An uninoculated gap 2-3mm wide
should separate the test and control areas
• Interpretation done by comparing the
size of inhibition zone

Factors Affecting Size of Zone of

Inhibition
• Inoculum density Larger zones with
light inoculum and vice versa
• Timing of disc application If after
application of disc, the plate is kept for
longer time at room temperature, small
zones may form