BIOSAFETY IN THE LABORATORY

BIOSAFTEY
 The prevention of large-scale loss of biological integrity, focusing both on ecology and
human health.
 It is a system for the safe handling of toxic and dangerous biological and chemical
substance.
 In medicine - It refers to the levels of lab containment protocols, measured as Bio Safety
Level (BSL) 1,2,3,4 in rising order of danger.
 
BIOSAFETY LEVELS
 Precautions to be taken by people researching or trying to identify organisms.
 Labs must adhere to these specific safety regulation.
 A biosafety level is the level of the bio containment precautions, required to undertake
while handling dangerous biological agents in an enclosed facility.
 
CONTAINMENT
 "Containment" - safe methods, facilities and equipment for managing infectious materials
in the laboratory environment where they are being handled or maintained.
 The purpose of containment - reduce or eliminate exposure of laboratory workers, other
persons, and the outside environment to potentially hazardous agents.
 The use of vaccines may provide and increased level of personal protection.
 
BARRIERS
 Prevent invaders from crossing
 
UNIVERSAL PRECAUTION
 An approach to infection control to treat all human blood and certain human body fluids
as if they were known to be infectious for HIV, HBV and other bloodborne pathogens.
 
PERSONAL PROTECTIVE EQUIPMENT (PPE)
 Protective clothing, helmets, goggles, or other garments or equipment designed to protect
the wearer's body from injury or infection.
 Specified by exposure control plan or by standard operating procedure
 
A BIOSAFETY CABINET (BSC)
 Also called as biological safety cabinet or microbiological safety cabinet
 It is enclosed, ventilated laboratory workspace for safely working with materials
contaminated with (or potentially contaminated with) pathogens requiring a defined
biosafety level
 BSCs first became commercially available in 1950
 
INFECTIONS OF SPECIAL CONCERN
 Tuberculosis
 Hepatitis B
 HIV
 Enteric Infections
 
ROUTES OF INFECTION
 Inoculation
 Ingestion

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  Inhalation
 
'BIOSAFETY: IN MICROBIOLOGY LABS: PREVENTING LAB-ACQUIRED INFECTIONS
FROM-
o Bacteria
o Viruses
o Fungi
o Parasites
o Prions
o Recombinant DNA
SOURCE-
o Various specimens
o Human blood
o Unfixed tissue
o Human cell lines
TO-
o Lab Personnel
o Community
 
BARRIERS
Primary Barriers
 Physical barriers of personal protective equipment for lab worker
 Gloves masks, goggles, aprons, suits, special breathing apparatuses
 
Secondary Barriers
 Structural aspects of the laboratory that make working environment safer against
infection
o Sink for handwashing
o Special air ventilation patterns
o Sterilization equipment
 
UNIVERSAL SAFETY PRECAUTIONS
 Consider all the specimens potentially infectious for HIV and other blood borne
infections
 All specimens should be placed in a leak-proof impervious container for transport
 Use gloves while handling all samples, especially when there is contact with body fluids,
non-intact skin or mucous membrane.
 If there is likelihood of spattering, use face mask with glasses and gowns. Wrap around
gowns should be preferred. These should not be used outside the lab.
 Cover cuts or abrasions present over skin with waterproof bandage.
 Decontaminate the laboratory work surfaces immediately in case of spillage of blood or
any other body fluids
 Follow no needle recapping strategy
 All sharps should be collected and disposed away properly
 Never pipette by mouth. Use mechanical pipetting devices
 There should always be a system working efficiently for management of hospital
generated waste.
 It is advisable for the laboratory personnel to be vaccinated against Hepatitis-B
 Facilities should be available easily for post exposure prophylaxis in case of exposure to
HIV & HBV.

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STANDARD MICROBIOLOGICAL PRACTICES
Do's
o Controlled access to the laboratory
o Frequent handwashing
o Mechanical pipetting
o Appropriate waste management & sterilization and disinfection measures
o Training to the workers
Don'ts
o Eating, drinking, smoking, handling contact lenses
o Storing food
o Mouth pipetting
 
BIOSAFETY LEVELS
 Precautions to be taken by people researching or trying to identify organisms.
 Labs must adhere to these specific safety regulation.
 A biosafety level is the level of the bio containment precautions, required to undertake
while handling dangerous biological agents in an enclosed facility.
 
LEVELS OF CONTAINMENT
BSL 1
 Microorganisms that don't consistently cause disease in healthy adults
o E.coli, polyoma virus
o Basic laboratory
o Standard microbiological practices
 STANDARD PRACTICES REQUIRED:
o Frequent handwashing
o Door that can be kept closed when working;
o Limits on access to the lab space when working;
o No smoking, eating, drinking, storage food in laboratory;
o Care to minimize splashes and actions that may create aerosols (tiny droplets);
o Decontamination of work surfaces after every use after any spills;
 

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BIOSAFETY LEVEL 2
 Agents associated with human disease
o Generally required for any human-derived blood, bodily fluids, tissues in which
infectious agent may be unknown
o Agents include measles virus, Salmonella species, pathogenic Toxoplasma,
Clostridium botulinum, hepatitis B Virus
 

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 Primary Hazards:
o Accidental needle sticks
o Exposure to eyes and nose (mucous membranes)
o Ingestion of infectious materials
 Agents do not cause lethal infections, are not transmissible via airborne route
o (do not cause infection if tiny droplets become airborne and are inhaled, which
might occur if the material were spattered)
 Agents are pathogens for which immunization or antibiotic treatment is available
 Extreme care should be taken with contaminated needles and sharp lab instruments
 Standard practices include BSL-1 plus:
o Policies to restrict access to lab;
o Biohazard warning signs posted outside lab;
o Surveillance of laboratory personnel with appropriate immunizations offered;
o Biosafety manual with definitions of needed waste decontamination or medical
surveillance policies; supervisory staff who gave experience of working with
infectious agents and specific training for laboratory personnel in handling these
agents
 
BIOSAFETY LEVEL 3
 Microorganisms that cause serious disease, transmitted by inhalation
o M. Tuberculosis, yellow fever virus, hantavirus, Y. Pestis (plague)
o Containment lab: double door entry; directional airflow; all work in biosafety
cabinet
 Standard Practices include BSL1 plus:
o Policies to restrict access to lab;

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 o Biohazard warning signs posted outside lab;
o Surveillance of laboratory personnel with appropriate immunization offered;
o Biosafety manual with definitions of needed waste decontamination or medical
surveillance policies;
o Supervisory staff who have experience of working with infectious agents and
specific training for laboratory personnel and handling these agents
o Strictly controlled access to the lab:
o Specific training for lab personnel in handling potentially lethal agents;
o Decontaminating all waste
o Changing contaminated protective lab clothing, decontaminating lab clothing
before laundering;
o Institutional policies regarding specimen collection and storage from workers to
prevent exposure
 Primary barriers: biosafety cabinets or other approved containment devices
 Personal protective equipment: lab coats, gloves, face protection as needed
 Protective clothing removed when personnel leave laboratory area
 Cabinets thoroughly decontaminated daily and monitored for radiation for personal
protection
 Secondary barriers: BSL-1 barriers plus autoclave for glassware
 
PRIMARY BARRIERS
 Similar to BSL-2 personal protective equipment
 Respiratory barriers
SECONDARY BARRIERS
 All BSL-2 barriers
 Corridors separated from direct access to lab
 Access through self-closing double doors
 Air handling systems to ensure negative air flow (air flows the lab)
 Air pumped into lab not re-circulated in building
 
BIOSAFETY LEVEL 4
 Microorganisms that cause lethal disease, with no known treatment or vaccine
o Ebola Virus, Marburg Virus
o Maximum containment lab; positive pressure ventilated suits (moon suits)
 Dangerous and exotic agents with high risk life threatening disease and are aerosol-
transmission
 Related agents with unknown risk of transmission
 Agents (all viruses) include Marburg virus, Ebola virus, viruses that cause Congo-
Crimean hemorrhagic fever, Lassa fever
 
PRIMARY BARRIERS
 Biosafety cabinets used at other biosafety levels
 Full-body, air supplied, positive pressure personnel suit
SECONDARY BARRIERS
 All physical barriers at BSL-3
 Isolated zone or a separate building;
 Dedicated supply and exhaust, vacuum, decontamination systems;
 A recommended absence or windows (or sealed and resistant to breakage)
 

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VIEWING THE MICROBIAL WORLD
USING METRIC SYSTEM TO EXPRESS THE SIZES OF MICROBES
 Metric units are used to express the sizes of microbes
 The basic unit of length in the metric system is the meter (m); it is equivalent to 39.4 inches
 The sizes of bacteria and protozoa are usually expressed in terms of micrometers (um). A
micrometer is one millionth of a meter.
 A typical spherical bacterium (coccus) is approximately 1 um in diameter.
 A typical rod-shaped bacterium (bacillus) is approximately 1 um wide x 3 um long.
 The size of viruses are expressed in terms of nanometers (nm). A nanometer is equal to one
billionth of a meter.
 Most of the viruses that cause human disease range in size from 10 to 300 nm.
 One exception is Ebola virus, a cause of viral hemorrhagic fever. Ebola viruses can be as long as
1,000 nm (1 um)
 When using a microscope, the sizes of microorganisms are measured using an ocular micrometer
o This must be calibrated using a stage micrometer for each microscope objective
o Stage micrometer acts as a scale of measurement
 
MICROSCOPES
 The human eye, a telescope, a pair of binoculars, a magnifying glass, and a microscope are
various types of optical instruments
 A microscope is an optical instrument that is used to observe tiny objects, objects so small that
they cannot be seen with the unaided human eye.
 Each optical instrument has a limit as to what can be seen using that instruments; this limit is
referred to as the resolving power or resolution of the instruments
 The resolving power of the unaided human eye is approximately 0.2 mm
 
SIMPLE MICROSCOPES
 A simple microscope is one that contains only one magnifying lens.
 A magnifying glass cloud be considered a simple microscope when using a magnifying glass,
images appear 3 to 20 times larger than the object's actual size.
 Leuuwenhoek's simple microscope had a maximum magnifying power of about x300 (about 300
times
 Not as widely used in research and labs
 Often uses external light source
 
COUMPOUND MICROSCOPE
 A compound microscope contains more than one magnifying lens
 Because visible light is the source of illumination, a compound microscope is also referred to as a
compound light microscope
 Compound light microscopes usually magnify objects about 1,000 times
 The resolving power of a compound light microscope is approximately 0.2 u,
o About 1,000 times better than the resolving power of the unaided human eye.
 It is the wavelength of visible light (0.45 um) that limits the size of objects that can be seen
 Objects cannot be seen if they are smaller than half of the wavelength of visible light
 Today's laboratory microscope contains two magnifying lens systems:
o The eyepiece or ocular lens (usually x10)
o The objective lens (x4, x10, x40 and x 100) are the four most commonly used objective
lenses

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 Total magnification is calculated by multiplying the magnifying power of the ocular lens by the
magnifying power of the objective lens being used.
×10 ocular x x4objective = x40 total magnification
×10 ocular x x10 objective = x100 total magnification
×10 ocular x x40 objective = x400 total magnification
×10 ocular x x100 objective = x1,000 total magnification
 Photographs taken through the lens system of the compound light microscope are called
photomicrographs
 Because objects are observed against a bright background or "bright filed" the compound light
microscope is sometimes referred to as a brightfield microscope
 If the condenser is replaced with what is known as a darkfield condenser, illuminated objects are
seen against a dark background or "dark field"; the microscope is then called a darkfield
microscope
 Other types of compound microscope include
o Phase contrast microscope
o Fluorescence microscope
 

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PHASE CONTRAST AND FLUORESCENCE MICROSCOPES
 Phase contrast microscopes are used to observe unstained living microorganisms
o Organisms are more easily seen because the light refracted by living cells is different
from the light refracted by the surrounding medium
 Fluorescence microscopes contain a built in ultraviolet light source
o When the UV light strikes certain dyes and pigments, these substances emit a longer-
wavelength lights, causing them to glow against a dark background
o Different parts of the same cell can be stained with different dyes to allow for complex
analysis of proteins within the cell
 
ELECTRON MICROSCOPES
 Electron microscopes enable us to see extremely small microscopes such as rabies and smallpox
viruses
 Living organisms cannot be observed using an electron microscope - the processing procedures
kill the organisms
o Microscope uses a vacuum
o An electron beam is used as the source of illumination, and magnets are used to focus the
beam
o Electron microscopes have a much higher resolving power than compound light
microscopes
 Wavelength of the beam is 100,000 times shorter than visible light
o There are two types of electron microscopes - transmission and scanning
 
TRANSMISSION ELECTRON MICROSCOPE
 Electron beam produced at the top of a tall column
 This microscope uses an electron gun to fire a beam of electrons through an extremely thin
specimen (<1 um thick)
 Some electrons transmit through the specimen while others are blocked creating an image
 An image of the specimen is produced on an phosphor-coated screen

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  Magnification is approximately 1,000 times greater than with the compound light microscope
 Resolving power is approximately 0.2 nm
 Can visualize internal cell structures
 
 

 
 
SACANNING ELECTRON MICROSCOPE
 Composed of a shorter column
 Electrons are bounced off the surface of a specimen and captured by detectors that create an
image that appears on a monitor
 This is used to observe the outer surfaces of specimens
 Resolving power of this microscope is about 100 times less than that of transmission electron
microscope
 
ELECTRON MICROSCOPES
 Micrographs collected are black and white images
o If color is used in photograph, it was artificially enhanced
 2-dimensional (2-D) micrographs obtained
 

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ATOMIC FORCE MICROSCOPES
 Provides a 3-dimensional (3-D) image silicon or silicon nitride cantilever with a sharp tip scans
surface or specimen when tip and a cantilever are in close proximity, deflection is caused due to
atomic forces between the two
 Deflection is measured using a laser reflected off the cantilever onto a photodiode creating an
image
 
 

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MORPHOLOGY OF BACTERIA
INTRODUCTION
 Bacteria is unicellular, free-living, microscopic microorganisms capable of performing all the
essential functions of life
 They possess both deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA)
 Bacteria are prokaryotic microorganisms that do not contain chlorophyll
 They occur in water, soil, air, food and all natural environment
 They can survive extremes of temperature, pH, oxygen, and atmospheric pressure
 
SIZE OF BACTERIA
 Bacteria are very small microorganisms which are visible under the microscope
 They are having the size range in microns
 Bacteria are stained by staining reagents and then visualized under high power of magnification
(1000x) of compound microscope.
 An electron microscope is used for clear visualization of internal structure of bacteria
 
BACTERIAL STRUCTURE
FLAGELLA
 Are long, slender, thin hair-like cytoplasmic appendages, which are responsible for the motility of
bacteria
 These are the organs of locomotion
 They are 0.01 to 0.02 um in diameter, 3 to 20 um in length
 Flagella are made up of a protein-flagellin
 The flagellum has three basic parts
o Filament
o Hook
o Basal body
 Filament is the thin, cylindrical, long outermost region with a constant diameter
 The filament is attached to a slightly wider hook
 The basal body is composed of a small central rod inserted into a series of rings
 Gram negative bacteria contain four rings as L-ring, P-ring, S-ring, M-ring whereas gram positive
bacteria have only S and M rings in basal body.
Flagella may be seen on bacteria body in the following manner
1. Monotrichous - these bacteria have single polar flagellum (e.g. vibrio cholera)
2. Lophotrichous - these bacteria have two or more flagella only at one end of the cell. (e.g.
pseudomonas fluorescence)
3. Amphitrichous - these bacteria have single polar flagella or tuft of flagella at both polse. (e.g.
aquaspirillum serpens)
4. Peritrichous - several flagella present all over the surface of bacteria (e.g. escherichia coli,
salmonella typhi)
 
PILI OR FIMBRAE
 Pili are like microfibrils, 0.5 to 2 um in length and 5 to 7 nm in diameter
 They are thinner, shorter and more numerous than flagella
 They are present only on gram negative cells
 They are composed of protein known as pillin.
 They are unrelated to motility and are found on motile and non-motile cells
 Fimbriae and pili, these are two terms are used interchangeably but they can be distinguished
 Fimbriae can be evenly distributed over the entire surface of the cell or they occurs at the poles of
the bacterial cell. Each bacteria possess 100 to 200 fimbriae
 Pili are usually longer than fimbriae and number only one or two per cell

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  Function: Pili play an important role in attachment to surface. Hence pili is also called organ of
adhesion.
 
PLASMA MEMBRANE
 The cytoplasmic (plasma) membrane is a thin (5 to 10 nm).
 It separates the cell wall and cytoplasm
 It composed of phospholipids (20 to 30%) and proteins (60 to 70%)
 Prokaryotic plasma membranes are less rigid than eukaryotic membrane due to lack of sterols
Function:
1. It acts as a semipermeable membrane controlling the inflow and outflow of metabolites to and
from the protoplasm
2. It provides the mechanical strength to the bacterial cell
3. It helps in DNA replication
4. It contains enzyme, permease, which plays an important role in the passage of selective nutrients
through the membranes.
 
MESOSOMES
 Mesosomes are respiratory sites of bacteria
 The mesosomes are attached to the bacterial chromosomes and is involved in DNA segregation
during cell division
 They are predominant in Gram positive bacteria
 
NUCLEOID
 The bacterial chromosomes is not surrounded by nuclear membrane so it is called nucleoid
 The bacterial chromosomes are made up of double strand circular DNA
 
INTRA CYTOPLASMIC INCLUSION
 Many species of bacteria cytoplasmic inclusion bodies which appears as round granules
 They are made up of either glycogen or starch
 They appear reddish when stained with polychrome methylene blue or toluidine blue
 
CAPSULE
 Bacteria synthesize loose amorphous organic exopolymer which is deposited outside and tightly
to cell wall called capsules
 Capsules may be composed of complex polypeptides or polysaccharides. Water (98%) is the
main component of bacterial capsule
 Some time the capsular material is loosely associated with the bacterium, it can be easily washed
away. The loose layer is called slime layer
 Capsulated bacteria produces rough colonies on the surface of agar media.
Functions:
1. They protect the cell from drying
2. They protects the bacterial cell against anti-bacterial agents and phages
 
CELL WALL
 Cell wall is rigid structure which gives definite shape to cell, situated between the capsule and
cytoplasmic membrane.
 It is about 10-20 nm in thickness and constitute 20-30% of dry weight of cell
 The cell wall cannot be seen by direct light microscopy and does not stain easily by different
staining reagents
 The cell wall of bacteria contains diaminopimelic acid (DAP), muramic acid and teichoic acid.
These substances known as peptidoglycan or murein or mucopeptide

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  Peptidoglycan is the major constituent of the cell wall of gram positive bacteria (50 to 90 %)
where as in gram negative bacterial cell wall its presence is only 5-10%
Functions:
1. Cell wall involves in growth and cell division of bacteria
2. It gives shape to the cell
3. It gives protection to internal structure and acts as supporting layer
4. To prevent rupture of bacteria caused by osmotic pressure differences between intra cellular and
extra cellular environment
5. To provide support for flagella
6. To regulate a certain degree of passage of molecules into and out of the cell
7. It serve as the sites of attachment for most bacterial viruses.

RIBOSOMES
 Are the center of protein synthesis
 They are slightly smaller than eukaryotic ribosomes
 The sedimentation constant is 70s
 this 70s ribosomes are made up of two subunits namely a large subunits 50s and a small subunits
30s
 During active protein synthesis the ribosomes are associated with mRNA and such associations
are called polysomes
 
SPORE
 The process of endospore formation is known as sporulation and it may take 4 to 8 hours in a
vegetative cell
 Endospores are thick-walled, highly refractile bodies that are produced one per cell
 Each bacterial spore on germination forms a single vegetative cell. Therefore, sporulation in
bacteria is a method of preservation and not reproduction
 Spores are extremely resistant to dessication, staining, disinfecting chemicals, radiation and heat
 They remain viable for centuries and help bacteria to survive for long period under unfavorable
environment. Endospore can remain dormant for thousand of years.
 Spores of all medically important bacteria are destroyed by moist heat sterilization (autoclave) at
121 C for 20 minutes

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MORPHOLOGICAL CLASSIFICATION
 Bacteria can be classified into six major groups on morphological basis.
 
TRUE BACTERIA
Cocci - these are spherical or oval cells. On the basis of arrangement of individual organisms they can be
described as
 Monococci (Cocci in singles) - Monococcus spp.
 Diplococci (Cocci in pairs) - Streptococcus pneumoniae
 Staphylococci (Cocci in grape-like-clusters) - Staphylococcus aureus
 Streptococci (Cocci in chains) - Streptococcus pyogenes
 Tetrad (Cocci in group of four) - streptococcus pyogenes
 Tetrad (Cocci in group of four) - Micrococcus spp.
 Sarcina (Cocci in group of eight)
 
Bacilli - these are rod shaped bacteria. On the basis of arrangements of organisms, they can be described
as
 Diplobacilli
 Streptobacilli
 Palisades
 Chinese-letter form
 Coccobacili
 Comma-shaped
 
Spirochaetes
 These are relatively longer, slender, non-branched microorganisms of spiral shape having several
coils
 
Mycoplasmas
 These bacteria lack in rigid cell wall (cell wall lacking) and are highly pleomorphic and of
indefinite shape. They occur in round or oval bodies in interlacing filaments.
 
Rickettsiae and Chlamydiae
 They are very small, obligate parasites, and at one time were considered closely related to the
viruses. Now, these are regarded as bacteria
 
BACTERIAL GROWTH CURVE
Microbial Growth
 Growth indicates that organism is in active metabolism

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  In microbial world. Particularly bacteria growth refers to change in population thru binary fission
 Population increases by geometric progression
o 1 cells divides into 2 cells - divides into 4 cells and so on
What is Microbial Growth?
 Involves an increase in the number of cells rather than in size of individual cells
2 levels of growth:
i. A cell synthesizes new components, increase its size
ii. Increase number of cells in the population
 Increase in cellular constituents that may result in:
o Increase in cell number
 E.g. When microorganisms reproduce by budding or binary fission
o Increase in cell size
 E.g. Some microorganisms have nuclear divisions that are not accompanied by
cell divisions
 Microbiologists usually study population growth rather than growth of individual cells
 
THE BACTERIAL DIVISIONS/REPRODUCTION
 Bacteria normally reproduce by a method called binary fission
 Binary Fission - the process of bacteria reproduction where one cell become two
 
BACTERIAL GENERATION (DOUBLING) TIME
Examples
 Escherichia Coli - 20 minutes
 Mycobacterium tuberculosis 18 hours
 Mycobacterium leprae - 14 days
 
2 SATGES IN BACTERIAL GROWTH CURVE
 When a few bacteria are inoculated into a liquid growth medium, it is possible to plot a bacterial
growth curve that shows the growth of cells over time
 
STAGES OF BACTERIAL GROWTH
 Bacterial populations follow a sequential series of growth phases: the lag, log, stationary, death
phase
 Knowledge of the bacterial growth curve is critical to understand population dynamics and
population control in the course of infectious diseases, and in food preservation
 
LAG PHASE
 Bacteria are the first introduced into an environment or media culture
 Bacteria are trying to adapt to nutrients
 Lag phase preparing to grow in size and synthesize
 Varies in length
o In some cases can be very short or even absent
 
LOG PHASE
 Also called log phase
 Rate of growth is constant
 Population number of cells undergoing binary fission doubles at a constant interval called
generation time
 Continue as ling as cells have adequate nutrients & good environment
 

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STATIOANRY PHASE
 If exponential growth continued unchecked, startingly large numbers of cells could arise
 In this stage microbial death is equal to microbial growth
Possibly reasons for entry into stationary phase
 Nutrient limitation
 Limited oxygen availability
 Toxic waste accumulation
 Critical population density reached
 
DEATH PHASE
 Decline in the number of viable cells
 Cells dying, usually at exponential rate
 Death; loss of ability to reproduce
 In some cases, death rate slows due to accumulation of resistant cells
 Slower than log phase
 
MEASUREMENTS OF CELL NUMBERS
Direct cell counts
a. Counting chambers
b. Electronic counters
c. On membrane filters
Viable cell counts
a. Plating methods (spread, pour plate)
b. Membrane filtration methods
 
COUNTING CHAMBERS
 Easy, inexpensive, and quick
 Useful for counting both eucaryotes and procaryotes
 Cannot distinguish living from dead cells
 
ELECTRONIC CHAMBERS
 Such as coulter counter
 Microbial suspension forced through small hole or orifice
 Movement of microbe through orifice impacts electric current that flows through orifice
 Cannot distinguished living from dead cells
 Quick and easy to use
 Useful for large microorganisms and blood cells, but not prokaryotes
 
MEMBRANE FILTERS
 Cells filtered through special membrane that provides dark background for observing cells
 Cells are stained with fluorescent dyes
 Useful for counting bacteria
 With certain counting bacteria
 With certain dyes, can distinguished living from dead cells
 
4 MICROBIAL ENVIRONMENTAL FACTOTRS AFFECT MICROBIAL GROWTH
i. pH
ii. Temperature
iii. Gas requirement
iv. Pressure
v. And other environmental factors (radiation, water activities etc.)

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pH
 Negative logarithm of the hydrogen ion concentration
 Affect the activity & integrity of enzymes & structural components of a cell
 Optimum pH for most microbes ranges approximately from 6 to 8
 Acidophiles
o Growth optimum between pH 0 and pH 5.5
 Neutrophiles
o Growth optimum between pH 5.5 and pH 7
 Alkalophiles
o Growth optimum between pH 8.5 and pH 11.5
 
TEMPERATURE
 Organisms exhibit distinct cardinal growth temperatures
o Minimum
o Maximum
o Optimum
 
Psychrophiles
 Grow well at 0 C
 Optimum growth at 15 C or lower
 Cold loving microbes
 Cannot grow above 20 C
Psychrotroph
 Can grow at 0-7 C
 Optimum between 20-3- C
 Max around 35 C
Mesophiles
 Growth optimum around 20-40 C
 Moderate temp. Loving microbes
Thermophiles
 Growth range is 45 C and 80 C
 Optimum between 55-65 C
 Heat loving microbes
Hyperthermophiles
 Optimum growth between 80 C and 100 C
 May grow to temperatures of 120 C
 
GAS REQUIRMENT
 Depends the kinds of microorganisms, some microorganisms need oxygen while others not need
oxygen to grow
 
PRESSURE
 Microbes obtain almost all their nutrients in solution from surrounding water
 Barophile organisms
o Adapted to life under high pressure
o Require or grow more rapidly in the presence of increased pressure
o Example: bottom dwellers in the ocean
 

19 | M I C R O B I O L O G Y L A B
 
IDENTIFICATION OF MICROORGANISMS
DIFFERENT STAINING PROCEDURE
1. Vital staining
o Refers to a stain that can be applied on living cells without killing them. Vital signs have
been useful for diagnostic and surgical
2. Supravital Staining
o It is a method of staining used in microscopy to examine living cells that have been
removed from an organism. It differs from intravital staining, which is done by injecting
or otherwise introducing the stain into the body. Thus a supravital stain may have a
greater toxicity, as only a few cells need to survive it a short while
3. Simple Staining
o It involves directly staining the bacterial cell with a positively charged dye in order to see
bacterial detail, in contrast to negative staining where the bacteria remain unstained
against a dark background
4. Negative Staining
o In microscopy, it is an established method, often used in diagnostic microscopy, for
contrasting a thin specimen with an optically opaque fluid. In this technique, the
background is stained, leaving the actual specimen untouched, and thus visible. This
contrasts with positive staining, in which the actual specimen is stained
5. Structural Staining
o It can be used to identify and study the structure of bacteria. They are useful in observing
endospores, capsules, and flagella
6. Differential Staining
o It is a staining process which uses more than one chemical stain. Using multiple stains
can better differentiate between different microorganisms or structures/ cellular
components of a single organism
 
FUNCTIONS OF THE DIFFERENT MICROSCOPIC METHODS
1. Wet mount – it is a procedure performed in the laboratory to observe motile organisms. It is
commonly used to examine material collected from the vaginal wall of a female patient.
o In this method, a drop of water is used to suspend the specimen between the slide and
cover slip. Place a sample on the slide. Using a pipette, place a drop of water on the
specimen.
o Then place on edge of the cover slip over the sample and carefully lower the cover slip
into place using a toothpick or equivalent.
 
Wet Mount –Unstained preparation in which specimens can be observed directly e.g. urine or
emulsified suspension. e.g. stool.
o A drop of urine or other specimen does not require emulsification is taken on
glass slide.
o Cover slip is placed on a glass slide and specimen like stool, which require emulsification are
emulsified in saline.
Uses:
a. To assess and enumerate inflammatory cells.
b. For examination of urine deposits.
c. For examination of parasites such as Trichomonas, Amoeba, and Guardia, etc.
 
2. Hanging drop Preparation

20 | M I C R O B I O L O G Y L A B
 o A drop of liquid culture or specimens such as stool is placed and cover slip in inverted
over a cavity slide so that remains hanging.
o This preparation is then observed under low power to adjust edge of the drop under
high power to study the motility of organisms.
Uses:
a. A drop of liquid culture or specimens such as stool is placed and cover slip in inverted over a
cavity slide so that remains hanging.
b. This preparation is then observed under low power to adjust edge of the drop under high power
to study the motility of organisms.
 
3. Gram Stain
o It is the most commonly used staining method devised by Christian Gram. It is
differential staining method, which differentiates organisms into Gram-positive and
Gram negative to their Gram reaction.
Preparation of smear:
o In case of liquid material e.g. urine, sputum, pus, CSF, broth culture, etc. a loopful of material is
taken with help of inoculating loop and spread thinly on the slide.
o In case of solid material, e.g colonies on culture plates, a minute quantity of material is obtained
by just touching the growth on culture plate and emulsified in a drop of sterile water or saline on
a glass slide.
o The smear prepared as above is dried in the air and fixed by passing the dried slide three times
slowly through the flame.
o The fixed smear is used for staining.
 
4. Acid-Fast Stain (Ziehl-Neelsen Satin) - Ziehl-Neelsen
o The organisms which are not easily stained by ordinary staining method, are stained by
acid-fast stain.
o A method commonly used is Ziehl Neelsen staining method.
o It is differential staining metho It is named for two German doctors who modified the
stain: the bacteriologist Franz Ziehl (1859–1926) and the pathologist
 
PURPOSE OF THE DIFFERENT SEROLOGICAL TEST FOR DIAGNOSIS OF INFECTION AND
IDENTIFICATION OF THE CAUSATIVE AGENTS.
 
1. Widal Test
o It is a simple test, inexpensive, and takes only a few minutes to perform. The Widal test
measures the capacity of antibodies against LPS and flagella in the serum of individuals
with suspected typhoid fever to agglutinate cells of S. Typhi; the test was introduced
over a century ago and it is still widely used.
 
2. Venereal Disease Research Laboratory (VDRL) Test
o The venereal disease research laboratory (VDRL) test is designed to assess whether a
patient has syphilis, a sexually transmitted infection (STI). Syphilis is caused by the
bacterium Treponema pallidum.
 
3. Elisa Test (Enzyme linked immunoassay)
o It is a commonly used laboratory test to detect antibodies in the blood. An antibody is a
protein produced by the body's immune system when it detects harmful substances,
called antigens.

21 | M I C R O B I O L O G Y L A B
 

4. Latex Agglutination Test

o The latex agglutination test is a laboratory method to check for certain antibodies or
antigens in a variety of body fluids including saliva, urine, cerebrospinal fluid, or blood.
 
5. Antistreptolysin O (ASO) Titer Test
o The antistreptolysin O (ASO) titer test is a blood test that checks for a strep infection.
When you come into contact with harmful bacteria, your body produces antibodies to
defend itself against these bacteria. Your body produces antibodies specific to the
bacteria they fight
 
6. Neutralization Test
o The neutralization test measures the ability of the patient's antibody to neutralize
infectivity and protect cells from infection, so it is considered a gold standard for the
assessment of protective antibody.
 
7. Serological Test
o A serological test is another method for diagnosis of infection and identification of
causative agent. Identification is done on the basis of detection of specific antibody to
the infectious agent. The commonly serological test are:
 Widal test for enteric fever
 VDRL test for syphilis
 ELISA test for HIV, Hepatitis B virus and other infections
 Latext agglutination test for Hepatitis B virus and other infections
 ASO test for streptococcal infections’
 Neutralization test for diagnosis of a number of viral infections.
 
 
 
 

22 | M I C R O B I O L O G Y L A B
BACTERIAL CULTURE MEDIA
MICROBIOLOGICAL CULTURE
 Method of cultivating microbial organisms by letting them reproduce in predetermined
culture media under controlled laboratory conditions
 
HISTORY OF CULTURE MEDIA
 Louis pasteur- used simple broths made up of urine or meat extracts
 Robert Koch- realized the importance of solid media and used potato pieces to grow
bacteria
 Fannie Eilshemius, wife of walter hesse (He was an assistant to robert Koch) that agar
was used to solidify culture media
 
AGAR
 Is a gelatinous substance derived from sea weeds
 In the past centuer agar has been used as a solid substrate to contain culture ,edium for
microbiological work
 Gelatin had some inherent problems:
 It existed as liquid at normal incubating temperature (35-37 C)
 Digested by certain bacteria
 Used for preparing solid medium
 Obtained from sea weeds
 No nutritive value
 Not affected by the growth of bacteria
 Melts at 98C and sets at 42 C
 2% agar is employed in solid medium
 
FIVE BASIC TECHNIQUES TO GROW AND EXAMINE AND CHARCTERIZE
MICROORGANISMS
INOCUALTION
 Inoculation(producing a culture)
 To cultivate or culture, one introduces a tiny sample(the inoculum)into a container of a
nutrient medium which provides an environment in which they multiply
 Selection of media with specialized functions can improve later steps of isolation and
identification.
INCUBATION
 To adjust the proper growth conditions of a sample
 Promotes multiplication of the microbes over a period of hours, days and even weeks.

23 | M I C R O B I O L O G Y L A B
  Produces a culture- the visible growth of the microbe in the medium

ISOLATION
 The end result of inoculation and incubation in macroscopic form
 The isolated microbes may take the form of separate colonies (discrete mounds of cells)
on solid media, or turbidity in broths
METHODS FOR ISOLATING BACTERIA
 Streak method -a small droplet of culture or sample is spread over the surface of the
medium according to a pattern that gradually thins out the sample and separates the cells
spatially over several sections of the plate
 Loop Dilution/ pour plate - the sample is inoculated serially into a series of cooled but
still liquid agar tubes so as to dilute the number of cells in each successive tube in the
series.
 Inoculated tubes are then plated out into sterile plates and are allowed to solidify. The
end result is that the number of cells per volume is so decreased that cells have ample
space to grow into separate colonies
 Spread plate technique -A small volume of liquid diluted sample is pipetted onto the
surface of the medium and spread around evenly by a sterile tool (like a hockey stick).
 Like the streak method, cells are pushed into separate areas on the surface so that they
can form individual colonies
INSPECTION
 Cultures are observed macroscopically for obvious growth characteristics(color,
texture,size) that could be useful in analyzing specimen contents.
 Slides are made to assess microscopic details such as shapes, size, and motility. Staining
techniques may be used to gather specific information on microscopic morphology.
IDENTIFICATION
 A major purpose of the 5 I’s is to determine the type
of microbe, usually to the level of species.
 Specialized tests include biochemical tests to determine metabolic activities specific to
the microbe
 
CULTURE MEDIA
 Culture media contains nutrients and physical growth parameters necessary for microbial
growth.
 All microorganisms cannot grow in a single culture medium and in fact many can’t
grow in any known culture medium.
 Organisms that cannot grow in artificial culture medium are known as obligate
parasites.
 Mycobacteium leprae, rickettsias, Chlamydias, and Treponema pallidum are obligate
parasites.
 

24 | M I C R O B I O L O G Y L A B
 CLASSIFICATION OF CULTURE MEDIA ON THE BASIS OF CONSISTENCY
 SOLID MEDIUM
o Solid medium contains agar at a concentration of 1.5-2.0% or some other,
mostly inert solidifying agent.
o Solid medium has physical structure and allows bacteria to grow in physically
informative or useful ways (e.g. as colonies or in streaks).
o Solid medium is useful for isolating bacteria or for determining the colony
characteristics of the isolate.
 Example: Nutrient Agar, Blood agar
 SEMISOLID MEDIA
o They are prepared with agar at concentrations of 0.5% or less.
o They have soft custard like consistency and are useful for the cultivation of
microaerophilic bacteria or for determination of bacterial motility.
 LIQUID (BROTH) MEDIUM
o These media contains specific amounts of nutrients but don’t have trace of
gelling agents such as gelatin or agar.
 Example: Selenite F broth- for the isolation of Salmonella, shigella
 Alkaline peptone water-for vibrio cholerae
 
ON THE BASIS OF COMPOSITION
 SYNTHETIC OR CHEMICALLY DEFINED MEDIUM
o A chemically defined medium is one prepared from purified ingredients and
therefore whose exact composition is known.
 NON SYNTHETIC OR CHEMICALLY UNDEFINED MEDIUM
o Non-synthetic medium contains at least one component that is neither purified
nor completely characterized nor even completely consistent from batch to
batch.
o Often these are partially digested proteins from various organism sources.
Nutrient broth, for example, is derived from cultures of yeasts.
o A simple non-synthetic medium is capable of meeting the nutrient requirements
of organisms requiring relatively few growth factors .
o A complex non-synthetic medium support the growth of more fastidious
microorganisms.
ON THE BASIS OF PURPOSE.FUNCTIONAL USE/APPLICATION
 GENERAL PURPOSE MEDIA/BASIC MEDIA
o Basal media are basically simple media that supports most non-fastidious
bacteria.
o Peptone water, nutrient broth and nutrient agar are considered as basal medium.
These media are generally used for the primary isolation of microorganisms.

25 | M I C R O B I O L O G Y L A B
  ENRICHED MEDIUM (ADDED GROWTH FACTORS)
o Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal
medium makes them enriched media.
o Enriched media are used to grow nutritionally exacting (fastidious) bacteria.
o Blood agar, chocolate agar, Loeffler’s serum slope etc are few of the enriched
media. Blood agar is prepared by adding 5- 10% (by volume) blood to a blood
agar base. Chocolate agar is also known as heated blood agar or lysed blood
agar.
 SELECTIVE AND ENRICHMENT MEDIA
o are designed to inhibit unwanted commensal or contaminating bacteria and help
to recover pathogen from a mixture of bacteria.
o While selective media are agar based, enrichment media are liquid in
consistency. Both these media serve the same purpose. Any agar media can be
made selective by addition of certain inhibitory agents that don’t affect the
pathogen of interest.
o Various approaches to make a medium selective include addition of antibiotics,
dyes, chemicals, alteration of pH or a combination of these.
 
SELECTIVE MEDIA
 Principle: Differential growth suppression
 Selective medium is designed to suppress the growth of some microorganisms while
allowing the growth of others.
 Selective medium are agar based (solid) medium so that individual colonies may be
isolated.
EXAMPLES OF SELECTIVE MEDIA
 Thayer Martin Agar used to recover Neisseria gonorrhoeae contains antibiotics;
vancomycin, colistin and nystatin.
 Mannitol Salt Agar and Salt Milk Agar used to recover S.aureus contains 10% NaCl.
 Potassium tellurite medium used to recover C.diphtheriae contains 0.04% potassium
tellurite.
 MacConkey’s Agar used for Enterobacteriaceae members contains bile salt that inhibits
most gram positive bacteria; for gram negative bacteria
 Pseudosel Agar (Cetrimide Agar) used to recover P. aeruginosa contains cetrimide
(antiseptic agent).
 Crystal Violet Blood Agar used to recover S. pyogenes contains 0.0002% crystal violet.
 Lowenstein Jensen Medium used to recover M.tuberculosis is made selective by
incorporating malachite green.
 Wilson and Blair’s Agar for recovering S. typhi is rendered selective by the addition of
dye brilliant green.

26 | M I C R O B I O L O G Y L A B
  Selective media such as TCBS Agar used for isolating V. cholerae from fecal specimens
have elevated pH (8.5- 8.6), which inhibits most other bacteria.
 
ENRICHMENT CULTURE MEDIA
 Enrichment medium is used to increase the relative concentration of certain
microorganisms in the culture prior to plating on solid selective medium. Unlike
selective media, enrichment culture is typically used as broth medium.
 Enrichment media are liquid media that also serves to inhibit commensals in the clinical
 specimen. Selenite F broth, tetrathionate broth and alkaline peptone water) are used to
recover pathogens from fecal specimens.
 
 DIFFERENTIAL/INDICATOR MEDIUM: DIFFERENTIAL APPEARANCE
o Certain media are designed in such a way that different bacteria can be
recognized on the basis of their colony colour.
o Various approaches include incorporation of dyes, metabolic substrates etc, so
that those bacteria that utilize them appear as differently coloured colonies. Such
media are called differential media or indicator media.
o Differential media allow the growth of more than one microorganism of interest
but with morphologically distinguishable colonies.
EXAMPLES OF DIFFERENTIAL MEDIA
1. Mannitol salts agar (mannitol fermentation = yellow)
2. Blood agar (various kinds of hemolysis i.e. α, β and γ hemolysis)
3. Mac Conkey agar (lactose fermenters, pink colonies whereas non- lactose fermenter
produces pale or colorless colonies.
4. TCBS (Vibrio cholerae produces yellow colonies due to fermentation of sucrose)
5. Mannitol Salt Agar (MSA) is a selective, differential and indicator medium which is
used to isolate and identify Staphylococcus aureus from the clinical specimen.
6. Blood agar is an enriched, bacterial growth medium. Fastidious organisms, such as
streptococci, do not grow well on ordinary growth media. Blood agar is a type of growth
medium (trypticase soya agar enriched with 5% sheep blood) that encourages the
growth of bacteria, such as streptococci, that
7. otherwise wouldn’t grow.
 
8. MacConkey agar was developed in 20th century by Alfred Theodore MacConkey.
9. It was the first formulated solid differential media.
10. MacConkey agar is a selective and differential culture media commonly used for the
isolation of enteric Gram-negative bacteria. It is based on the bile salt- neutral red-
lactose agar of MacConkey.
11. It is primarily used for detection and isolation of members of family of
12. enterobacteriaceae and Pseudomonas spp.
 

27 | M I C R O B I O L O G Y L A B
 TRANSPORT MEDIA
o Clinical specimens must be transported to the laboratory immediately after
collection to prevent overgrowth of contaminating organisms or commensals.
o This can be achieved by using transport media. Such media prevent drying
(desiccation) of specimen, maintain the pathogen to commensal ratio and inhibit
overgrowth of unwanted bacteria.
o Some of these media (Stuart’s & Amie’s) are semi-solid in consistency. Addition
of charcoal serves to neutralize inhibitory factors.
o Clinical specimens must be transported to the laboratory immediately after
collection to prevent overgrowth of contaminating organisms or commensals.
This can be achieved by using transport media. Such media prevent drying
(desiccation) of specimen, maintain the pathogen to commensal ratio and inhibit
overgrowth of unwanted bacteria. Some of these media (Stuart’s & Amie’s) are
semi-solid in consistency. Addition of charcoal serves to neutralize inhibitory
factors.
o Cary Blair transport medium and Venkatraman Ramakrishnan (VR) medium are
used to transport feces from suspected cholera patients.
o Sach’s buffered glycerol saline is used to transport feces from patients suspected
to be suffering from bacillary dysentery.
o Pike’s medium is used to transport streptococci from throat specimens.
ANAEROBIC MEDIA
o Anaerobic bacteria need special media for growth because they need low oxygen
content, reduced oxidation –reduction potential and extra nutrients.
o Robertson Cooked Meat (RCM) medium that is commonly used to grow
Clostridium spps contains a
o 2.5 cm column of bullock heart meat and 15 ml of nutrient broth.
ASSAY MEDIA
o These media are used for the assay of vitamins, amino acids and antibiotics. E.g.
antibiotic assay media are used for determining antibiotic potency by the
microbiological assay technique.
o Other types of medium includes;
 Media for enumeration of Bacteria,
 Media for characterization of Bacteria,
 Maintenance media etc.
 
DISPOSAL OF CULTURE MEDIA
 Sterilize all biohazardous waste before disposal.

 The use of a biological safety cabinet is recommended when culturing or processing

specimens for fungi, mycobacteria, or for any procedure that may create dangerous

aerosols.

28 | M I C R O B I O L O G Y L A B
  After use, all media, specimens, and containers must be sterilized by incineration or in an
autoclave before disposal (121 degrees C. for 30 minutes is recommended as a
minimum).
 Care should be exercised in the opening of tubes with tight caps to prevent the breakage
of the glass.
 Care should be taken to avoid contact with skin, eyes, or mucous membranes when
handling culture media or any laboratory reagent, stain, fixative, or chemical. If contact
occurs, flush immediately with running water. Contact a physician, hospital, or poison
control center if overexposure or irritation exists.
 
PROPER MAINTENANCE OF CULTURE MEDIA
 REFRIGERATION
o Pure cultures can be successfully stored at 0-4°C either in refrigerators or in
cold-rooms.
o This method is applied for short duration (2-3 weeks for bacteria and 3-4 months
for fungi) because the metabolic activities of the microorganisms are greatly
slowed down but not stopped.
 PARAFFIN METHOD
o This is a simple and most economical method of maintaining pure cultures of
bacteria and fungi. In this method, sterile liquid paraffin in poured over the slant
(slope) of culture and stored upright at room temperature.
o The layer of paraffin ensures anaerobic conditions and prevents dehydration of
the medium. This condition helps microorganisms or pure culture to remain in a
dormant state and, therefore, the culture is preserved for several years.
 CRYOPRESERVATION
o Cryopreservation (i.e., freezing in liquid nitrogen at- 196°C) helps survival of
pure cultures for long storage times.
o In this method, the microorganisms of culture are rapidly frozen in liquid
nitrogen at -196°C in the presence of stabilizing agents such as glycerol, that
prevent the formation of ice crystals and promote cell survival.
 LYOPHILISATION (FREEZE DRYING)
o In this method, the culture is rapidly frozen at a very low temperature (-70°C)
and then dehydrated by vacuum.
o Under these conditions, the microbial cells are dehydrated and their metabolic
activities are stopped; as a result, the microbes go into dormant state and retain
viability for years.
o Lyophilized or freeze-dried pure cultures and then sealed and stored in the dark
at 4°C in refrigerators. Freeze- drying method is the most frequently used
technique by culture collection centres.
 
 
 

29 | M I C R O B I O L O G Y L A B
  
 
 
ANTIMICROBIAL AGENTS
INTRODUCTION
 Another aspect of controlling the growth of microorganisms involves the use of
 drugs to treat (and, hopefully, to cure) infectious diseases.
 Although we most often hear the term chemotherapy used in conjunction with cancer (i.e., cancer
chemotherapy), chemotherapy actually refers to the use of any chemical (drug) to treat any
disease or condition.
 The chemotherapeutic agents used to treat infectious diseases are collectively referred to as
antimicrobial agents. Thus, an antimicrobial agent is any chemical (drug) used to treat an
infectious disease, either by inhibiting or killing pathogens in vivo.
 
DEFINITION OF TERMS
 Antibacterial Agents - drugs used to treat bacterial diseases
 Antifungal Agents – drugs used to treat fungal diseases
 Antiprotozoal Agents -drugs used to treat protozoal diseases
 Antiviral Agents - used to treat viral diseases
 Static Agents - an agent that is static in action which will inhibit the growth of microorganisms.
 Cidal Agents - an agent that is cidal in action will kill microorganisms and viruses.
 Antibiotic - is a substance produced by a microorganism that is effective in killing or inhibiting
the growth of other microorganisms.
 Antibiotics are produced by certain moulds and bacteria, usually those that live in soil.
 The antibiotics produced by soil organisms give them a selective advantage in the struggle for
the available nutrients in the soil. Penicillin and cephalosporins are examples of antibiotics
produced by moulds. bacitracin, erythromycin, and chloramphenicol are examples of antibiotics
produced by bacteria.
 Chemotherapeutic agents - drugs to kill cancer cells, They work by slowing or stopping the
growth of cancer cells.
 Mechanism of action - In medicine, a term used to describe how a drug or other substance
produces an effect in the body.
 
CHARACTERISTICS OF AN IDEAL ANTIMICROBIAL AGENT
1. Kill of inhibit the growth of pathogens
2. Cause no damage to the host
3. Cause no allergic reaction in the host
4. Be stable when stored in solid or liquid form
5. Remain in specific tissues in the body long enough to be effective
6. Kill the pathogens before they mutate and become resistant to it

30 | M I C R O B I O L O G Y L A B
 

DIFFERENT ANTIMICROBIAL AGENTS BASED ON CHEMICAL NATURE

PENICILLIN / B-M]LACTAM DRUGS
 Penicillins are referred to as -lactam drugs because their molecular structure includes a four-
sided ring structure known as a -lactam ring.
 Penicillins interfere with the synthesis of bacterial cell walls and have maximum effect on
bacteria that are actively dividing
AMINOGLYCOSIDES
 Aminoglycosides are bactericidal broad spectrum drugs that inhibit bacterial protein synthesis.
 Aminoglycosides are effective against a wide variety of aerobic Gram-negative bacteria, but are
ineffective against anaerobes. They are used to treat infections with members of the family
Enterobacteriaceae (e.g., Escherichia coli and Enterobacter, Klebsiella, Proteus..
MACROLIDES
 Macrolides inhibit protein synthesis. They are considered bacteriostatic at lower doses and
bactericidal at higher doses.
 The macrolides include erythromycin, clarithromycin, and azithromycin. They are effective
against chhlamydias, mycoplasmas, T. pallidum.
NARROW SPECTRUM ANTIBIOTICS
 Destroy only gram positive bacteria
o Example: COLISTIN and NALIDIXIC ACID.
 
BROAD SPECTRUM ANTIBIOTICS
 Antibiotics that are destructive to both Gram-positive and Gram- negative bacteris
o Examples: AMPICILLIN, CCHLORAMPHENICOL AND TETRACYCLINE
 
HOW ANTIMICROBIAL AGENTS WORKS
 The 5 most common mechanisms of action of antimicrobial agents are;
1. Inhibition of cell wall synthesis
2. Damage to cell membranes
3. Inhibition of nucleic acid synthesis (either DNA or RNA synthesis)
4. Inhibition of protein synthesis
5. Inhibition of enzyme activity
 
ANTIBACTERIAL DRUGS BASED ON MECHANISM OF ACTION
INHIBITOR OF CELL WALL SYNTHESIS
Selective inhibitors- they target the structure and function not found in eukaryotic cells.
 
PENICILLINS/BETA LACTAMS
 Most of them are derivatives of 6-amino penicillanic acid. (eg: penicillin G or benzyl penicillin).
 Crucial feature –beta lactam ring.
 Penicillin resistant bacteria have pencillinase activity.

31 | M I C R O B I O L O G Y L A B
  Structure of penicillin resembles the terminal D-alanyl-D - alanine found on the peptide side
chain of the peptidoglycan subunit.

CEPHALOSPORINS
 Isolated in 1948 from the fungus Cephalosporium.
 Contain beta lactam structure that is very similar to penicillin.
 Inhibit the trans-peptidation reaction during peptidoglycan synthesis.
 They are given to penicillin allergic patients.
 
VANCOMYCIN
 Glycopeptide antibiotic, produced by bacterium Streptomyces orientalis.
 Cup shaped molecule peptide linked to diasaccharide.
 
PROTEIN SYNTHESIS INHIBITORS
 Inhibit protein synthesis, binding with bacterial chromosome and other component of protein
synthesis.
 Discriminate between eukaryotic and bacterial ribosomes
 
CLASSIFICATION OF PROTEIN SYNTHESIS INHIBITORS
TERTACYCLINES
 Democlocycline - declomycin
 Doxycycline - vibramycin
 Minocycline - minocin
 Tetracycline - sumycin
GLYCYLCYCLINES
 TIGECYCLINE - TYGACIL
AMINOGLYCOSIDES
 Amikacin - amikin
 Gentamicin - garamycin
 Neomycin - neo-fradin
 Streptomycin - streptomycin
 Tobramycin - tobrex
MSCROLIDES/KETOLIDES
 Azithromycin - zithromax
 Clarithromycin - biaxin
 Erythromacin - e-mycin
 Telithromycin - ketek
OTHERS
 Chloramphenicol - chloromycetin
 Clindamycin - cleocin
 Linezolid - zyvox
 Quinupristin/dalfopristin - synercid
 
AMINOGLYCOSIDES
 Contain a cyclohexane ring and amino sugars.

32 | M I C R O B I O L O G Y L A B
  Streptomycin, kanamycin, neomycin and tobramycin synthesized by different species of
Streptomyces.
 gentamycin from related genus Micromonospora purpura.
 can be quite toxic - cause hearing and renal damage, loss of balance, nausea and allergic reaction.
 
PROTEIN SYNTHESIS INHIBITORS
1. Streptomycin - is an aminoglycoside antibiotic indicated to treat multi-drug resistant
mycobacterium tuberculosis and various non-tuberculosis infections.
*Streptomycin irreversibly binds to the 16S rRNA and S12 protein within the bacterial 30S
ribosomal subunit. As a result, this agent interferes with the assembly of initiation complex
between mRNA and the bacterial ribosome, thereby inhibiting the initiation of protein synthesis.
2. Gentamicin - is bactericidal and is a broad spectrum antibiotic (except against streptococci and
anaerobic bacteria).
*Its mechanism of action involves inhibition of bacterial protein synthesis by binding to 30S
ribosomes.
3. Tobramycin - tobramycin works by binding to a site on the bacterial 30S and 50S ribosome,
preventing formation of the 70S complex. As a result, mRNA cannot be translated into protein,
and cell death ensues. Tobramycin also binds to RNA-aptamers, artificially created molecules to
bind to certain targets.
4. Neomycin - is bactericidal in action. Similar to other aminoglycosides, it inhibits bacterial
protein synthesis through irreversible binding to the 30 S ribosomal subunit of susceptible
bacteria.
5. Amikacin - belongs to the class of medicines known as aminoglycoside antibiotics. It works by
killing bacteria or preventing their growth.
6. Chloramphenicol - inhibits bacterial protein synthesis by binding to the 50S ribosomal subunit.
In addition to hematopoietic toxicity, the gray baby syndrome is one of the most notable adverse
reactions associated with this agent.
7. Tetracyclines - were discovered in the 1940s, are a family of antibiotics that inhibit protein
synthesis by preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor. Peptidyl
transferase is inhibited by chloramphenicol.
This class of drugs inhibit the synthesis of cell walls in susceptible microbes by inhibiting peptidoglycan
synthesis. They bind to the amino acids within the cell wall preventing the addition of new units to the
peptidoglycan.
INHIBITORS OF MEMBRANE FUNCTIONS
1. Polymyxins - are natural polypeptide antibiotics that were first discovered in 1947 as products of
Bacillus polymyxa; only polymyxin B and polymyxin E (colistin) have been used clinically.
*interact with lipopolysaccharide (LPS) of the outer membrane of Gram-negative bacteria
and are subsequently taken up via the 'self-promoted uptake' pathway.
2. Imidazoles - alter the cell membrane permeability of susceptible yeasts and fungi by blocking the
synthesis of ergosterol (demethylation of lanosterol is inhibited), the primary cell sterol of fungi.
are commonly used for localized surface infections are used for invasive, life-threatening fungal
infections.
3. Gramicidin - inhibits RNA synthesis by purified RNA polymerase.

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INHIBITORS OF NUCLEIC ACID SYNTHESIS
1. Quinolones and fluoroquinolones inhibit DNA replication by targeting the bacterial enzymes
DNA gyrase, aka topoisomerase II, and topoisomerase IV. DNA gyrase untwists the DNA during
replication to relieve torsional stress, and topoisomerase IV cuts the daughter chromosomes apart
after replication.
2. Ethambutol is bacteriostatic against actively growing TB bacilli. It works by obstructing the
formation of cell wall. Mycolic acids attach to the 5'-hydroxyl groups of D-arabinose residues of
arabinogalactan and form mycolyl- arabinogalactan-peptidoglycan complex in the cell wall.
3. Isoniazid - The antimicrobial activity of INH is selective for mycobacteria, likely due to its
ability to inhibit mycolic acid synthesis, which interferes with cell wall synthesis, thereby
producing a bactericidal effect.
4. Rifampicin - blocks initiation of RNA synthesis by specifically inhibiting bacterial RNA
polymerase. It does not interact with mammalian RNA polymerases, making it specific for
Gram- positive bacteria and some Gram-negative bacteria.
 
INHIBITORS OF METABOLIC PATHWAYS
1. Other antibiotics act on selected cellular processes essential for the survival of the bacterial
pathogens. For example, both sulfonamides and trimethoprim disrupt the folic acid pathway,
which is a necessary step for bacteria to produce precursors important for DNA synthesis.
2. Sulfonamides target and bind to dihydropteroate synthase, trimethophrim inhibit dihydrofolate
reductase; both of these enzymes are essential for the production of folic acid, a vitamin
synthesized by bacteria, but not humans.
3. Sulfonamides and Trimethoprim target the folic acid biochemical pathway of bacteria. These
antibacterial compounds are termed folic acid pathway inhibitors. Sulfonamides interfere with
the formation of folic acid, an essential precursor for nucleic acid synthesis.
 
ANTI VIRAL AGENTS FOR DISEASES
1. Idoxuridine - is an antiviral topical medication used to treat viral infections of the eye, such as
herpesvirus-1 in cats. When idoxuridine is prescribed in the treatment of herpesvirus in cats, it is
referred to as 'extra-label' or 'off label' use.
2. Amnantadine - Amantadine is used to treat the symptoms of Parkinson's disease (PD; a disorder
of the nervous system that causes difficulties with movement, muscle control, and balance) and
other similar conditions. It is also used to control movement problems that are a side effect of
certain medications used to treat Parkinson's disease.
3. Acyclovir - is used to treat the symptoms of chickenpox, shingles, herpes virus infections of the
genitals (sex organs), the skin, the brain, and mucous membranes (lips and mouth), and
widespread herpes virus infections in newborns.
4. Zidovudine (ZDV), also known as azidothymidine (AZT), is an antiretroviral medication used to
prevent and treat HIV/AIDS. It is generally recommended for use in combination with other
antiretrovirals.
5. Ribavirin is an antiviral medication that is used to treat chronic hepatitis C. Ribavirin is not
effective when used alone. This medicine must be used in combination with interferon alfa or
peginterferon alfa. Ribavirin is sometimes given to people taking other antiviral medications to
treat hepatitis C.

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 6. Adenine arabinoside - effective for systemic therapy of herpes zoster infections in
immunocompromised persons
7. Trifluorothymidine - an antiviral medicine. It is used to treat eye infections caused by a virus,
such as herpes infection.
 
 
 
 
 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
 
 

 
 
 
 
 

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