ACTIVITY
HOT AIR OVEN
LABORATORY EQUIPMENT
1. ANALYTICAL BALANCE
✔Analytical balances are highly sensitive lab
instruments designed to accurately
measure mass.
✔ Their readability has a range between
0.1mg - 0.01mg.
✔ Analytical balances have a draft shield or
weighing chamber to prevent the very small
samples from being affected by air currents.
2. AUTOCLAVE
✔ Also known as steam sterilizers, and are
typically used for healthcare or industrial
applications.
✔ It is a machine that uses steam under
pressure to kill harmful bacteria, viruses,
fungi, and spores on items that are placed
inside a pressure vessel.
3. CENTRIFUGE
✔ It is a device used to separate
components of a mixture on the basis of
their size, density, the viscosity of the
medium, and the rotor speed.
4. COLONY COUNTER
✔ Colony counters are used to estimate a
liquid culture's density of microorganisms
by counting individual colonies on an agar
plate, slide, mini gel, or Petri dish.
5.
✔ It is widely used in the medical industry
to sterilize the equipment and other
materials that are used in a laboratory.
✔ It is used for delivering the heat
treatment to the product.
6. INCUBATOR
✔ It is an insulated enclosure in which
temperature, humidity, and other
environmental conditions can be regulated
at levels optimal for growth, hatching, or
reproduction.
7. WATER BATH
✔ It is a device used in the laboratories to
incubate samples in water maintained at a
constant temperature.
✔ Temperature may be controlled digitally
or by a dial and once set, the water bath
cycles on and off to ensure constancy of the
temperature.
8. LAMINAR FLOW CABINET
✔ Also known as tissue culture hood and is
a carefully enclosed bench designed to
prevent contamination of semiconductor
wafers, biological samples, or any particle
sensitive materials.
9. MAGNETIC STIRRER
✔ A device widely used in laboratories and
consists of a rotating magnet or a stationary
electromagnet that creates a rotating
magnetic field.
✔ This device is used to make a stir bar,
immerse in a liquid, quickly spin, or stirring
or mixing a solution.
10. HOMOGENIZERS
✔ It is a piece of laboratory or industrial
equipment used for the homogenization of
various types of material, such as tissue,
plant, food, soil, and many others.
11. DEEP FREEZER
✔ Testing equipment that are used to
preserve and store medical equipment,
blood samples for a long period of time.
ACTIVITY 1
BIOSAFETY IN THE LABORATORY
BIOSAFETY
• Biosafety is 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
substances
• 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 LEVEL
> 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.
CONTAMINENT
• “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 an
increased level of personal protection.
BARRIERS
• Prevent invaders from crossing
UNIVERSAL PRECAUTION
• Universal precautions is 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)
• is protective clothing, helmets, goggles, or FROM-
other garments or equipment designed to
> Bacteria
protect the wearer's body from injury or
infection. > Viruses
• Specified by exposure control plan or by > Fungi
standard operating procedure.
> Parasites
> Prions
A BIOSAFETY CABINET (BSC)
> Recombinant DNA
- Also called as biological safety cabinet or
microbiological safety cabinet.
SOURCE-
- It is an enclosed, ventilated laboratoey
workspace for safely working with materials > Various specimens
contaminated with (potentially
> Human blood
contaminated) pathogens requiring a
defined biosafety level > Unfixed tissue
- BSCs first became commercially available > Human cell lines
in 1950

TO-
INFECTIONS OF SPECIAL CONCERN
> Lab personnel
- Tuberculosis
> Community
- Hepatitis B
- HIV
BARRIERS
- Entric infections
- Primary barriers
- Secondary barrirs
ROUTES OF INFECTION
- Inoculation
>PRIMARY BARRIERS: Physical barriers or
- Ingestion personal protective equipment for lab
worker.
- Inhalation
Examples: Gloves, mask, goggles, aprons

BIOSAFETY: In microbiology labs:

preventing lab-acquired infections
>SECONDARY BARRIERS: structural aspects
of the laboratory that make working
environment safer against infection
• Sinks for hand washing
• Special containment areas
• Special air ventilation patterns
• Sterilization equipment
UNIVERSAL SAFETY PRECAUTION
1. Consider all the specimens potentially
infectious for HIV and other blood borne
infections
2. All specimens should be placed in a leak-
proof impervious container for transport
3. Use gloves while handling all samples,
especially when there is contact with body
fluids, non-intact skin or mucous
membrane.
4. 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.
5. Cover cuts or abrasions present over skin
with waterproof bandage.
6. Decontaminate the laboratory work
surfaces immediately in case of spillage of
blood or any other body fluids
7. Follow 'no needle recapping' strategy
8. All sharps should be collected and
disposed away properly.
9. Never pipette by mouth. Use mechanical
pipetting devices.
10. There should always be a system
working efficiently for management of
hospital generated waste.
11. It is advisable for the laboratory
personnel to be vaccinated against
Hepatitis-B
12. Facilities should be available easily for
post exposure prophvlaxis in case of
exposure to HIV & HBV
STANDARDS MICROBIOLOGICAL
PRACTICES
Do's
•controlled access to the laboratory
•Frequent hand wash
•Mechanical pipetting
•Appropriate waste management &
Sterilization and disinfection measures
•Training to the workers
Don'ts
•Eating, drinking, smoking, handling contact
lenses,
•storing food
•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 CONTAMINENT
• BSL1 - microorganisms that don't
consistently cause disease in healthy adults
• E. coli, polyoma virus
• Basic laboratory
• Standard Microbiological Practices
Standard practices required:
• Frequent hand washing
• Door that can be kept closed when
working;
• Limits on access to the lab space when
working;
• No smoking, eating, drinking, storage of
food in laboratory;
• Care to minimize splashes and actions
that may create aerosols (tiny droplets);
• Decontamination of work surfaces after
every use after any spills;
THE MICROORGANISMS
BSL 1
- canine hepatitis virus, non-pathogenic
Escherichia coli, other non-infectious
bacteria, B.subtilis
Remarks: Minimal protection required
BSL 2
- C. difficile, most Chlamydiae, hepatitis A,
B, and C virus, HIV, orthomyxoviruses (other
than smallpox), influenza A, Lyme disease,
Salmonella, mumps, measles, scrapie,
MRSA, and VRSA, B. anthracis
Remarks: Can cause only mild disease to
humans, or are difficult to common via
aerosol in a lab setting
BSL 3
- Yersinia pestis, Francisella tularensis,
Leishmania donovani, Mycobacterium
tuberculosis, Chlamydia psittaci, West Nile
virus, Venezuelan equine encephalitis virus,
Eastern equine encephalitis virus, SARS
coronavirus, Coxiella burnetii, Rift Valley
fever virus, Rickettsia rickettsii, several
species of Brucella, rabies virus, and yellow
fever virus
Remarks: Can cause serious or potential
lethal disease after inhalation humans but
for which treatment DOES exist
BSL 4
- Bolivian and Argentine hemorrhagic
fevers, Marburg virus, Ebola virus, Lassa
virus, Crimean-Congo hemorrhagic fever,
and various other hemorrhagic diseases
Remarks: Dangerous and exotic agents to
pose a high individual risk of aerosol-
transmitted laboratory infections, these
cause severe fatal disease in humans for
what vaccines or other treatments are NOT
available
BIOSAFETY LEVEL 2
• Agents associated with human disease
• Generally required for any human-derived
blood, bodily fluids, tissues in which
infectious agent may be unknown
• Agents include measles virus, Salmonella
species, pathogenic Toxoplasma,
Clostridium botulinum, hepatitis B virus
BIOSAFETY LEVEL 3 - microorganisms that
cause serious disease, transmitted by
inhalation
• M. tuberculosis, yellow fever virus
hantavirus, Y pestis (plague)
• Containment lab: double door entry;
hirectional airflow: all work in biosafety
cabinet
PERSONAL PROTECTIVE QUIPMNENT
• designed to protect the wearer's body
from injury or infection. The hazards
addressed by protective equipment include
physical, electrical, heat, chemicals,
biohazards, and airborne particulate
matter.
Universal Precautions
• Universal precautions refers to the
practice, in medicine, of avoiding contact
with patients' bodily fluids, by means of the
wearing of nonporous articles such as
medical gloves, goggles, and face shields.
ACTIVITY 2: THE MICROSCOPES
Viewing the Microbial World
- Using the 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
(µm). A micrometer is one millionth of a
meter.
• A typical spherical bacterium (coccus) is
approximately 1 µm in diameter.
• A typical rod-shaped bacterium (bacillus)
is approximately 1 µm wide × 3 µm long.
- Using the Metric System to Express the
Sizes of Microbes (cont.)
• The sizes 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
diseases 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 µm).
• When using a microscope, the sizes of
microorganisms are measured using an
ocular micrometer.
► This must be calibrated using a stage
micrometer for each microscope objective
► Stage micrometer acts as a scale of
measurement
MICROSCOPE
• 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 instrument;
this limit is referred to as the resolving
power or resolution of the instrument.
• The resolving power of the unaided
human eye is approximately 0.2 mm.
1. Simple Microscopes
• A simple microscope is one that contains
only one magnifying lens.
• A magnifying glass could be considered a
simple microscope
• When using a magnifying glass, images
appear 3 to 20 times larger than the
object’s actual size.
• Leeuwenhoek’s simple microscopes had a
maximum magnifying power of about ×300
(about 300 times)
• Not as widely used in research and labs
• Often uses external light source
2. Compound Microscopes
• 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 µm
• About 1,000 times better than the
resolving power of the unaided human eye.
• It is the wavelength of visible light (~0.45
µm) 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:
– The eyepiece or ocular lens (usually ×10)
– The objective lens (×4, ×10, ×40, and ×100
are the four most commonly used objective
lenses)
Compound Microscopes (cont.)
• 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 × ×4 objective = ×40 total
magnification
– ×10 ocular × ×10 objective = ×100 total
magnification
– ×10 ocular × ×40 objective = ×400 total
magnification
– ×10 ocular × ×100 objective = ×1,000 total
magnification
• Photographs taken through the lens
system of the compound light microscope
are called photomicrographs.
Compound Microscopes (cont.)
• Because objects are observed against a
bright background or “bright field,” 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 microscopes
include
– Phase-contrast microscopes
– Fluorescence microscopes
- Darkfield and Fluorescence Micrographs
- Phase-Contrast and Fluorescence
Microscopes
• Phase-contrast microscopes are used to
observe unstained living microorganisms.
– 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 (UV) light source.
– When the UV light strikes certain dyes and
pigments, these substances emit a longer-
wavelength light, causing them to glow
against a dark background.
– Different parts of the same cell can be
stained with different dyes to allow for
complex analysis of proteins within the cell
3. Electron Microscopes
• Electron microscopes enable us to see
extremely small microbes such as rabies
and smallpox viruses.
• Living organisms cannot be observed
using an electron microscope⎯the
processing procedures kill the organisms.
• Microscope uses a vacuum
• An electron beam is used as the source of
illumination, and magnets are used to focus
the beam.
• Electron microscopes have a much higher
resolving power than Compound light
microscopes.
• Wavelength of the beam is 100,000 times
shorter than visible light
• There are two types of electron
microscopes ⎯ transmission and scanning.
3.1 Transmission Electron Microscope
(TEMs)
• 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 µm thick).
• Some electrons transmit through the
specimen while others are blocked creating
an image
• An image of the specimen is produced on
a phosphor-coated screen.
• Magnification is approximately 1,000
times greater than with the compound light
microscope.
• Resolving power is approximately 0.2 nm.
• Can visualize intermal cell structures
3.2 Scanning Electron Microscope (SEM)
• 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
• If color is used in photograph, it was
artificially enhanced
• 2-dimensional (2-D) micrographs obtained
Staphylococcus aureus (Blue) and Red Blood
Cells as Seen by Light Microscopy
4. Atomic force microscopes
• Provides a 3-dimensional (3-D) image
• Silicon or silicon nitride cantilever with a
sharp tip scans surface of specimen
• When tip and 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
ACTIVITY 3
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 ⮚These cells divide in one planes and
and then visualized under high power of remain attached, to form chains.
magnification (1000X) of compound
• Eg: streptococcus lactis.
microscope.
• Eg : streptococcus pyogenes
⮚An electron microscope is used for clear
visualization of internal structure of
bacteria.
TETRADS/ Tetracocci
SHAPE OF BACTERIA
⮚ groups of four cells in a square
On the basis of shape bacteria are classified arrangement.
as
⮚ They divide in two planes and live in
1. Cocci groups of four.
2. Bacilli ⮚ Eg: Gaffyka tetragena.
3. Vibrios ⮚ Eg: micrococcus lutues
4. Spirilla
5. Spirochetes Staphylococci/CUS
6. Actinomycetes ⮚grapelike clusters of cells
7. Mycoplasma
⮚Cocci cells divide in three planes in an
irregular pattern. These cells produce
bunches of cocci as in grapes.
1. COCCI
⮚Eg: staphylococcus aureus, staphylococcus
⮚Cocci are small, spherical or oval cells. In
albus.
greek 'Kokkos' means berry.
Eg: micrococcus
1. SARCINAE
⮚Diplococci arrange in pairs
⮚Sarcinae cells divide in three planes in a
⮚They split in one plane and remains in regular pattern.
pair. Eg: diplococcus pneumoniae neisseria
gonorrhea ⮚These cells produces a cuboidal
arrangement of group of an eight cells.

⮚Eg: Micrococcus tetragena.

STREPTOCOCCI/CUS

⮚rows or chains of such cells are called

streptococci
2. BACILLI ⮚They have a helical shape and rigid body.
⮚ They are rod shaped cells. ⮚Eg: Spirillum ruprem.
⮚ Eg: Bacillus anthracis.

⮚ It is derived from greek word "Bacillus" 5. SPIROCHETES

meaning stick.
⮚ They are slender and flexuous spiral
⮚ In some of the bacilli the length of cell forms
may be equal to width. Such bacillary forms
⮚ contains distinctive diderm (double-
are known as coccobacilli.
membrane) bacteria, most of which have
⮚ Eg: Bracella. long, helically coiled (corkscrew-shaped or
spiraled).
⮚ E. Coli
⮚ Example : Treponema pallidum
Diplobacilli: Two bacilli arranged side by
side with each other.
> Example: 6. ACTINOMYCETES

Corynebacterium diptheriae ⮚The characteristic shape is due to the

presence of rigid cell wall. Eg:
Streptobacilli
Streptomyces.
⮚Arrange in chain (rods)
⮚They are branching filamentous bacteria.
⮚Example: bacillus anthracis
⮚Eg: Streptomyces species.

3. VIBRIOS
7. MYCOPLASMA
⮚They are comma shaped curved rods.
⮚They are cell wall deficient bacteria and
⮚Eg: Vibrio comma. hence do not possess stable morphology.

⮚several species of which can cause
foodborne infection, usually associated with
eating undercooked seafood.
Typically found in salt water.
⮚They occur as round or oval bodies with
interlacing filaments.
⮚Eg: mycoplasma pneumoniae

4. SPIRILLA

⮚They are longer rigid rods with several

curves or coils.
TERMINOLOGY
1. Endospore:
⮚This complex developmental process
which often initiated in response to nutrient
deprivation. It allows the bacterium to
produce a dormant and highly resistant cell
to preserve the cell's genetic material in
times of extreme stress.
⮚It is produced within the bacterial cell.
⮚Bacteria producing endospore are:
Bacillus, Clostridium, Sporosarcina
2. Pathogenicity of the Bacteria
A microbe that is capable of causing disease
is referred to as a pathogen, while the
organism being infected is called a host. The
ability to cause disease is referred to as
pathogenicity, with pathogens varying in
their ability. An opportunistic pathogen is a
microbe that typically infects a host that is
compromised in some way, either by a
weakened immune system or breach to the
body’s natural defenses, such as a wound.
The measurement of pathogenicity is called
virulence, with highly virulent pathogens
being more likely to cause disease in a host.
3. Immunogenicity
⮚the ability of cells/tissues to provoke an
immune response and is generally
considered to be an undesirable
physiological response.
4. Antibiotic sensitivity
⮚This means the antibiotic is effective
against the bacteria.
⮚Susceptible means they can't grow if the
drug is present.
⮚Resistant means the bacteria can grow
even if the drug is present.
5. Chromaticity
⮚the quality of color characterized by its
dominant or complementary wavelength
and purity taken together.
6. Antigenicity
⮚describes the ability of a foreign material
(antigen) to bind to, or interact with, the
products of the final cell-mediated response
such as B-cell or T-cell receptors.
7. Virulence
⮚defined as the relative ability of a
microorganism to overcome host defenses,
or the degree of pathogenicity within a
group or species
8. Plasmolysis
⮚ is the process in which cells lose water in
a hypertonic solution. The reverse process,
deplasmolysis or cytolysis, can occur if the
cell is in a hypotonic solution resulting in a
lower external osmotic pressure and a net
flow of water into the cell.
 9. Binary Fission
⮚asexual reproduction by a separation of
the body into two new bodies. In the
process of binary fission, an organism
duplicates its genetic material, or
deoxyribonucleic acid (DNA), and then
divides into two parts (cytokinesis), with
each new organism receiving one copy of
DNA.
ACTIVITY 4
DIFFERENT STAINING PROCEDURE
1. VITAL STAINING
- refers to a stain that can be applied on
living cells without killing them. Vital stains
have been useful for diagnostic and
surgical.
2. SUPRAVITAL STAINING
– 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
- 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
– 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
– it can be used to identify and study the
structure of bacteria. They are useful in
observing endospores, capsules, and
flagella.
6. DIFFERENTIAL STAINING
– 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.
❖ 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.
❖ 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.
⮚ A drop of urine or other specimen does
not require emulsification is taken on glass
slide.
⮚ Cover slip is placed on a glass slide and
specimen like stool, which require
emulsification are emulsified in saline.
Uses
1. To assess and enumerate inflammatory
cells.
2. For examination of urine deposits.
3. For examination of parasites such as
Trichomonas, Amoeba, and Guardia, etc.
2. Hanging drop Preparation
❖ 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.
❖ This preparation is then observed under
low power to adjust edge of the drop under
high power to study the motility of
organisms.
Uses
1. To study motility of Vibrio Cholerae in
stool sample.
2. To study motility of organisms as fluid
culture.
3. Gram Stain
-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:
❖ 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.
❖ 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.
❖ The smear prepared as above is dried in
the air and fixed by passing the dried slide
three times slowly through the flame.
❖ The fixed smear is used for staining.
4. Acid-Fast Stain (Ziehl-Neelsen Stain) -
Ziehl–Neelsen
❖ The organisms which are not easily
stained by ordinary staining method, are
stained by acid-fast stain.
❖ A method commonly used is Ziehl
Neelsen staining method.
❖ It is differential staining method
 It is named for two German doctors who
modified the stain: the bacteriologist
Franz Ziehl (1859–1926) and the
pathologist Friedrich
PURPOSES OF THE DIFFERENT
SEROLOGICAL TEST FOR DIAGNOSIS OF
INFECTION AND IDENTIFICATION OF THE
CAUSATIVE AGENTS.
1. Widal test
–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
- 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 - ELISA stands for enzyme-
linked immunoassay.
- 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.
4. Latex Agglutination Test
– 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
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
- 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.
SEROLOGICAL TEST
–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.
ACTIVITY 5
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
bacterua
• Fannie Eilshemius, wife of walter hesse
(whis 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
AGAR CHARACTERISTICS
• Used for preparing solid medium
• Obtaine 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 characterize microorganisms
• INOCULATION
• INCUBATION
• ISOLATION
• INSPECTION
• IDENTIFICATION
1. INOCULATION
– 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.
2. INCUBATION
– To adjust the proper growth conditions of
a sample
– Promotes multiplication of the microbes
over a period of hours, days and even
weeks.
– Produces a culture- the visible growth of
the microbe in the medium
3. 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
Methods for isolating bacteria
• 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
4. 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.
5. 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.
CLASSIFICATION OF CULTURE MEDIA
• on the basis of consistency
• on the basis of composition
• on the basis of purpose/ functional use/
application
• on the basis of consistency
1. Solid medium
• Solid medium contains agar at a
concentration of 1.5-2.0% or some other,
mostly inert solidifying agent.
• Solid medium has physical structure and
allows bacteria to grow in physically
informative or useful ways (e.g. as colonies
or in streaks).
• Solid medium is useful for isolating
bacteria or for determining the colony
characteristics of the isolate.
• Example: Nutrient Agar, Blood agar
2. Semisolid media
• They are prepared with agar at
concentrations of
0.5% or less.
• They have soft custard like consistency
and are useful for the cultivation of
microaerophilic bacteria or for
determination of bacterial motility.
3. Liquid (Broth) medium
• 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
CLASSIFICATION OF CULTURE MEDIA
• on the basis of composition
1. Synthetic or chemically defined medium
A chemically defined medium is one
prepared from purified ingredients and
therefore whose exact composition is
known.
2. Non synthetic or chemically undefined
medium
• Non-synthetic medium contains at least
one component that is neither purified nor
completely characterized nor even
completely consistent from batch to batch.
• Often these are partially digested proteins
from various organism sources. Nutrient
broth, for example, is derived from cultures
of yeasts.
2. Non synthetic or chemically undefined
medium
• Non Synthetic medium may be simple or
complex depending up on the supplement
incorporated in it.
– A simple non-synthetic medium is capable
of meeting the nutrient requirements of
organisms requiring relatively few growth
factors
– A complex non-synthetic medium support
the growth of more fastidious
microorganisms.
CLASSIFICATION OF CULTURE MEDIA
• on the basis of purpose/ functional use/
application
1. General purpose media/ Basic media
• Basal media are basically simple media
that supports most non-fastidious bacteria.
• Peptone water, nutrient broth and
nutrient agar are considered as basal
medium. These media are generally used
for the primary isolation of microorganisms.
2. Enriched medium (Added growth
factors):
• Addition of extra nutrients in the form of
blood, serum, egg yolk etc, to basal medium
makes them enriched media.
• Enriched media are used to grow
nutritionally exacting (fastidious) bacteria.
• 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.
3. Selective and enrichment media
• are designed to inhibit unwanted
commensal or contaminating bacteria and
help to recover pathogen from a mixture of
bacteria.
• 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.
• Various approaches to make a medium
selective include addition of antibiotics,
dyes, chemicals, alteration of pH or a
combination of these.
CLASSIFICATION OF CULTURE MEDIA
• A. selective medium
– 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.
• A. selective medium
Examples of selective media include:
1. Thayer Martin Agar used to recover
Neisseria
gonorrhoeae contains antibiotics;
vancomycin, colistin and nystatin.
2. Mannitol Salt Agar and Salt Milk Agar
used to recover S.aureus contains 10%
NaCl.
3. Potassium tellurite medium used to
recover C.diphtheriae contains 0.04%
potassium tellurite.
• A. selective medium
• Examples of selective media include:
4. MacConkey’s Agar used for
Enterobacteriaceae members contains bile
salt that inhibits most gram positive
bacteria; for gram negative bacteria
5. Pseudosel Agar (Cetrimide Agar) used to
recover P. aeruginosa contains cetrimide
(antiseptic agent).
6. Crystal Violet Blood Agar used to recover
S. pyogenes contains 0.0002% crystal violet.
CLASSIFICATION OF CULTURE MEDIA
• A. selective medium
• Examples of selective media include:
7. Lowenstein Jensen Medium used to
recover M.tuberculosis is made selective by
incorporating malachite green.
8. Wilson and Blair’s Agar for recovering S.
typhi is rendered selective by the addition
of dye brilliant green.
9. 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.
• Certain media are designed in such a way
that different bacteria can be recognized on
the basis of their colony colour.
• 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.
• Differential media allow the growth of
more than one microorganism of interest
but with morphologically distinguishable
colonies.

CLASSIFICATION OF CULTURE MEDIA CLASSIFICATION OF CULTURE MEDIA cont.

• B. Enrichment culture medium • on the basis of purpose/ functional use/

application
• Enrichment medium is used to increase
the relative concentration of certain • 4. Differential/ indicator medium:
microorganisms in the culture prior to differential appearance:
plating on solid selective medium.
• Examples of differential media include:
- Unlike selective media, enrichment culture
1. Mannitol salts agar (mannitol
is typically used as broth medium.
fermentation = yellow)
• Enrichment media are liquid media that
2. Blood agar (various kinds of hemolysis i.e.
also serves to inhibit commensals in the
α, β and γ hemolysis)
clinical specimen. Selenite F broth,
tetrathionate broth and alkaline peptone 3. Mac Conkey agar (lactose fermenters,
water) are used to recover pathogens from pink colonies whereas non- lactose
fecal specimens. fermenter produces pale or colorless
colonies.
4. TCBS (Vibrio cholerae produces yellow
CLASSIFICATION OF CULTURE MEDIA
colonies due to fermentation of sucrose)
• on the basis of purpose/ functional use/
application
• 4. Differential/ indicator medium:
differential appearance:
CLASSIFICATION OF CULTURE MEDIA cont.
• Mannitol Salt Agar (MSA) is a selective,
differential and indicator medium which is
used to isolate and identify Staphylococcus
aureus from the clinical specimen.
• 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
otherwise wouldn’t grow.
CLASSIFICATION OF CULTURE MEDIA cont.
• MacConkey agar was developed in 20th
century by
Alfred Theodore MacConkey.
• It was the first formulated solid
differential media.
• 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 saltneutral red-lactose agar of
MacConkey.
• It is primarily used for detection and
isolation of
members of family of
enterobacteriaceae and Pseudomonas spp.
CLASSIFICATION OF CULTURE MEDIA
• on the basis of purpose/ functional use/
application
• 5. Transport media:
• 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.
• 5. Transport media:
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.
CLASSIFICATION OF CULTURE MEDIA cont.
• on the basis of purpose/ functional use/
application
• 5. Transport media:
• Cary Blair transport medium and
Venkatraman Ramakrishnan (VR) medium
are used to transport feces from suspected
cholera patients.
• Sach’s buffered glycerol saline is used to
transport feces from patients suspected to
be suffering from bacillary dysentery.
• Pike’s medium is used to transport
streptococci from throat specimens.
CLASSIFICATION OF CULTURE MEDIA cont.
• on the basis of purpose/ functional use/
application
• 6. Anaerobic media:
• Anaerobic bacteria need special media for
growth because they need low oxygen
content, reduced oxidation –reduction
potential and extra nutrients.
• Robertson Cooked Meat (RCM) medium
that is commonly used to grow Clostridium
spps contains a 2.5 cm column of bullock
heart meat and 15 ml of nutrient broth.
CLASSIFICATION OF CULTURE MEDIA
• on the basis of purpose/ functional use/
application
• 7. Assay media
• 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.
• 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.
• 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 MAINTENACE OF CULTURE MEDIA
1. Refrigeration:
✓ Pure cultures can be successfully stored
at 0-4°C either in refrigerators or in cold-
rooms.
✓ 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.
2. Paraffin Method:
✓ 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.
✓ 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.
4. Lyophilisation (Freeze-Drying)
• In this method, the culture is rapidly
frozen at a very low temperature (-70°C)
and then dehydrated by vacuum.
• 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.
• 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.

3. Cryopreservation:

✓ Cryopreservation (i.e., freezing in liquid

nitrogen at196°C) helps survival of pure
cultures for long storage times.

✓ 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.