Anaerobic Jar

Maximum air is sucked out from outlet

by vacuum pump.

Hydrogen is added from inlet.

Hydrogen combines with remaining

oxygen in presence of room
temperature catalyst (Palladium coated
alumina or asbestos).

Anaerobic condition is indicated by

absence of color in methylene blue in
the indicator tube..
Lowenstein Jensen Medium
Contains Beaten hen’s egg,
Asparagine, KH2PO4, Magnesium
citrate, MgSO4, Malachite green,
Glycerol.

Bluish green medium in screw capped

bottles

Sterilized by Inspissation.

Type of medium - Semisynthetic,

enriched, selective and indicator
medium.
Loeffler’s Serum Slope
Sterilized by Inspissation.

Use - For culture of C.

diphtheriae.

Type of Medium -
Enriched
Medium

Identification - White slope

with water of
condensation usually in
screw capped bottle or
stoppered tube.
Hot Air Oven
Temp - 160 C X 60 minutes

Principal - Sterilization by dry heat

(Oxidative destruction)

Articles sterilized - All metal articles,

All glass articles, fats, oils, greases,
heat stable powders like sulpha &
talcum.

Articles not sterilized - Articles

having metal & glass components,
culture media.
Blood Agar

What is the content of this medium?

The base medium is a nutrient agar that is

autoclaved and allowed to cool to 45-50 C. Then,
5% defibrinated blood (usually from sheep) is
added to the medium and it is dispensed into petri
plates. Once it solidifies, it is ready for use.

Use - For growing fastidious bacteria.

To demonstrate hemolysis.

Type of medium - Enriched and indicator

medium
Alpha hemolysis on blood agar
Blood agar showing
Alpha hemolytic
colonies
(Greenish
discoloration with
partial hemolysis).

Example of bacteria
producing Alpha
haemolysis - Viridans
Group of Streptococci
Beta hemolysis on blood agar
Blood agar showing Beta
hemolytic colonies
(Complete zone of
clearing).

Example of bacteria
producing Beta hemolytic
- Streptococcus
pyogenes,
Staphylococcus aureus.
Satellitism - H. influenzae
Satellitism of H. influenzae. Note the
larger colonies near the streak of
Staphylococcus aureus and they
become smaller away from it and in
fact disappear far away from it.

Source of Factor X - Blood in the

blood agar

Source of Factor V - Staphylococcus

aureus
Target hemolysis - Clostridium
perfringens

Double zone of
hemolysis seen in
Clostridium
perfringens.
Swarming growth on Blood Agar

Swarming or spreading in wave form is

seen with motile organisms
particularly Proteus mirabilis.

Cause of swarming - The flagella of

bacteria are naturally set to spin
counter-clockwise causing the bacteria
to be propelled forward. Only as toxin
concentration goes up or nutrient
concentration goes down does the cell
switch to clockwise rotation
Plastic disposable syringe
Plastic disposable syringe is one time
use instrument.

How sterilized - By Ethylene oxide

(Cold sterilization)

Decontamination and disposal after

use - Burn the needle in Needle
burner, decontaminate by immersing
in 1% Hypochlorite, transfer to yellow
colored bag and send to incineration
for final disposal.
Lactose fermenting colonies on
MacConkey’s Medium

MacConkey’s medium
showing pink (Lactose
fermenting) colonies. The
crenated margins
(Maple leaf appearance)
suggests possibility of E.
coli colonies.
Lactose non-fermenting colonies on
MacConkey’s Medium

MacConkey’s medium with Lactose non-

fermenting colonies. Organisms which can
form such colonies include Proteus,
Salmonella, Shigella.
Autoclave
Autoclave -

Principal - Steam under pressure. Microbes destroyed by

Coagulative destruction.

Temperature & Holding time - 121 C X 15-20 Minutes.

Pressure - 15 lbs/sq inch or 1 kg/cm

Articles sterilized - Linen, rubber & plastic goods,

Glass articles, Metal articles, Articles with glass and
metal components, simple nutritive culture media.

Articles not sterilized - Fats, Oils, Greases. Sugar / egg

/serum/blood containing media
Inculation Loop

Inoculation loop -
Usually made from Nichrome
alloy.

Sterilized by Red Heat.

Uses -
1. Transfer of clinical specimen
to culture media.
2. Transfer of bacterial growth
from medium to slides / to
other culture media
TCBS Medium
Thiosulphate Citrate
Bile Salt Sucrose
Medium.

Type of medium -
Selective & Indicator
medium

Use - For isolation of

Vibrio cholerae which
grow on this medium
and due to sucrose
fermentation show up
as yellow colonies.
Seitz Filter
Seitz filter is made up of
stainless steel and has
asbestos disc. Used for making
fluids bacteria free.

Can stop bacteria but not

viruses.

Uses - Preparation of toxins,

antitoxins, antibiotic solutions
etc.
Swab stick

Sterile swab stick -

Uses -
❏ For collection of clinical
specimen like pus, throat,
nasal etc
❏ For making lawn culture for
Antibiotic sensitivity test

How to sterilize?
❖ Hot air oven - preferred
❖ Autoclave
Oxidase Test
This test depends on the presence of
cytochrome oxidase in bacteria that will
catalyze the transport of electrons between
electron donors and redox dye.
Tetramethyl-p-phenylene diamine
dihydrochloride in the reagent is reduced
to deep purple color.

This test is used for the screening of

Pseudomonas, Vibrio, Neisseria, Brucella
and Pasteurella, which give positive test.
Enterobacteriaceae are oxidase negative.
Nutrient Agar
Urease Test
Urease test principle

Many organisms especially those that infect the urinary tract,

have a urease enzyme which is able to split urea in the
presence of water to release ammonia and carbon dioxide. The
ammonia combines with carbon dioxide and water to form
ammonium carbonate which turns the medium alkaline, turning
the indicator phenol red from its original orange yellow color to
bright pink.

Name of urease positive organisms

1. Helicobacter pylori
2. Proteus spp
3. Yersinia spp
4. Klebsiella spp
5. Brucella spp
6. Cryptococcus spp
Citrate utilization Test
Simmons citrate agar tests the ability of organisms to
utilize citrate as a carbon source. Simmons citrate
agar contains sodium citrate as the sole source of
carbon, ammonium dihydrogen phosphate as the sole
source of nitrogen, other nutrients, and the pH
indicator bromthymol blue. This test is part of the
IMViC tests and is helpful in differentiating
theEnterobacteriaceae .

If the medium turns blue, the organism is citrate

positive. If there is no color change, the organism is
citrate negative. Some citrate negative organisms
may grow weakly on the surface of the slant, but
they will not produce a color change.

Example of citrate + bacterium - Klebsiella

Example of citrate - bacterium - E. coli, S.
typhi
Catalase Test
The catalase test is used to differentiate
staphylococci (catalase-positive) from
streptococci (catalase-negative).

The enzyme, catalase, is produced by bacteria

that respire using oxygen, and protects them from
the toxic by-products of oxygen metabolism.
Catalase-positive bacteria include strict aerobes
as well as facultative anaerobes, although they all
have the ability to respire using oxygen as a
terminal electron acceptor. Catalase-negative
bacteria may be anaerobes, or they may be
facultative anaerobes that only ferment and do not
respire using oxygen as a terminal electron
acceptor (ie. Streptococci).
Chocolate Agar
Chocolate agar is a non-selective, enriched
growth medium. [1] [2] It is a variant of the
blood agar plate, containing red blood cells
that have been lysed by slowly heating to
80 °C. Chocolate agar is used for growing
fastidious respiratory bacteria, such as
Haemophilus influenzae and Neisseria
meningitidis.[3] These bacteria need growth
factors, like NAD (factor V) and hemin
(factor X), which are inside red blood cells;
thus, a prerequisite to growth is lysis of the
red blood cells. The heat also inactivates
enzymes which could otherwise degrade
NAD. The agar is named for the color and
contains no actual chocolate.
Coagulase Test
Staphylococcus aureus is known to produce
coagulase, which can clot plama into gel in tube or
agglutinate cocci in slide. This test is useful in
differentiating S.aureus from other oagulase-negative
staphylococci.
Most strains of S.aureus produce two types of
coagulase, free coagulase and bound coagulase.
While free coagulase is an enzyme that is secreted
extracellularly, bound coagulase is a cell wall
associated protein. Free coagulase is detected in tube
coagulase test and bound coagulase is detected in
slide coagulase test. Slide coagulase test may
be used to screen isolates of S.aureus and tube
coagulase may be used for confirmation. While there
are seven antigenic types of free coagulase, only one
antigenic type of bound coagulase exists. Free
coagulase is heat labile while bound coagulase is heat
stable.
Triple Sugar Iron Agar
Triple Sugar Iron medium is a differential medium that can
distinguish between a number of Gram-negative enteric bacteria
based on their physiological ability (or lack thereof) to:
a. metabolize lactose and/or sucrose
b. conduct fermentation to produce acid
c. produce gas during fermentation
d. generate H2S.
The medium contains 1.0% each of sucrose and lactose and 0.1%
glucose. If only glucose is fermented, acid produced in the butt
will turn it yellow, but insufficient acid products are formed to
affect the methyl red in the slant. However, if either sucrose or
lactose are fermented, sufficient fermentation products will be
formed to turn both the butt and the slant yellow. If gas is formed
during the fermentation, it will show in the butt either as bubbles
or as cracking of the agar. If no fermentation occurs (as for an
obligate aerobe), the slant and butt will remain red.The medium
also contains ferrous sulfate. If the bacterium forms H2S, this
chemical will react with the iron to form ferrous sulfide, which is
seen as a black precipitate in the butt (a black butt).
GPB with terminal bulging spores
Clostridium tetani is a Gram-positive, spore-
forming, rod-shaped bacterium that is
resistant to temperature modifications,
moisture, and chemical disinfectants. C.
tetani is strictly anaerobic and dies in the
presence of oxygen; however, spores
produced by this species are able to
withstand oxygenated environments and
other harmful environments. The
endospores are produced in a swollen
sporangium and their shape generally
resembles a 'drumstick'.
Gram Negative Bacilli
Examples include all enterobacteria like E.
coli, Klebsiella, Proteus and non
enterobacteria like Pseudomonas, Vibrio
cholerae
Robertson’s Cooked Meat Medium
The Medium provides a favorable environment for the
growth of anaerobes since the muscle protein in the
heart tissue granules is a source of amino acids and
other nutrients. The muscle tissue also provides
reducing substances, particularly glutathione. The
sulfhydryl groups, which exert the reducing effect, are
more available in denatured protein; therefore, the meat
particles are cooked for use in the medium.

Growth is indicated by turbidity and, with some

organisms, by the presence of gas bubbles in the
medium. Disintegration and blackening of the meat
particles indicates proteolysis.
Gram Negative Diplococci
Gram negative diplococci
extracellular and intracellular in
neutrophils. If the smear was
taken from urethral discharge, it
strongly suggestive of N.
meningitidis.
 Guinea Pig
Uses -
1. Isolation of M. tuberculosis & Leptospira
2. Virulenec test for MTB & C. diphtheriae
3. For differenciation between epidemic &
endmic typhus
4. For producing experimental infections like
Diphtheria & Anthrax.
5. Source of complent in CFT
Rabbit
Uses -
1. Differenciation between MTB & M, bovis
2. Toxigenicity test of C. diphtherae
3. Study of amall pox & Herpes viruses
4. For small scale production of antisera
5. Used in past for pregnanacy test
6. Used in past production of antirabic vaccine.
 Rat
Uses -
1. Studies on Hypersensitivity testing
2. Carcinogenesis study
3. Transplantation studies
4. Tetatnus toxin study
5. Experimental diabetes induction.
Mouse
Uses -
1. Isolation of S. pneumoniae & Borrelia
2. Production of experimental infections like
Plague, Anthrax
3. Study of carcinogenesis
4. Used in past pregnancy test.