General microbiology spotters By Dr Sudheer Kher MD HOD Microbiology
Dr. Sudheer Kher
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,
Bluish green medium in screw capped
Sterilized by Inspissation.
Type of medium - Semisynthetic,
enriched, selective and indicator
Loeffler’s Serum Slope
Sterilized by Inspissation.
Use - For culture of C.
Type of Medium -
Identification - White slope
with water of
condensation usually in
screw capped bottle or
Hot Air Oven
Temp - 160 C X 60 minutes
Principal - Sterilization by dry heat
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
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
Alpha hemolysis on blood agar
Blood agar showing
Example of bacteria
haemolysis - Viridans
Group of Streptococci
Beta hemolysis on blood agar
Blood agar showing Beta
(Complete zone of
Example of bacteria
producing Beta hemolytic
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
Source of Factor V - Staphylococcus
Target hemolysis - Clostridium
Double zone of
hemolysis seen in
Swarming growth on Blood Agar
Swarming or spreading in wave form is
seen with motile organisms particularly
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
How sterilized - By Ethylene oxide
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
showing pink (Lactose
fermenting) colonies. The
crenated margins (Maple
suggests possibility of E.
Lactose non-fermenting colonies on
MacConkey’s medium with Lactose non-
fermenting colonies. Organisms which can
form such colonies include Proteus,
Principal - Steam under pressure. Microbes destroyed by
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
Inoculation loop -
Usually made from Nichrome
Sterilized by Red Heat.
1. Transfer of clinical specimen
to culture media.
2. Transfer of bacterial growth
from medium to slides / to
other culture media
Bile Salt Sucrose
Type of medium -
Selective & Indicator
Use - For isolation of
Vibrio cholerae which
grow on this medium
and due to sucrose
fermentation show up
as yellow colonies.
Seitz filter is made up of
stainless steel and has
asbestos disc. Used for making
fluids bacteria free.
Can stop bacteria but not
Uses - Preparation of toxins,
antitoxins, antibiotic solutions
Sterile swab stick -
❏ For collection of clinical
specimen like pus, throat,
❏ For making lawn culture for
Antibiotic sensitivity test
How to sterilize?
❖ Hot air oven - preferred
This test depends on the presence of
cytochrome oxidase in bacteria that will
catalyze the transport of electrons between
electron donors and redox dye.
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.
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
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
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
The catalase test is used to differentiate
staphylococci (catalase-positive) from streptococci
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 is a non-selective, enriched
growth medium.   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. 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.
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
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
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 H2
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 H2
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
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.
Guinea Pig Rabbit
1. Isolation of M. tuberculosis & Leptospira
2. Virulenec test for MTB & C. diphtheriae
3. For differenciation between epidemic &
4. For producing experimental infections like
Diphtheria & Anthrax.
5. Source of complent in CFT
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.
1. Isolation of S. pneumoniae & Borrelia
2. Production of experimental infections like
3. Study of carcinogenesis
4. Used in past pregnancy test.
1. Studies on Hypersensitivity testing
2. Carcinogenesis study
3. Transplantation studies
4. Tetatnus toxin study
5. Experimental diabetes induction.