General Microbiology Spotters

Dr. Sudheer Kher

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 SlopeSterilized 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 OvenTemp - 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 MediumThiosulphate 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 TestThis 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 TestUrease 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 organisms1. Helicobacter pylori2. Proteus spp3. Yersinia spp4. Klebsiella spp5. Brucella spp6. Cryptococcus spp

Citrate utilization TestSimmons 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 - KlebsiellaExample of citrate - bacterium - E. coli, S. typhi

Catalase TestThe 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 AgarChocolate 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 TestStaphylococcus 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 AgarTriple 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 sucroseb. conduct fermentation to produce acidc. produce gas during fermentationd. 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 BacilliExamples include all enterobacteria like E. coli, Klebsiella, Proteus and non enterobacteria like Pseudomonas, Vibrio cholerae

Robertson’s Cooked Meat MediumThe 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 Rabbit

Uses -1. Isolation of M. tuberculosis & Leptospira2. Virulenec test for MTB & C. diphtheriae3. For differenciation between epidemic &

endmic typhus4. For producing experimental infections like

Diphtheria & Anthrax.5. Source of complent in CFT

Uses -1. Differenciation between MTB & M, bovis2. Toxigenicity test of C. diphtherae3. Study of amall pox & Herpes viruses4. For small scale production of antisera5. Used in past for pregnanacy test6. Used in past production of antirabic vaccine.

Rat Mouse

Uses -1. Isolation of S. pneumoniae & Borrelia2. Production of experimental infections like

Plague, Anthrax3. Study of carcinogenesis4. Used in past pregnancy test.

Uses -1. Studies on Hypersensitivity testing2. Carcinogenesis study3. Transplantation studies4. Tetatnus toxin study5. Experimental diabetes induction.