Anaerobic Bacteria Cultivation: Introduction, Principle, Procedure and Res

Anaerobic Bacteria Cultivation: Introduction, Principle, Procedure and Result Interpretation

Introduction of Anaerobic Bacteria Cultivation

Obligate anaerobes can not grow in the presence of oxygen i.e. air. These organisms (obligate anaerobic bacteria) die rapidly on exposure to air, therefore for maintaining anaerobiosis various methods have been devised for anaerobic culture.

Principle for Anaerobic Bacteria Cultivation

The mechanism of anaerobiosis achieves by the following methods-

Displacement of oxygen
Absorption of oxygen
Combustion of oxygen or their combinations

A deep nutrient agar tube is the simplest method. Anaerobes grow in the depth of the medium, and the number of colonies becomes fewer towards the surface. Strict anaerobes will not grow within a centimeter of the surface. Using reducing agents like glucose, ascorbic acid, cysteine, thioglycollate in the medium. Cooked meat particle also acts as s good reducing agent due to having glutathione e.g. Robertson’s cooked meat medium. Another simple method of anaerobiosis is the application of a candle jar. A burning candle can not continue due to the exhaustion of oxygen inside the jar. In laboratories, combustion involves the combining of oxygen with hydrogen to form water in the presence of a catalyst like palladium or palladinized asbestos. Anaerobic jars are a constant feature of anaerobic culture. They include the McIntosh and Fildes anaerobic jar, which has inlets to admit hydrogen and carbon dioxide, a vacuum pump for evacuating oxygen, and a catalyst fitted into the lid. Similarly, a simpler but more expensive technique is the Gaspak system. This utilizes a transparent polycarbonate jar with a lid bearing a screened catalyst chamber. The catalyst, consisting of pellets of sodium borohydride, cobalt chloride, citric acid, and sodium bicarbonate is contained in the sachet. Water is added to the sachet and it is immediately placed in the jar, which is then sealed tightly. The resulting reaction liberates hydrogen and carbon dioxide. An indicator is also added to demonstrate anaerobiosis.

Requirements for Culture of anaerobic bacteria

Anaerobic culture system
McIntosh and Fildes anaerobic jar
Gaspak system
Robertson’s cooked meat medium/ Thioglycollate broth
Blood agar plates
Control strains
Clostridium sporogenes (anaerobes)- a 48 hours thioglycollate broth culture
Pseudomonas aeruginosa (obligate aerobes)- a 48 hours thioglycollate broth culture

Procedure for Culture of anaerobic bacteria

Inoculate the specimen in Robertson’s cooked meat medium and incubate for 48 hours. Divides each blood agar plate into two equal parts. Inoculate a loopful of each control organism into half-blood agar. Similarly, also inoculate test specimen of 48 hours incubated Robertson’s cooked meat medium into a half-blood agar plate. Stack the plates into the anaerobic jar, introduce the catalyst and quickly seal the lid. Incubate the plates at 37°C for 48 hours. Incubate one plate of control strains aerobically. Remove the plates and examine for growth.

Quality control

Pseudomonas aeruginosa does not grow anaerobically. Clostridium sporogenes does not grow aerobically.

Observations

When anaerobiosis is complete, obligate anaerobes like Clostridium sporogenes will grow, while obligate aerobes like Pseudomonas aeruginosa will not grow.

Result and Interpretation

P. aeruginosa will not grow on the blood agar plate incubated anaerobically, while Cl. sporogenes will grow on the blood agar plate. P. aeruginosa is a strict aerobe that can not grow in the absence of oxygen. P. aeruginosa will show growth on the aerobically incubated while Cl. sporogenes will not grow because Cl. sporogenes is a strict anaerobe that can not grow if oxygen is present.

Keynotes on Culture of anaerobic bacteria

For screening anaerobes, a 5 µg metronidazole disk can use. Organisms are sensitive toward this disk. Whereas 10 µg gentamycin uses for the anaerobes and they are resistant. The anaerobic condition should be checked by using an alkaline methylene blue indicator. List of anaerobic bacteria-

Anaerobic rods

Gram-positive rods

Bifidobacterium
Propionibacterium
Eubacterium
Lactobacillus
Actinomyces
Clostridium
Mibilnicus

Gram-negative rods

Bacteroides
Fusobacterium
Prevotella
Porphyromonas
Leptotrichia

Anaerobic cocci

Gram-positive cocci

Peptostreptococcus
Coprococcus
Ruminococcus

Gram-negative cocci

Veilonella
Acidaminococcus
Megasphaera

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