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

Introduction of Anaerobic Bacteria Culture

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 Culture/Cultivation

The mechanism of anaerobiosis achieves by the following methods-

1. Displacement of oxygen
2. Absorption of oxygen
3. 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, and 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)- 48 hours of thioglycollate broth culture
• Pseudomonas aeruginosa (obligate aerobes)- 48 hours of 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 specimens 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 them 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

Pseudomonas aeruginosa will not grow on the blood agar plate incubated anaerobically, while  Clostridium 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 Clostridium sporogenes will not grow because  Clostridium 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
S.No	Gram-positive rods
1	Bifidobacterium
2	Propionibacterium
3	Eubacterium
4	Lactobacillus
5	Actinomyces
6	Clostridium
7	Mibilnicus
	Gram-negative rods
1	Bacteroides
2	Fusobacterium
3	Prevotella
4	Porphyromonas
5	Leptotrichia
	Anaerobic cocci
	Gram-positive cocci
1	Peptostreptococcus
2	Coprococcus
3	Ruminococcus
	Gram-negative cocci
1	Veilonella
2	Acidaminococcus
3	Megasphaera

Related Photos

Pus specimen for anaerobic bacteria culture

Pus sample in Thioglycollate broth

Anaerobic bacterial growth on Robertson cooked meat (RCM) medium

McIntosh Filde Anaerobic Jar for Anaerobic bacteria culture

Clostridium growth around gentamicin disk but no growth near metronidazole disk in anaerobically incubated blood agar plate

Clostridium Gram-positive rods or bacilli in Gram staining of culture

Further Reading

1. Bailey & Scott’s Diagnostic Microbiology. Editors: Betty A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
2. https://clsi.org/media/1440/m56a_sample.pdf
3. https://stacks.cdc.gov/view/cdc/7666/cdc_7666_DS1.pdf
4. Clinical Microbiology Procedures Handbook – Lynne S. Garcia
5. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
6. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
7. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melbourne, Sans Francisco 1996.
8. Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier