Anaerobiosis

Anaerobiosis. gas pack, mcintosh jar & Filde's jar, reducing agents

1. Anaerobiosis Dr. Dinesh Jain SMS MC Jaipur

2. Types of anaerobes Obligate anaerobic bacteria- bacteria that can grow in the absence of free oxygen, but fails to multiply in the presence of oxygen eg .- Bacteroides fragilis , Clostridium perfringens , C. novyi , Porphyromonas , Fusobacterium . Aerotolerant anaerobes- A naerobic bacteria that will show limited or scanty growth in room air or in a 5-10% CO 2 incubator, but show good growth under anaerobic conditions. eg .- C. carnis , C. histolyticum , C. tertium etc. Microaerophilic bacteria- Organism require O 2 as a terminal electron acceptor, yet these do not grow on the surface of solid media in an aerobic incubator (21% O 2 ) & grow minimally if at all under anaerobic condition eg .- Campylobacter. jejuni (grow in 5% O 2 ).

3. Features suggestive of anaerobic infection Foul smelling discharge. Necrotic gangrenous tissue and abscess formation. Free gas in tissue. Black discoloration of exudates ( Bacteroides melaninogenicus ). Sulphur granules in discharge ( Actinomyces spp.). Bacteraemia or endocarditis with no growth on aerobic blood cultures

4. SPECIMEN COLLECTION The best specimen for anaerobic culture is an aspirate obtained by needle and syringe Disinfect skin surface with 70% alcohol, allow to dry. Aspirate specimen directly into the syringe. Remove air from syringe. Aseptically transfer material into an anaerobic transport vial for fluids.

5. SPECIMEN COLLECTION Tissue samples and biopsies are also very good specimens for anaerobic culture. Punch biopsies, bone, or any other tissue specimen may be collected surgically. Place specimen in a sterile container without formalin

6. specimens that are not suitable for anaerobic cultures Specimens from sites in which anaerobic bacteria are normal flora (e.g., throat, rectal swabs, urine, bronchial washes, cervico -vaginal mucosal swabs, sputum).

7. specimens that are not suitable for anaerobic cultures Voided and catheterized urine. Gastric contents, small bowel contents, feces, colocutaneous fistula and colostomy contents should not be cultured for anaerobic bacteria Specimens not submitted in anaerobic transport media.

8. Handling & transport of clinical specimens Immediate delivery of the specimen is crucial. If delay is anticipated, an anaerobic transport system should be utilized especially for swabs and small volume specimens. ANAEROBIC ORGANISMS ARE COLD SENSITIVE. SAMPLES FOR ANAEROBIC CULTURE SHOULD NOT BE REFRIGERATED. THEY MUST BE STORED AT ROOM TEMPERATURE

9. Anaerobiosis It is an anaerobic bacteria culture method used to grow anaerobes from a clinical specimen. Obligate anaerobes grow only in the absence of oxygen. They are destroyed when exposed to the atmosphere for as briefly as 10 minutes

10. METHODS OF ANAEROBIASIS 1.Producing a vacuum 2. Oxygen displacement 3. Oxygen absorption 4. Reducing agents 5. Anaerobic chambers–(have catalyst, desiccant, H2, CO2, N2 + indicator; airtight gloves)

11. METHODS OF ANAEROBIASIS Producing a vacuum

12. METHODS OF ANAEROBIASIS: McIntosh & Filde’s Jar Stout glass or metal jar with a lid Lid has an inlet for gas,outlet&2 terminals Alumina pellets coated with palladium (catalyst) under the lid Inoculated plates kept inside the jar Lid is clamped tight Air is evacuated

13. METHODS OF ANAEROBIASIS Outlet tube—vacuum pump to take out air Inlet—Hydrogen passed in—normal atmospheric pressure Catalyst: Hydrogen + Oxygen = Water Reduced methylene blue is indicator. Colorless is anaerobic and blue when exposed to oxygen

14. METHODS OF ANAEROBIASIS Oxygen displacement Remove the air inside the jar/cabinet with vacuum pump, by producing negative- pressure upto 660 mm of Hg. The jar is filled with either a mixture of gases 80% N2, 10% H2, 10% CO2 or by adding 10% CO2 and 90% H2 gas separately. Hydrogen gas reacts with oxygen in the presence of catalyst to form water. The commonly used catalyst is palladium coated alumina pellets .

15. METHODS OF ANAEROBIASIS:

16. Wright’ tube method

17. METHODS OF ANAEROBIASIS Oxygen absorption Use of copper coated steel wool to remove oxygen The plates are incubated in a airtight plastic bag Steel wool is activated immediately before use by being dipped in acidified copper sulphate solution. Place in the bag an open tube or bottle containing equal volumes of magnesium carbonate and sodium bicarbonate for the release of carbondioxide . About 1.5 g of each chemical is required

18. METHODS OF ANAEROBIASIS Complete anaerobic conditions are usually obtained within 4 hours of sealing the bag. Just before sealing the anaerobic bag, use a piece of rubber tubing and syringe to draw out some of the air.

19. METHODS OF ANAEROBIASIS: Gas Pak Pellets of sodium borohydride , cobolt chloride, citric acid and sodium bicarbonate Generate hydrogen and Carbon dioxide in presence of waterH2 + O2 = H2O Inocculated plates in airtight Jar Gas-Pak + Water added; lid closed

20. Roll Streak System It uses PRAS media prepared in tubes with rubber stoppers. Tubes of agar media are cooled in a rolling machine after autoclaving, which results in a thin coating of the inner surfaces of the tubes with solidified medium. Both PRAS liquid media & roll streak tubes requires addition of a reducing agent, such as L- cystine -hydrochloride , which is added just before autoclaving to maintain a low oxidation-reduction potential. All inoculating & subculturing of the PRAS solid & liquid media are performed under a stream of O 2 -free CO 2 , which minimizes exposure to air & help to maintain a reduced oxidation-reduction potential in the media before & after growth.

21. 1.Exclude oxygen by flushing the tube with the desired gas 2. Place 4.5ml of pre-reduced anaerobic agar medium into tube 3. Seal the tube with the butyl rubber stopper and screw cap 4.Autoclave the tube 5.Inoculate with a syringe 6.Prepare on roll tube spinner 7.Incubate in water bath

22. METHODS OF ANAEROBIASIS: By Reducing agents Glucose0.5%-1% Thioglycollate 0.01%-0.02% Cooked meat pieces Cystein < 0.05% Ascorbic acid 0.1% Sodium sulphite 0.025% Metallic iron

23. METHODS OF ANAEROBIASIS: Anaerobic Glove Box System Self contained system that allows to process specimens & perform test for isolation & identification without exposure to O 2 . Glove boxes suitable for cultivation, can be constructed from various materials, including steel, acrylic plastic, vinyl plastic or fiberglass.

24. METHODS OF ANAEROBIASIS: Economical to operate b/c it permits the use of conventional plating media & cost of gases for operation of the system is minimal. Once setup, the major expense is for the 85% N 2 , 10%H 2 , 5%CO 2 gas mixture used to replace the air in the entry lock when materials are passed into the glove box chamber

25. METHODS OF ANAEROBIASIS:

26. Newer anaerobic systems Don’t require either catalyst or the addition of water to activate these systems. AnaeroPack , absorbs O 2 and generates CO 2 , but doesn’t generate H 2. It appear to be an excellent alternative to the GasPak and other established anaerobic incubation systems. Another type of commercially available catalyst free-system ie . Anaerocult (Merck, Germany), makes use of iron filings in a sachet to which water is added, producing an O 2 free, CO 2 -rich atmosphere.

27. Oxoid AneroGen system The active oxygen-removing component in AneroGen sachets is ascorbic acid. The sachets are designed for use in 2.5 litre and 3.5 litre capacity anaerobic jars. The paper sachet is placed in the jar immediately before it is closed. No water is needed to activate the chemical. Within 30 minutes of closing the jar, the oxygen level is reduced to below 1% (carbon dioxide level is 9–13%).

28. Anaerobic Holding Jar Convenient adjunct to the jar & glove box systems that allows primary plating, inspection of cultures, & subcultures of colonies at the bench with only minimal exposure to atmosphere. Inexpensive, commercial-grade N 2 can be used in the holding jar system. Open the small needle valve & set to gas tank regulator at prescribed pressure. Alternatively, CO 2 passed through a tube of heated copper catalyst (Sargent furnace) can be used in the holding jars instead of N 2 .