Anaerobiosis Methods : Introduction, various types and their uses in

Anaerobiosis Methods : Introduction, Various Types and Their Uses in Bacteriology

Anaerobiosis Methods

Anaerobiosis Methods for anaerobic organisms that can live and multiply only in the absence of oxygen. As you know, isolating the causative agent of anaerobic infection is really cumbersome. It means that leave the practice which is not a solution. This is not my personal opinion but according to researchers’ data; “many reports associate 50-60% of important infections with anaerobic bacteria.”This is the reason, we concern with anaerobic infections and therefore the importance of anaerobiosis methods.

List of anaerobic bacteria-

Anaerobic bacilli

Gram-positive bacilli-

Bifidobacterium
Propionibacterium
Eubacterium
Lactobacillus
Actinomyces
Mobilincus
Clostridium

Gram-negative bacilli

Bacteroides
Fusobacterium
Prevotella
Porphyromonas
Leptotrichia

Anaerobic cocci

Gram-positive cocci

Peptostreptococcus
Coprococcus
Ruminococcus

Gram-negative cocci

Veillonella
Acidaminococcus
Megasphaera

Examples of anaerobic bacteria and the disease they cause –

Clostridium perfringens : Gas gangrene
Clostridium tetani : Tetanus
Clostridium botulinum: Botulism (complete paralysis of respiratory and other muscles )
Clostridium difficille: Colitis

Actinomyces spp. : Oral , cervicofacial , thoracic , pelvis and abdominal infection , dental caries

Propionibacterium spp. : Inflammatory process in acne

Bifidobacterium spp. : Bacteremia, diarrhea

Eubacterium spp. : Infection of abdomen, pelvis, or genitourinary tract

Oxygen is directly toxic to the cell. Since the organism does not form catalase, H₂O₂ produced is toxic to the cell. Growth is dependent upon low oxidation-reduction potential, which is not possible in the presence of air.

Various anaerobiosis Methods

By displacement of oxygen anaerobiosis method
Cultivation in a vacuum, in a vacuum desiccator – tried but proved to be unsatisfactory. Displacement of oxygen by inert gases like hydrogen or nitrogen. Drawbacks include repeated evacuation and re-filling.
Absorption of oxygen by chemicals (Chemical method) anaerobiosis method: (a) Pyrogallic acid (Buchner 1888): In a large tube (Buchner‘s tube ) containing a solution of NaOH, pyrogallic acid is added. The tube is placed inside an airtight jar loaded with inoculated plates. (b) A mixture of chromium salt and H₂SO₄ (Marshall, 1990): Two chemicals react in the presence of oxygen to produce Chromous sulfate. (c) Brewer’s technique (1942): Petri dish cover together with an agar media containing thioglycollate and methylene blue for the surface cultivation of anaerobes. Methylene blue – indicator sodium thioglycollate – a reducing substance that removes oxygen. (d) Absorption of oxygen by activated iron, Parker(1955): Use of activated iron wool. Activated by immersing in an acid solution of CuSO₄, then draining ( e) Using microorganisms to achieve anaerobiosis: Slow and ineffective. One plate – with Pseudomonas aeruginosa and – Second plate– specimen of the anaerobic organism. Two plates are placed one over the other and sealed along the rims and incubated. (f) Exclusion of atmospheric oxygen: A tube of deep nutrient broth is inoculated and covered with a ½ inch layer of sterile melted vesper ( mixture of petroleum jelly and paraffin) (g). Use of semisolid agar (0.05–0.2%): Media should be deep inoculated where O₂ concentration is very low. (h) Gas – Pak: Simple and effective. – Commercial products in the form of a disposable. a packet of aluminum foil containing pellets of sodium borohydride and cobalt chloride and of citric acid and sodium bicarbonate. Generates hydrogen and CO₂inside the jar when water is added. catalyst (alumina pellets coated with palladium) H₂+ O₂ → H₂O- Inoculated plates are placed inside a large, air-tight jar, incubated at 37°C.
By displacement and combustion of oxygen anaerobiosis method: Anaerobiosis obtained by McIntosh and Filde’s anaerobic jar is a most dependable and widely used method. Principle: Spongy palladium or platinum kept inside the jar acts as a catalyzing agent which causes a slow combination of hydrogen and oxygen to form water. Procedure: It’s a glass or metal-made jar with a tight-fitting metal lid. The lid can be clamped airtight with a screw and fitted with two tubes with taps, one for the introduction of gas inside and the other for the vacuum valve. A capsule containing alumina pellets coated with palladium is suspended under the lid. Inoculated culture plates are kept inside the jar along with the indicator. The lid is screwed tight. The inlet tube is closed and the outlet tube is connected to a vacuum pump and at least 3 quarters of the air of the jar is removed. The outlet tap is closed and the inlet tap is connected to a hydrogen supply. The pressure inside the chamber equals the atmospheric pressure. Indicator: reduced methylene blue (mixture of NaOH, methylene blue, and glucose ) colorless anaerobically and regains blue color on exposure to oxygen.
By incorporating reducing agents in the media method of anaerobiosis – The reducing agents used are -1% glucose, 0.1% thioglycollate, 0.05% cysteine, 0.1% ascorbic acid, and cooked meat pieces. Two mostly used liquid culture media are (a) Thioglycollate broth – contains nutrient broth and 1% thioglycollate and uses for anaerobic culture in blood Indicator methylene blue or resazurin. (b) Robertson’s cooked meat media -Consists of nutrient broth and pieces of fat-free cooked minced meat of beef heart. Unsaturated fatty acids in the meat utilize oxygen for auto-oxidation. The reaction is catalyzed by hematin in the meat. Glutathione and cysteine in the meat (reducing substances) also utilize oxygen. Saccharolytic anaerobes (C. welchii)- red. Proteolytic anaerobes (C. tetani) – black media must be boiled in a water bath before inoculation at 80°C for ½ hour to drive out oxygen. For strict anaerobiosis, the surface of the media is covered with a layer of 1 cm of sterile liquid paraffin. Transport of specimen-Three different kinds of anaerobic transport systems are available- Rubber- stoppered collection vial containing an agar indicator system -Vial is gassed out with oxygen-free CO₂ or nitrogen. The specimen is injected through the rubber stopper after all air is expelled from the syringe and needle. for only a swab specimen A special collection device with an oxygen-free atmosphere is required. When re-inserting the swab, care must be taken not to tip the container which would cause the oxygen-free CO₂ or nitrogen to spill out and be displaced by air. Tissue can be placed in a small amount of liquid (saline) to keep it from drying out and placed in an anaerobic pouch. All specimens should be held at room temperature. Refrigeration can oxygenate the specimen. Incubation -1. Anaerobic jars most frequently used system (Beckton Dickinson and company, Oxoid USA ). Uses a clear heavy plastic jar with a lid clamped down to make it airtight. It uses a commercially available H₂ and CO₂ generator envelope which is activated by adding water or by the moisture from agar plates. The production of heat and moisture on the walls of the jar indicates that the catalyst and generator envelope are functioning properly. Indicators are blue or pink initially, become colorless with a low concentration of oxygen. Holding jar Used during specimen processing and examination of culture. Anaerobic jar with loosely fitted lids attached by rubber tubing of nitrogen gas Un-inoculated and inoculated plates are kept, pending use for culture set-up or incubation or examination. Anaerobic chamber made of molded or flexible clear plastic- Use of gloves or sleeves forming airtight seals around the arms to handle items inside the chamber A gas mixture of 5-10% CO₂, 5% Hydrogen, and 85 -90% nitrogen and a palladium catalyst maintains anaerobiosis. It has a positive pressure inside the chamber. The catalyst reduces O₂ to H₂O₂ removing atmospheric O₂. CO₂ is included because many anaerobes require it for growth. Do not exceed 5% hydrogen because of hazardous conditions. Add anaerobic indicator to the chamber in an empty Petri dish to prevent drying. Humidity is controlled by the absorption of H₂O formed in the catalytic conversion with silica gel crystals. Change catalyst daily. Incubate at 35 – 37°C. The Hungate procedure – Anaerobic method to prevent access of air to media during preparation, inoculation, and incubation. Surface colonies are grown in roll tubes in which a thin layer of agar coats the inside of the tube. Media must be transparent for the surface colonies to be visible. Anaerobic media plates should be freshly prepared. Plates stored for longer periods accumulate peroxides and cause dehydration of media. Prereduced, anaerobically sterilized (PRAS) media are produced, packaged, shipped, and stored under anaerobic conditions. Commonly used anaerobic media 1. Anaerobic blood agar media, 2. Bacteroides bile esculin agar (BBE), 3. Kanamycin Vancomycin blood agar, 4. Anaerobic phenyl ethyl alcohol agar (PEA), 5. Egg yolk agar, 6. Cycloserine cefoxitin fructose agar,7. Cooked meat broth 8. Peptone yeast extract glucose broth (PYG), and 9. Thioglycollate broth.

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