Methods of creation of anaerobic conditions. Physical methods.

Physical methods are based on creation of oxygen-free conditions of growth by the following means:

• inoculation of media containing reducers and easily oxidative substances;

• inoculation of microorganisms into depth of solid media;

• mechanical removal of air from incubating cameras/flasks;

• replacement of air by special gas mixture.

Removal or replacement of air is performed mechanically from anaerobic jars.

Anaerobic jar - is hermetically sealed metallic or plastic jar from which it is possible to pump out oxygen and replace it by special gases (helium, nitrogen, argon). Triple gas mixture which consists of nitrogen 80 %, carbon dioxide 10 %, and hydrogen 10 % is used.

Inoculation of media containing reducers and easily oxidative substances.

One of the best examples of broth with reducers is Kitt-Tarozzi medium. Usually peaces of animal or plants are used as reducers (e.g. liver, brain, blood, potato, etc. ). Those are binding diluted oxygen in nutrient media and also absorbing bacteria. In order to decrease of concentration of oxygen in nutrient medium, before inoculation it should be boiled for 10-15 min., then quickly cooled down and sealed with sterile liquid paraffin or petrolatum. Glucose, lactose and amino formic sodium are used as easily oxidative substances.

Inoculation of microorganisms into depth of solid media.

This is done by Weinberg and Venyal-Venyon methods.

Weinberg method is based on cultivation of anaerobes into tube glucose agar. Glucose agar is poured into tube in column fashion up to 2/3 of tube volume. Then it allowed to cool down to +42-45°C. Studied specimen is added to agar and thoroughly mixed. Tube is placed in tube stand. After solidification of media, good anaerobic conditions are created (especially in bottom part of the tube).

Venyal-Venyon method is based on mechanical protection of anaerobic cultures from atmospheric oxygen. For this purpose, a long (30 cm in length and 3-6 mm is diameter) glass tube. One side of tube is stretched out as a Pasteur pipette and on second side constriction is made. The wide side of tube is sealed with cotton plug. Studied material is inoculating into tubes with melted and cooled up to +50°C agar. Inoculated agar then is sucked into sterile Venyal- Venyon tubes. Capillary side of the tube is sealed by flaming and tubes are placed into incubator, thus creating suitable conditions for inoculating even strict anaerobes. For isolation of separate colony tube is cut with file in aseptic conditions, on the level of colony, cut, then colony is taken by sterile bacteriological loop and taken to tube with nutrient medium.

Chemical methods.

Chemical methods are based on absorption of oxygen in hermetically sealed flask (anaerobic or candle jar) by pyrogallol or sodium bisulfite.

Biological methods.

Biological methods (Fortner' method) are based on concomitant incubation of anaerobes and obligate aerobes. For this purpose approximately 1 cm wide strip is cut by sterile scalpel from solidified agar (by diameter of Petri dish). By this approach, 2 agar semi-disks are obtained per 1 plate. On one side of agar strip, aerobe is inoculated (e.g. Staphylococcus aureus or Serratia marcescens), on the other one - anaerobe. Edges of Petri dishes are sealed with liquid paraffin, and Petri dishes are placed in the incubator. In case of suitable conditions, aerobes are starting to multiply thus consuming oxygen. After 3-4 days, when all oxygen is consumed, anaerobes are starting to grow.

Combined methods are based on various combinations of physical, chemical and biological methods.