Most organisms need oxygen for growth and are incubated in normal atmospheric conditions. Some pathogens, e.g., the tetanus bacilli, will grow only in the absence of oxygen.
This is achieved by using Mcintosh and Fildes’ jar, a thick metal or glass jar with a metal lid which can be clamped down tightly by bolts. On this lid are 2 holes-one on air inlet and the other an outlet. There are also 2 electric terminals.
Image Source: frontiersin.org
ADVERTISEMENTS:
On the underside of the lid is a piece of asbestos saturated with palladium and covered by wire gauze. This is connected to the terminals, and acts as a catalyst in combing any oxygen still present after evacuation of the jar with the hydrogen which is passed into the jar.
The method is given below:
(a) Keep the plates upside down integer.
(b) Place in the jar indicator-equal parts of 10% NaOH, 6% glucose and 0.5% methylene blue, boiled until the solution becomes colourless. It should remain colourless throughout incubation. If it turns to its original blue colour during incubation, complete anaerobiosis (oxygen less state) has not been achieved.
ADVERTISEMENTS:
(c) Tightly clamp down the lid.
(d) Open the air outlet valve and close the air inlet valve.
(e) Attach the apparatus to an exhaust pump, and slowly evacuate the jar, (if a glass jar is used, it should be evacuated while enclosed in a padded box to avoid danger of explosion).
(f) Allow hydrogen obtained from hydrogen cylinders or Kipp’s apparatus in through the inlet valve after closing the outlet valve.
ADVERTISEMENTS:
(g) Attach the terminals to the main current and leave for 20 minutes. This heats the palladiumized asbestos to assist the combination of hydrogen with any remaining oxygen.
(h) Allow little more hydrogen in via the inlet valve.
(i) Put the jar in the incubator overnight. The present day Mclntosh- Filde’s jars have room temperature catalysts and need no electrical charge. They are left at room temperature for 15-30 minutes before allowing more hydrogen into the jar. There are other, less complicated methods of achieving anaerobiosis (i.e. an oxygenless state) e.g.
1. Boil a tube of nutrient broth and layer over it sterile vaseline. The boiling removes the oxygen and the vaseline prevents more entering as the broth cools. The tube is inoculated using a sterile pasteur pipette.
2. A sterile iron nail placed in glucose broth which has been treated as in method (1), will maintain anaerobic conditions for some time.
3. Robertson’s cooked meat medium and Brewer’s thioglycolate broth are frequently used in the culture of anaerobic organisms.
Some organisms are not anaerobic, but do grow better when the amount of oxygen has been reduced. One simple technique is to place the plates in a tin or wide mouthed bottle with tight fitting lids. A candle is lit inside the container and the lid replaced firmly. The candle flame will use off the oxygen and give an atmosphere of 5-10% CO2. The container is placed in the incubator.