Cultivation of Anaerobic Organisms | Microbial Biotechnology

The following points highlight the top two methods used for cultivation of anaerobic organisms. The methods are: 1. Shake Culture Method 2. Pyrogallic Acid: NaOH Method.

1. Shake Culture Method:

Principle:

The anaerobic organism does not require molecular oxygen for growth and hence will grow within the agar medium and E. coli above the medium when both are inoculated in the same medium.

Requirements:

1. 24-48 hour thioglycollate broth culture of Clostridium sporogenes.

2. Nutrient agar deep tubes.

3. Inoculating loop.

4. Bunsen flame.

5. Glass marking pencil.

6. Thioglycollate medium (pH 7.2):

Bacto yeast extract: 5.0 g

Bacto-casitone: 15.0

NaCl: 2.5g

L-cystine: 0.25 g

Thioglycollic acid: 0.30 ml

Bacto agar: 0.75 g

Methylene blue: 0.002 g

Procedure:

1. Melt nutrient agar deep tubes, cool to 45°C inoculate each tube with one cul­ture, shake gently by striking against fingers.

2. Cool the tubes rapidly under running tap water, wipe the surface and incubate at 37°C for 24-48 hours.

Clostridium will grow in the deeper regions while E. coli will grow on the surface.

2. Pyrogallic Acid: NaOH Method:

Principle:

Pyrogallic acid and NaOH absorbs O₂ from the tube and creates an anaerobic atmosphere for the growth of the organism.

Requirements:

1. 24—48 hour cultures of Clostridium and E. coli.

2. Nutrient agar deep tubes (2).

3. Pyrogallic acid crystal.

4. 4% NaOH.

5. Rubber stopper.

6. Pasteur pipettes.

7. Glass rod.

8. Inoculating loop.

9. Bunsen flame.

10. Glass marking pencil.

11. Incubator.

Procedure:

1. Streak inoculates both bacteria in nutri­ent agar tubes separately and ignite the cotton plug after replacing it passing over the Bunsen flame.

2. With the glass rod push the burning cot­ton plug into the tube until it just touches the slant.

3. Place pyrogallic acid crystals to fill the space above the cotton plug.

4. Add 2 ml of 4% NaOH on top and im­mediately place the rubber stopper tightly to the tube.

5. Incubate the tubes in an inverted posi­tion at 37°C for 24-48 hours (Fig. 2.3).

Anaerobic Clostridium will grow whereas E.coli will not since there was no free oxy­gen for the growth of the latter.