How to determine the sulphate reducing bacteria in a sample

Sulphate Reducing Bacteria 

Sulfate reducing bacteria (SRB) are anaerobic organisms which require an oxygen free environment for growth. They reduce sulfate to hydrogen sulfide.  They are responsible for the  foul hydrogen sulfide odors and are direct agents for corrosion. The hydrogen sulfide immediately reacts with metallic or dissolved iron to form black iron sulfide.

Being anaerobic, SRB can not be enumerated by the same technique used for heterotrophs. SRB are enumerated by a method known as Most Probable Number (MPN) technique. This technique was originally used for counting coliforms in potable water analysis. This method is modified to count sulfate-reducing bacteria by replacing Mc Conkey’s broth by sulfite broth.

Summary Of Method

The test procedure for the enumeration of Sulfate Reducing Bacteria consists of inoculating the test sample into a test tube filled with a liquid medium. The test tube is incubated and observed. Production of black ferrous sulfide indicates the presence of sulfate reducing bacteria.

 

Equipments

Incubator

Autoclave

Pipettes (10ml)

Dilution Flasks

Non Absorbent Cotton

Test tube with screw cap ( 25 x 150 mm & 15 x 150 mm) Liquid Paraffin

McCardy’s MPC Table

Glassware is sterilised at 121oC at 15 lbs / inch2 for 20 min. in an autoclave.

 

Procedure

1. A medium, suitable for the growth and enumeration of Sulfate Reducing Bacteria, is prepared as follows:-

(a) Dissolve

 10.0 gram Tryptone

 1.0 gram Sodium Sulfite &

 10.0 ml ferric citrate 5% solutions in distilled water and make up to 1000 ml.

(b) For preparation of double strength medium, dissolve above ingredients in distilled water and make upto 500 ml.

2. Dispense 10 ml each of double strength medium in 5 clean & dry 25 x 150 mm screw capped tubes.

3. Dispense 10 ml each of normal strength medium in 10 clean & dry 15 x 150 mm screw capped tubes.

4. Autoclave all the screw capped tubes containing medium in a autoclave at 15 lb / inch2 pressure at 121oC for 20 minutes.

5. Cool the tubes.

6. Add 10 ml of the test sample to a set of tubes containing double strength medium.

7. Add 1 ml of the test sample to a set of tubes containing normal strength medium and add

0.1 ml of test sample to remaining 5 tubes of normal strength medium.

8. Add sterile liquid paraffin in each tube so as to obtain 0.5 cm layer on the top.

9. Incubate the tubes in an incubator at 37o C for 3 to 5 days.

10. Blackening of liquid in the tubes in typical of sulfate reducing bacteria and may occur in 3 to 5 days.

11. Compare the number of positive tubes containing blackish spots to standard McCardy’s MPN table to obtain number of sulfate reducing bacteria per 100 ml of sample.

12. Record the final result of sulfate reducing bacteria as number of SRB per 100 ml.