Hydrogen Sulphide (H2S) Production Test

Objective:

            To determine the ability of microorganism reduces the sulphur containing compounds to hydrogen sulphide during metabolism.

Principle:

            Some bacteria utilize sulphur containing amino acids such as cysteine from the proteins by enzyme desulferase; it loses the sulfur atom then reduced by addition of hydrogen atom from water to form hydrogen sulphide.  This organism also reduces the inorganic sulphur compounds such as sodium thio sulfate, sulfate and sulfite to gaseous hydrogen sulphide.

            The SIM (Silfide, Indole, and Motility) agar contains the peptone a source of cysteine and sodium thiosulfate as sulfur substrates, ferrous ammonium sulfate which behaves as the H₂S indicator. If H₂S produced it combines with ferrous ammonium sulfate, forming insoluble black ferrous sulfide precipitate.   

Materials required:

24 Hours old bacterial cultures (E.coli. Proteus sp., Klebsiella sp. and Salmonella sp.)

SIM agar medium

Procedure:

- Prepare and sterilize the SIM agar deep tubes, adjust the pH to 7.4

- Inoculate the bacterial cultures by straight stabbing to a depth of 2-3 cm.

- Incubate the tubes at 37°C for 24 - 48 hours

Result:

Salmonella sp. and Proteus sp. gives positive result.

E.coli and Klebsiella sp. gives negative result.

Interpretation:

Salmonella sp. and Proteus sp.  shows blackening color due to the production of hydrogen sulphide and gives positive reaction. E.coli and Klebsiella sp. are not showing blackening color.

SIM Agar Medium Composition:

Beef extract    3.0 g

Peptone  30.0 g

Ferrous ammonium sulphate   0.2 g

Sodium thiosulphate 0.025 g

Agar   3.0g

Final pH ( at 25°C) 7.3±0.2

Distilled water    1000ml