Sulfur Reduction Test- Principle, Procedure, Results, Uses

Objectives of Sulfur Reduction Test

• To determine the ability of the organism to reduce sulfur for the production of hydrogen sulfide.
• To differentiate members of Enterobacteriaceae, especially the sulfur-reducing Salmonella and Proteus from the non-reducing Morganella morganii and Providencia rettgeri.

Principle of Sulfur Reduction Test

The Sulfur Reduction Test is performed using the SIM medium. SIM medium also tests for indole production and motility. It is a semi-solid medium that is formulated with casein and animal tissue as sources of amino acids, an iron-containing compound, and sulfur in the form of sodium thiosulfate. The medium contains ferrous ammonium sulfate and sodium thiosulfate, which together serve as indicators for the production of hydrogen sulfide. Hydrogen sulfide production is detected when ferrous sulfide, a black precipitate, is produced as a result of ferrous ammonium sulfate reacting with H₂S gas. Sulfur reduction to H2S is an anaerobic activity and can be accomplished by bacteria in two different ways, depending on the enzymes present. Sulfur can be reduced to H₂S (hydrogen sulfide) either by catabolism of the amino acid cysteine by the enzyme cysteine desulfurase or by reduction of thiosulfate in anaerobic respiration. If hydrogen sulfide is produced, a black color forms in the medium, indicative of the positive reaction. No black color formation on the medium is indicative of negative test results implying that the organism is not capable of reducing the sulfur on the medium.

Culture Media (SIM Medium)

Composition per liter:

Pancreatic digest of casein…………………………..20.0g

Peptic digest of animal tissue………………………..6.1g

Agar…………………………………………………………..3.5g

Fe(NH4)2(SO4)2·6H2O………………………………….0.2g

Na2S2O3·5H2O…………………………………………….0.2g

pH 7.3 ± 0.2 at 25°C

Procedure of Sulfur Reduction Test

1. Inoculate the organism into a labeled tube by means of stab inoculation in the SIM medium.
2. Incubate the inoculated tubes at 37°C for 24-48 hours.
3. Observe for the formation of black precipitate on the medium.

Result of Sulfur Reduction Test

• Positive result: blackening on the medium
• Negative result: no blackening on the medium

Limitations

• When inoculating semi-solid media, it is important that the inoculating needle is removed along the exact same line used to inoculate the medium. A fanning motion may result in growth along the stab line that may result in false-positive interpretation.
• Motility and H₂S results must be interpreted prior to the addition of Kovacs Reagent.

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Quality Control

Salmonella enterica ATCC14028: H2S positive, black color along the stab line
Escherichia coli ATCC25922: H2S negative, no black color along the stab line
Morganella morganii ATCC 25830: H2S negative, no black color along the stab line

References

1. Tille P.M (2014)Bailey and Scott’s diagnostic microbiology, Thirteen edition, Mosby, Inc., an affiliate of Elsevier Inc., 3251 Riverport Lane, St. Louis, Missouri 63043
2. Aneja K.R (2003), Experiments in Microbiology, Plant Pathology and Biotechnology, fourth revised edition, New Age International (P) limited, Ansari road, Daryaganj, New Delhi-110002.