Sulphur Reduction Test Introduction

Many of the biochemical tests are performed in order to distinguish the different species of bacteria, based on their enzymatic activity. One such biochemical test is Sulphur reduction test.

These biochemical tests play an important role in identifying the microbes. These tests are performed based on the fermentation activity of the certain substances such as carbohydrates.

In recent times, Microbial tests performed biochemically shortens the time to identify the certain microbes. It can also be performed economically with less cost and it also has the accuracy in its results.

What is Sulphur Reduction Test?

Sulphur reduction test is usually performed in the Sulphide Indole Motility medium, which is a conjugation of different medium which tests the three variety of parameters such as Sulphur Reduction, indole production and the motility.

As the name says, this test is commonly used to test a microbe for the ability to synthesize the gas hydrogen sulfide.

Sulphur Reduction Test Objective

 The aim of the test is to detect the ability of the organism to reduce Sulphur.

 To differentiate the species, of gram-negative enteric bacilli based on their production of sulphide.

Sulphur Reduction Test Principle

Here, the organism that produces enzyme thiosulfate reductase, which reduces the Sulphur into hydrogen sulfide gas.

This occurs when strain degrades the amino acid cysteine while the degradation process of protein or during the process of protein degradation or during the process of anaerobic respiration, which shuttles the electrons to Sulphur instead of oxygen.

In Sulphur indole motility tubes, the medium contains casein and the animal proteins as the source of amino acid, sodium thiosulphate, as a source of sulfur and the ferrous ammonium sulphate, which acts as the hydrogen sulphide indicator.

Cysteine is a Sulphur containing amino acids that is present in the SIM medium.

The enzyme cysteine desulfurizes and thiosulfate reductase catalysis the reactions of hydrolysis and it produces hydrogen sulphide.

Thus, the produced hydrogen sulphide gas, combines with the ferrous ammonium sulfates and forms an insoluble, black color ferrous sulfide precipice.

This black color acts as an indicator during the presence of a hydrogen sulphide.

In the laboratory, a fresh medium of the cultured organisms is inoculated in the center of the medium in a single stab with the help of a straight needle.

After the process of incubation, the tube is further observed for the production of hydrogen sulphide gas, which is observed by the blackening of the medium.

SIM medium is generally used for this Sulphur reagent test, it consists of nutrients, iron and sodium thiosulphate.

One of the vital nutrient present in the medium is peptone, which is made up of amino acids like those of tryptophan.

However, if the organism has the capability to reduce Sulphur into hydrogen sulfide, the reduced hydrogen sulphide will combine with the iron and results in the formation of the ferric sulphide, which is shown as a black precipitate.

This is the reason for blackening of the medium and it indicates the reduction of the Sulphur in the medium and gives a positive result.

Sulphur Reduction Test Reagent

SIM Agar: For preparation of a Sulphide, indole moetilty agar, the pancreatic digest of casein is taken into about 20-gram, peptic digest of an animal tissue about 6 grams, agar-3.5 grams.

Sulphur Reduction Test Procedure

Initially an 18-to-24-hour culture is collected using a straight needle. Here the stab is taken in a single depth of about half an inch from the middle of the tube.

Then it is incubated at a temperature of about 35 to 37ºC and it is observed regularly for a maximum of 7 days. Blackening of the medium is observed, it is noted on the line of inoculation.

Sulphur Reduction Test

Positive Results: It results in darkening of the medium, where black precipitate is seen, or in some cases, it can also be seen in blackening of the line in the inoculation which indicates the presence of certain bacteria that produces hydrogen sulphide gas.

Negative Result: Negative result is often indicated by lack of black precipitate in the medium.

Sulphur Reduction Test Uses

 This test is often used to differentiate the Sulphur reducing members of the family Salmonella shigella and the Genera of protists from the species such as Negative Moranella morganii and providencia rettgeri.

 This test can also be used for producing the hydrogen sulfide which can be used for diagnosing and identifying the enteric bacteria.

Sulphur Reduction Test Limitation

 Although, there are many advantages of this test, there are some limitations such that the reaction given by the SIM medium is not enough to speciate the organism, hence there is a need of additional biochemical and serological tests.

 It should also be noted that inoculum is taken from the solid medium.

 As taking an inoculum from a liquid or a broth suspension delays the initiation of the growth process, which also leads to the errors in the results.