Biochemical Test

Sulfur Reduction Test Principle, Purpose, Procedure, Result

Most of the time, the Sulphur Reduction test is done in the Sulphide Indole Motility medium, which is a mix of different...

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Sulfur Reduction Test Principle, Purpose, Procedure, Result
Sulfur Reduction Test Principle, Purpose, Procedure, Result
  • Most of the time, the Sulphur Reduction test is done in the Sulphide Indole Motility medium, which is a mix of different mediums that tests three different things: Sulphur Reduction, indole production, and motility.
  • This test, as the name suggests, is often used to see if a microbe can make the gas hydrogen sulphide.
  • SIM medium (Sulfide Indole Motility medium) is a blend differential medium that tests three different limits: sulphur reduction, indole production, and mobility.
  • As the name suggests, it is most often used to see if an organism can make hydrogen sulphide gas (H2S). The letter “S” in SIM stands for sulphur.
  • The sulphur reduction test can help tell different types of organisms that live in the gut apart. The indole test is part of a group of tests called the IMViC series, which is used to tell the Enterobacteriaceae apart. The motility test can be used to test a wide range of living things. Overall, the SIM test is most helpful for telling the difference between Salmonella and Shigella.
  • The SIM medium has iron and sodium thiosulfate in it. Peptone, which has amino acids like tryptophan, is one of the nutrients.
  • If an organism can turn sulphur into hydrogen sulphide, the hydrogen sulphide will combine with iron to make ferric sulphide, which is a black precipitate. If the medium gets darker, it means that the amount of sulphur has gone down, which is a good thing.
  • Before you do the indole test, you should know the results of the sulphur and motility tests.
  • Some bacteria can make the enzyme tryptophanase, which breaks down the amino acid tryptophan. By deamination, the end products of this hydrolysis are indole, pyruvic acid, and ammonia.
  • Hydrochloric acid, p-dimethylaminobenzaldehyde (DMABA), and n-amyl alcohol are all in the Kovac’s reagent, which you add to the SIM medium to test for indole.
  • When DMABA is mixed with indole, a red quinoidal compound is made. The indole test is positive if the reagent turns red.

Objective of Sulfur Reduction Test

  • A method for determining an organism’s sulfur-reducing potential.
  • This study aimed to distinguish gram-negative enteric bacilli based on their sulphide producing capabilities.

Principle of Sulfur Reduction Test

  • SIM media is used to conduct the Sulfur Reduction Test.
  • Both indole synthesis and motility can be evaluated with SIM medium.
  • This semi-solid medium contains casein and animal tissue for amino acids, as well as a compound containing iron and sodium thiosulfate for sulphur.
  • Indicators for hydrogen sulphide generation are present in the media in the form of ferrous ammonium sulphate and sodium thiosulfate.
  • When ferrous ammonium sulphate reacts with H2S gas, a black precipitate called ferrous sulphide is formed, which can be used as a detectable indicator of hydrogen sulphide production.
  • Anaerobic sulphur reduction to H2S can be achieved in two distinct ways by bacteria, depending on the enzymes available.
  • Cysteine desulfurase, an enzyme involved in the catabolism of the amino acid cysteine, and thiosulfate, a byproduct of aerobic respiration, are both capable of reducing sulphur to H2S (hydrogen sulphide).
  • Whenever hydrogen sulphide is formed, the medium turns black to show that the reaction was successful.
  • If the media does not become black, it means the organism was unable to reduce the sulphur present in the medium and the test was therefore negative.

SIM Agar Composition and Preparation

ComponentsAmount (Composition per liter)
Pancreatic digest of casein20.0g
Peptic digest of animal tissue6.1g
Agar3.5g
Fe(NH4)2(SO4)2·6H2O0.2g
Na2S2O3·5H2O0.2g

pH 7.3 ± 0.2 at 25°C

About the SIM Agar 

  • It’s a differential media, to put it simply. Sulfur reduction, indole production, and agar swimming are all put to the test (be motile). SIM has become a standard method for distinguishing between different types of Enterobacteriaceae.
  • Cysteine desulfurase, an enzyme involved in the catabolism of the amino acid cysteine, and thiosulfate, a byproduct of aerobic respiration, are both capable of reducing sulphur to H2S (hydrogen sulphide). Hydrogen sulphide produces a dark hue in its surroundings. As for H2S production, Proteus mirabilis scores high. The far left bacterium in this image produces hydrogen sulphide.
  • Tryptophane, an amino acid, can be converted to indole by bacteria harbouring the enzyme tryptophanase. When Kovac’s reagent is added to indole, a red dye called rosindole is produced (indole +). The bacterium Escherichia coli tests positive for indole. E. coli, the bacteria seen second from the left, is indole positive.
  • An individual stab in the base of a SIM tube is all that is needed to inoculate the tube. The entire tube will appear cloudy if the organism growing within is mobile; the growth will spread outward from the point of stab. Both Pseudomonas aeruginosa and the Proteus mirabilis strain we use are able to move around on their own.

Sulfur Reduction Test Procedure

  1. At first, a straight needle is used to get a culture that has been growing for 18 to 24 hours.
  2. Stabbing the organism into the SIM medium in a labelled tube is one way to start it.
  3. Keep the tubes that have been seeded at 37°C for 24–48 hours.
  4. Keep an eye out for black particles to fall onto the medium.

Expected Results of Sulfur Reduction Test 

  • Positive: The presence of bacteria that make hydrogen sulphide is shown by a darkening of the medium (a black precipitate) or a darkening of the line of inoculation.
  • Negative: If there is no blackening, the H2S test is negative. 
Expected Results of Sulfur Reduction Test 
Expected Results of Sulfur Reduction Test Before the Kovac’s reagent has been added
Expected Results of Sulfur Reduction Test 
Expected Results of Sulfur Reduction Test With the Kovac’s reagent added

Quality Control

  • Salmonella enterica ATCC14028: has a black line along the stab line and is positive for H2S.
  • Escherichia coli ATCC25922: has no H2S and no black along the stab line.
  • Morganella morganii ATCC 25830: no H2S, no black colour along the stab line.

Sulfur Reduction Test Uses

  • It is used to tell the difference between Moranella morganii and Providencia rettgeri, which don’t reduce sulphur, and Salmonella, Shigella, and Proteus, which do.
  • The ability of bacteria to make hydrogen sulphide is a good way to tell if they are enteric or not.

Sulfur Reduction Test Limitations

  • It’s crucial to remove the inoculating needle from semi-solid media in the same direction that it was inserted. A false-positive reading could occur if growth along the stab line was stimulated by a fanning action.
  • Prior to adding Kovacs Reagent, findings for motility and H2S must be interpreted.
  • SIM Medium’s responses are insufficient for species differentiation. Confirmation requires additional biochemical and serological testing.

References

  • 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
  • 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.
  • https://microbiologyinfo.com/sulphur-reduction-test/
  • https://researchtweet.com/sulphur-reduction-test-principle-procedure-result/
  • http://www.uwyo.edu/molb2021/additional_info/summ_biochem/sim.html
  • https://www.austincc.edu/microbugz/sim_medium.php
  • https://www.studocu.com/in/document/rajiv-gandhi-university-of-health-sciences/general-microbiology-and-immunity/sulfur-reduction-test-useful-notes/32394261
  • https://www.sciencedirect.com/topics/earth-and-planetary-sciences/sulphur-reduction
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