MEDIUM SIM: FOUNDATION, PREPARATION AND USES - BIOLOGY - 2025

The SIM medium is a semisolid and differential agar, specially designed to help identify some bacteria, especially Enterobacteriaceae. It is composed of triptein, peptone, iron sulfate, ammonium sulfate, sodium thiosulfate, and agar.

This medium allows the execution of three important tests: the production of hydrogen sulfide (H ₂ S), the formation of indole and motility, hence the acronym SIM. Due to its great utility, it cannot be absent in a bacteriology laboratory.

A. Commercial SIM medium B. SIM H2S (-), Indole (-) and Motility (+) Test and SIM H2S (+), Indole (-), Motility (+) Test. Source: A. Photo taken by the author MSc. Marielsa Gil B. Mfloayza

Unlike other media, it must be semi-solid in order for the movement capacity of some bacteria to be detectable. In this sense, this test works very well for Enterobacteriaceae, but not in non-fermenting Gram-negative rods, where other methods are preferred, such as hanging drop.

The SIM medium allows to distinguish certain specific properties that characterize some bacteria in relation to others. For example, Escherichia coli is distinguished by being H ₂ S (-), Indole (+) and motility (+), while Proteus mirabilis is H ₂ S (+), indole (-), motility (+).

Basis

It is a culture medium that is considered differential, because its use distinguishes between microorganisms capable of producing hydrogen sulfide from those that do not; it also highlights those that form indole from tryptophan from those that do not, and finally differentiates motile bacteria from immobile ones.

Power source

Like any culture medium, it has elements that provide the necessary nutrients so that non-demanding microorganisms can develop. These elements are represented by peptones and triptein.

The development of the microorganism in the medium is essential to be able to observe the presence or absence of the characteristics that this medium evaluates.

Production of hydrogen sulfide

The letter S of the acronym SIM refers to the production of hydrogen sulfide (H ₂ S). Bacteria capable of forming hydrogen sulfide will take up the sulfur from sodium thiosulfate.

Once the H ₂ S -colourless gas- is formed, it reacts with the iron salt present in the medium, forming ferrous sulfide, clearly visible (black precipitate). Bacteria that do not form H ₂ S, leave the medium of the original color (beige).

The presence of the black precipitate can hinder the interpretation of motility. However, most H ₂ S- producing Enterobacteriaceae are known to be positively motile, such as Salmonella, Proteus, and Citrobacter. Furthermore, the black precipitate that covers almost the entire medium suggests positive motility.

Indole formation

The second letter of the acronym SIM is "I", which represents the formation of indole.

In this sense, triptein, in addition to being a source of nutrient, fulfills another fundamental function. This peptone is rich in an amino acid called tryptophan, therefore, it can show bacteria that produce tryptophanase.

This enzyme is responsible for cleaving the amino acid tryptophan, with the consequent formation of indole (colorless substance), pyruvic acid and ammonium.

That is why, to show this reaction, it is necessary to add a revealing substance (Ehrlich's reagent or Kovac's reagent). Either reacts with indole, forming a red-fuchsia ring-shaped substance on the surface of the agar. If the fuchsia ring appears, the indole test is interpreted as positive.

Bacteria that do not possess this enzyme will not form the ring and it is interpreted as a negative indole test.

It is important to note that the indole test should be the last to be interpreted, since once the reagent is added, the medium becomes cloudy, making it difficult to visualize motility.

Motility

Finally the letter "M" of the word SIM means motility. In order to evaluate motility, this medium is strategically semi-solid, as this characteristic is essential to be able to observe whether or not there is bacterial movement. Bacteria that possess flagella are those that give this positive test.

A positive test will be evident when turbidity is observed, both in the initial inoculum, and around it. Whereas, nonmotile bacteria only develop in the path of the initial inoculum.

Preparation

Medium SIM

Weigh 30 g of the dehydrated medium and dissolve in a liter of distilled water. The mixture is left to stand for 5 minutes and then heated to boiling, stirring frequently until completely dissolved.

Distribute the mixture in test tubes with cotton caps and autoclave at 121 ° C for 15 minutes. Remove the tube rack from the autoclave and allow to solidify in a vertical position, so that the medium is in the shape of a block.

For its conservation it is kept in the refrigerator until its use. The prepared medium must have a final pH of 7.3 ± 0.2.

At the time of inoculating the medium, it must be at room temperature. The middle color is beige.

Kovac's reagent

Measure 150 ml of amyl or isoamyl or butyl alcohol. (Use one of the three mentioned).

Dissolve 10 g of p-dimethylaminobenzaldehyde. Then slowly add 50 ml of concentrated hydrochloric acid.

The ready-to-use reagent is colorless or light yellow. It should be kept in an amber bottle and stored in a refrigerator. Do not use if it turns dark brown; that indicates that it is damaged. This reagent is preferred when it comes to Enterobacteriaceae.

Erlich's reagent

Weigh out 2 g of p-dimethylaminobenzaldehyde and dissolve in 190 ml of absolute ethyl alcohol and mix slowly with 40 ml of concentrated hydrochloric acid. Store in the same way the Kovac's reagent. Ehrlich's reagent is used most for non-fermenting and anaerobic bacteria.

Applications

SIM medium is highly used in bacteriology laboratories. Its advantage is that three essential characteristics can be observed in the same tube in the identification of Enterobacteriaceae.

Sown

The correct way to sow this medium is using the needle, with which a portion of the pure colony to be studied is taken and inserted vertically into the center of the medium. A single lunge should be performed. The puncture should not reach the bottom of the tube, the correct thing is to cover only two thirds of the depth.

Repeating the inoculum is not recommended, as this can lead to false interpretations of positive motility. The inoculated medium is incubated aerobically at 37 ° C for 24 hours.

Once the time has concluded, it is observed whether or not there was production of H ₂ S and the motility is read. Finally the indole is revealed, adding 3 to 4 drops of the Ehrlich or Kovac's reagent, mix gently and interpret.

Source: Koneman E, Allen S, Janda W, Schreckenberger P, Winn W. (2004). Microbiological Diagnosis. 5th ed. Editorial Panamericana SA Argentina.

QA

As a sterility control, one or two tubes are incubated without inoculation in an oven at 37 ° C for 24 hours. It is expected that after this time there is no growth or color change.

As quality control, known certified strains can be used, such as: Escherichia coli ATCC 25922, Enterobacter aerogenes ATCC 13048, Klebsiella pneumoniae ATCC 13883, Salmonella typhimurium ATCC 14028, Shigella sonnei ATCC 29930, Proteus vulgaris ATCC 13315.

The expected results are: Escherichia coli H ₂ S negative, indole and positive motility, Enterobacter aerogenes only positive motility, Salmonella typhimurium H ₂ S and positive motility, with negative indole. Proteus vulgaris all positive, while Klebsiella pneumoniae and Shigella are all negative.

Limitations

-Some strains of Morganella morganii, among other strains, can produce a brownish pigment in this medium due to the production of melanin, this should not be confused with the precipitate of ferrous sulfide. In inexperienced professionals this situation can generate false positives in the interpretation of the H ₂ S test.

-Strict aerobic bacteria will only grow on the surface of the tube, making it difficult to interpret motility.

References

1. BD Laboratories. BBL SIM Medium. 2008. Available at: bd.com
2. Neogen Laboratories. SIM Medium. Available at: foodsafety
3. Difco Francisco Soria Melguizo. SIM Medium. 2009.Available at:
4. Brizuela-Lab Laboratory. Medium SIM. Available at:.brizuela-lab.com
5. Britannia Laboratories. Medium SIM. 2015.Available at: studyres.es/doc
6. Koneman E, Allen S, Janda W, Schreckenberger P, Winn W. (2004). Microbiological Diagnosis. 5th ed. Editorial Panamericana SA Argentina.