AGAR MRS: FOUNDATION, PREPARATION AND USES - BIOLOGY - 2025

The agar MRS is a selective solid medium culture used for the isolation and enumeration of lactic acid bacteria, especially of the genus Lactobacillus. This agar was created in 1960 by Man, Rogosa and Sharpe, bearing the same name, but due to its complexity, the abbreviation MRS is often used.

It is composed of proteose peptone, meat extract, yeast extract, glucose, sorbitan monoleate, dipotassium phosphate, sodium acetate, ammonium citrate, magnesium sulfate, manganese sulfate, and agar.

Man Rogosa and Sharpe Agar (MRS) seeded with Lactobacillus sp. Source: Photo donated for this article by the authors: Báez E, González G, Hernández G, López E, Mega M. Photo taken during the investigation entitled: Evaluation of lactic acid bacteria and Bifidobacteria with probiotic potential in breast milk and infant stools.

This composition allows the proper development of lactic acid bacteria from clinical samples, such as feces, vaginal discharge, oral samples and breast milk, as well as dairy and meat foods.

It is not used routinely in clinical laboratories, because lactic acid bacteria are rarely involved in disease processes. However, in the area of ​​food microbiology, the use of MRS agar is more frequent.

On the other hand, this medium is used by some Research Centers whose objective is the study of lactic acid bacteria.

Basis

Man, Rogosa and Sharpe agar has a fairly complex composition. By breaking down the function of each of its components, its foundation can be explained.

Proteose peptone, meat extract, yeast extract and glucose are nutrients that provide the source of carbon, nitrogen, vitamins and minerals necessary for bacterial growth. Furthermore, glucose is the universal energy source used in most culture media.

On the other hand, to favor the growth of lactic acid bacteria, the presence of cofactors (cations) essential in the metabolism of Lactobacillus and related bacteria is necessary; These compounds are the sodium, magnesium, and manganese salts.

Likewise, sorbitan monoleate or polysorbate 80 are an important source of fatty acids as they are absorbed as nutrients.

Furthermore, sorbitan monoleate and ammonium citrate act by inhibiting the development of the accompanying flora, especially Gram negative bacteria, providing the selective character of this agar.

Finally, the agar-agar is the one that provides the solid consistency to the medium.

There are other variants of Man Rogosa Sharpe agar; one of them is supplemented with cysteine ​​(MRSc), very useful for the isolation of bifidobacteria, among other microorganisms. On the other hand, there is the MRS medium supplemented with neomycin, paromomycin, nalidixic acid and lithium chloride, special for the selective counting of bifidobacteria in dairy products.

Preparation

Weigh out 68.25 grams of the dehydrated medium and dissolve in one liter of distilled water. Let stand for 5 minutes. To dissolve completely, turn to a heat source, stirring frequently, and boil for 1 to 2 minutes. Sterilize in an autoclave at 121 ° C for 15 minutes.

When leaving the autoclave, allow to stand for a few minutes and distribute while still hot in sterile Petri dishes.

Allow to solidify and invert plates, order in plate racks and refrigerate until use. Allow plates to warm to room temperature before use.

The pH of the medium should be 6.4 ± 0.2. Some commercial houses recommend pH between 5.5 to 5.9.

The dehydrated medium is beige in color and prepared is dark amber.

Both the dehydrated medium and the prepared plates should be stored at 2-8 ° C.

Applications

MRS agar plates can be surface-seeded (exhaustion or Drigalski spatula). It can also be sown by depth. Plates should be incubated at 37 ° C in microaerophilicity (4% O ₂ and 5-10% CO ₂) for 24 to 72 hours.

The sowing method is chosen according to the purpose pursued (isolation or counting).

Characteristics of the colonies

The presumptive colonies of Lactobacillus grow whitish in color and have a mucoid or creamy appearance on this agar. They must subsequently be identified.

Isolation of lactic acid bacteria

For this, surface seeding is used. The samples to be sown require a previous procedure.

In the case of breast milk samples, it is recommended to centrifuge 1 ml of the sample at 14,000 rpm for 10 minutes, in order to remove the fatty layer. 900 µl is discarded, and in the remaining 100 µl the sediment is suspended and poured on the surface of the MRS agar. It should then be distributed evenly with a Drigalski spatula.

In the case of stool samples, one (1) gram of stool is weighed and homogenized in 9 mL of 0.1% sterile peptone water, corresponding to a 1/10 dilution. Then serial dilutions are made, until a final dilution of 10 ^{-4 is obtained}.

Finally, 100 µl of the 10 ^-2, 10 ^-3 and 10 ^-4 dilutions are taken and each dilution is seeded on MRS agar, distributing evenly with a Drigalski spatula.

Lactic acid bacteria count

In this case the sowing is done by depth.

For breast milk samples, 1 mL is taken and placed in a sterile conical plastic tube. MRS agar is added at an approximate temperature of 40 ° C to a final volume of 25 mL, obtaining a homogeneous mixture. Subsequently, it is poured into sterile Petri dishes in a uniform manner and allowed to stand until polymerization.

For stool samples, dilutions are made, as previously described. Take 1 mL of each dilution and place it in sterile conical plastic tubes. Molten MRS agar is added to a volume of 25 mL.

The mixture of each dilution is poured evenly into sterile Petri dishes. Finally, it is left to rest until polymerization.

Colony count on Man Rogosa and Sharpe (MRS) agar. Source: Photo donated for this article. Authors: Báez E, González G, Hernández G, López E, Mega M. Photo taken during the Research entitled: Evaluation of lactic acid bacteria and Bifidobacteria with probiotic potential in breast milk and infant feces.

Research level

Every day the study of lactic acid bacteria is gaining more interest; Researchers especially seek to learn about new strains and their potential as starter ferments for standardization in the manufacture of dairy products, among other uses.

In this sense, Alvarado et al. (2007) used MRS agar to carry out a study in which they isolated, identified and characterized lactic acid bacteria present in an artisan Andean smoked Venezuelan cheese.

In the cheese they found the presence of bacteria of the Lactococcus and Lactobacillus genera, and concluded that the mixtures of isolated strains are suitable as starter strains in the manufacture of cheeses from pasteurized milk.

On the other hand, Sánchez et al. (2017) used MRS agar to investigate the presence of lactic acid bacteria in the digestive tract of piglets, in order to use them as native probiotics that increase the productivity of healthy piglets.

With this medium they managed to isolate four species: Lactobacillus johnsonii, Lactobacillus brevis, Enterococcus hirae and Pediococcus pentosaceus.

Likewise, Báez et al. (2019) used MRS agar to evaluate lactic acid bacteria (LAB) and bifidobacteria with probiotic potential in breast milk and infant feces.

They managed to isolate 11 BAL and 3 Bifidobacteria sp in breast milk, and 8 BAL and 2 Bifidobacteria sp. in feces. All met certain parameters that prove them as bacteria with probiotic activity.

The authors concluded that both breast milk and feces from exclusively breastfed infants serve as natural sources of probiotic bacteria.

QA

To evaluate the quality of MRS agar, control strains such as:

Lactobacillus fermentum ATCC 9338, Lactobacillus casei ATCC 393, Bifidobacterium bifidum ATCC 11863, Lactobacillus plantarum MKTA 8014, Lactobacillus lactis MKTA 19435, Pediococcus damnosus MKTA 29358, Escherichia coli and Bacillus cerei.

The expected results are satisfactory growth for the first 6 bacteria, while E. coli and Bacillus cereus must be completely inhibited.

References

1. Alvarado C, Chacón Z, Otoniel J, Guerrero B, López G. Isolation, Identification and Characterization of Lactic Acid Bacteria from a Venezuelan Smoked Andean Artisan Cheese. Its Use as a Starter Culture. Cient. (Maracaibo) 2007; 17 (3): 301-308. Available at: scielo.org.
2. Sánchez H, Fabián F, Ochoa G, Alfaro Isolation of Lactic Acid Bacteria from the Digestive Tract of the Piglet. Rev. investiga. vet. Peru 2017; 28 (3): 730-736. Available at: scielo.org.
3. Báez E, González G, Hernández G, López E, Mega M. Evaluation of lactic acid bacteria and Bifidobacteria with probiotic potential in breast milk and feces of infants in the Acevedo municipality, Miranda 2017. Undergraduate work to qualify for the Bachelor's degree in Bioanalysis. University of Carabobo, Venezuela.
4. Britannia Laboratory. MRS agar. 2015.Available at: britanialab.com
5. Wikipedia contributors. MRS agar. Wikipedia, The Free Encyclopedia. January 10, 2018, 19:44 UTC. Available at: wikipedia.org Accessed February 17, 2019.
6. Roy D. Media for the isolation and enumeration of bifidobacteria in dairy products. Int J Food Microbiol, 200128; 69 (3): 167-82.