Lactobacillus MRS Broth Test – Principle, Procedure, and Results

• The MRS formulation was made by de Man, Rogosa, and Sharpe to replace a variable product (tomato juice) and give lactobacilli in general a good place to grow.
• So, MRS agar and broth are often used to grow and identify Lactic acid bacteria, especially Lactobacillus spp., because they are the best.
• “Lactic acid bacteria” includes species from the genera Lactobacillus, Streptococcus, Pediococcus, and Leuconostoc. All of these species can make large amounts of lactic acid.
• They are Gram-positive, negative for catalase and oxidase, and very picky about what they eat. Conditions with very little oxygen help growth a lot.
• Lactobacilli tend to grow best in MRS medium, but leuconostocs and pediococci may also grow. Changing the pH can change how selective something is. Lactobacilli can live in pH levels lower than streptococci (5.0–6.5), which is where pediococci and leuconostocs grow best.

Principle of MRS Broth Test

• MRS Broth is a better medium for lactobacilli. It helps them grow well and is especially useful for a number of fussy strains that don’t grow well on other general media. 
• Organisms that were found on MRS Agar can be used to test the medium. 
• It has an enzyme digest of animal tissue, beef extract, and yeast extract. These are the sources of carbon, nitrogen, and vitamins that MRS Broth medium uses to meet the general needs of growth. 
• The fermentable carbohydrate that is in the medium is dextrose. 
• Sodium acetate in broth slows down the growth of bacteria. Sodium acetate and ammonium citrate are both energy sources and selective agents that stop organisms from growing too much and causing an overgrowth of other organisms. 
• The buffering agent is called potassium phosphate. 
• Magnesium sulphate and manganese sulphate both contain cations, which are needed for metabolism. 
• Polysorbate 80 is a surfactant that helps lactobacilli take in nutrients. 
• Bubbles on the Durham tube in the broth medium show that gas is being made when sugar is fermented. 
• Turbidity on the medium shows that the organism is growing.
• When an organism is put into the broth medium, those that can ferment the dextrose sugar, get past the selective agents, and use the other nutrients show a lot of growth, which is a good sign. 
• Some organisms may also be able to make gas, which can be used to tell them apart from the rest. This is done with a Durham tube (eg. Leuconostoc sp. from Lactobacillus spp.).

Objectives of MRS Broth Test

• It is used in a lab to grow lactobacilli and count how many of them there are.
• To find out if an organism can make gas when it breaks down glucose.

Requirement

• Sterile sticks
• Incubator
• Gas detection options
  • Vaspar, liquid paraffin, or petroleum jelly, maintained at 56°C in liquid form.
  • Durham tube
• Test organism: Gram-positive coccobacilli that are catalase-negative, PYR-negative, vancomycin-resistant, and grow aerobically.
• MRS Broth

MRS Broth Composition

Final pH 6.5 +/- 0.3 at 25ºC.

MRS Broth Test Procedure

Plate Preparation

1. 52.25 gms of the medium are dissolved in one litre of distilled water. Mix and dissolve the ingredients by heating them and stirring them frequently.
2. Boil the mixture for one minute, or until it is all dissolved. Fill the right containers with the mixture and sterilise them for 12 minutes in an autoclave set to 121°C. Keep the prepared medium between 2°C and 8°C.
3. The colour is a bright yellow-orange. The dried medium should be beige, uniform, and easy to move around. If the medium changes in any way, it should be thrown away.

Procedure

1. Add one or two colonies from a blood agar plate culture that has been going for 18 to 24 hours.
2. Add a Durham tube. If you don’t have a Durham tube, cover the inoculated MRS broth with a plug of melted Vaspar or petroleum jelly if you don’t have a Durham tube. Cover the whole broth layer without letting air in.
3. Incubate at 35°C to 37°C in room temperature air for 24 to 48 hours. Check every day to see if there is gas in the Durham tube or solid plug.

Result of MRS Broth Test

• Positive:  Leuconostoc spp. grows, and the trapped gas bubble in the Durham tube shows that gas is being made. If a vaspar plug is used instead of a Durham tube, the plug can be seen to lift up and move away from the surface of the broth.
• Positive: Lactobacillus spp. has grown; there is no gas bubble in the Durham tube and the wax plug hasn’t moved.
• Negative: No growth (not shown).

Quality Control of MRS Broth Test

Before using each new lot or shipment of media, do quality control (QC) by using both positive and negative control. Check to see if the broth is clear. If there is a Durham tube and it has a bubble in it, turn it upside down.

• Positive  (growth with no-gas production): Lactobacillus lactis (ATCC19435)
• Positive  (growth with gas production):  Leuconostoc mesenteroides ATCC 10830
• Negative (no growth, no gas production): Enterococcus faecalis ATCC 29212

Limitations of MRS Broth Test

• Some strains may not grow well or at all on this medium because they need different amounts of nutrients.
• Lactobacilli are not the only thing that can grow in this medium. Isolates must be tested biochemically to make sure they are lactobacilli.

Uses of MRS Broth Test

• It’s used to figure out which types of Lactobacillus spp. and Leuconostoc spp. make gas.
• The test is done to confirm that organisms found on MRS Agar are what they are supposed to be.
• This test is often used to find Lactobacilli in the mouth, in dairy products, foods, faeces, and other places.

References

• https://www.itwreagents.com/download_file/product_infos/413785/en/413785_en.pdf
• 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
• Merck Microbiology Manual. 12th Edition. MRS broth.
• http://foodsafety.neogen.com/pdf/acumedia_pi/7406_pi.pdf
• https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/LactobacilliMRSBroth.html
• www.labm.com/products/m-r-s-broth.asp
• www.scharlabmagyarorszag.hu/katalogus/02-135_TDS_EN.pdf
• https://www.bd.com/europe/regulatory/Assets/IFU/Difco_BBL/288110.pdf