Lactobacillus MRS Broth test is used to test gas production by Gram-positive rods in a glucose-containing medium. MRS is an acronym for the authors of this publication (De Mann JD, Rogosa, and Sharpe), who suggested this medium for the cultivation of lactobacilli.
MRS broth test determines whether gram-positive bacilli form gas during glucose fermentation. MRS broth test is helpful to differentiate Leuconostoc spp., which produces gas from Lactobacillus spp, which is gas negative. Weissella confusa is also a gas producer, whereas streptococci and Pediococcus do not produce gas.
Table of Contents
Principle
MRS broth is a selective medium that uses sodium acetate and ammonium citrate to prevent overgrowth by contaminating organisms. It contains sources of carbon, nitrogen, and vitamins to support the growth of lactobacilli and other gram-positive organisms.
A: positive with gas; B: Positive but no gas. (Image source: Ref-1)
Growth is considered a positive result. A Durham tube can be added to the MRS broth to detect gas production. A rise in a petroleum jelly plug or displacement of broth with air in the Durham tube suggests gas production. Ordinary sugar fermentation tubes cannot detect these organisms because of a lack of sufficient gas production.
Materials
- 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
| Ingredients | Amount (Gram/liter) |
| Enzymatic digest of animal tissue | 10 g |
| Beef extract | 10 g |
| Yeast extract | 5 g |
| Dextrose | 20 g |
| Polysorbate 80 | 1 g |
| Ammonium citrate | 2 g |
| Sodium acetate (NaC2H3O2) | 5 g |
| Magnesium sulfate (MgSO4) | 0.1 g |
| Manganese sulfate (MnSO4) | 0.05 g |
| Dipotassium phosphate | 2 g |
| Agar | 15 g |
| Final pH: 6.5 |
Quality Control
Perform quality control (QC) on each new lot or media shipment, using positive and negative control before using them. Inspect the broth for lack of turbidity. Invert if there is a Durham tube and it contains a bubble.
- 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
Method
- Inoculate MRS broth lightly with one or two colonies from an 18 to 24-hour culture from a blood agar plate.
- Add a Durham tube.
If a Durham tube is not used, overlay the inoculated MRS broth with a plug of melted Vaspar or petroleum jelly. Cover the broth layer entirely without introducing air.
- Incubate for 24 to 48 hours at 35°C to 37°C in ambient air for up to 7 days.
Observe daily for gas trapped in the Durham tube or solid plug.
Results
- Positive: Leuconostoc spp. growth; gas production indicated by the trapped gas bubble in the Durham tube. Suppose vaspar plug is used instead of Durham tube, visible lifting of the plug and its complete separation from the broth surface is seen.
- Positive: Lactobacillus spp.—growth; no gas bubble in the Durham tube or no lifting of the wax plug.
- Negative: No growth (not shown).
Limitations
Since strains of Leuconostoc produce copious amounts of gas, the Durham tube is a safer alternative to petroleum jelly. However, the Durham tube method does not work as well.
References and further readings
- Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition
- Andrea J. Linscott, 2016. Clinical Microbiology Procedures Handbook, 4th Edition. ASM Press, Washington, DC. doi: 10.1128/9781683670438.