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Dehydrated Culture Media

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MRS (ISO) AGAR (DE MAN, ROGOSA and SHARPE)

Code: CM1153

An ISO formulated version of the MRS medium for the culture of lactic acid bacteria from food.

Typical Formula*

gm/litre

Enzymatic digest of casein

10

Meat extract

10

Yeast extract

4

Tri-ammonium citrate

2

Sodium acetate

5

Magnesium sulphate heptahydrate

0.2

Manganese sulphate tetrahydrate

0.05

Di-potassium hydrogen phosphate

2

Sorbitan mono-oleate

1.08

Glucose

20

Agar

12.4

TOTAL

66.73g/L

* Adjusted as required to meet performance standards

Directions

Suspend 66.73g in 1 litre of distilled water. Boil to dissolve the medium completely. Dispense into tubes, bottles or flasks and sterilize by autoclaving at 121°C for 15 minutes. Selectivity can be altered by pH adjustment.

Description

The MRS formulation was developed by de Man, Rogosa and Sharpe1 in 1960 to replace a variable component (tomato juice) and, at the same time, provide a medium which would support good growth of lactobacilli in dairy products, even those strains which showed poor growth in existing media. MRS medium is superior to the tomato juice medium of Briggs2 and the meat extract tomato juice medium of de Man. It gives more profuse growth of all strains of lactobacilli, especially the difficult and slow growing strains of Lactobacillus brevis and Lactobacillus fermenti.

MRS (ISO) Agar was designed to conform to the formulation specified in ISO 15214: 1998 Horizontal method for the enumeration of Mesophilic lactic acid bacteria – colony count technique at 30°C. This medium encourages the growth of the lactic acid bacteria, which includes species of the following genera: Lactobacillus, Streptococcus, Pediococcus and Leuconostoc. All these species can produce lactic acid in considerable amounts. They are Gram positive, catalase and oxidase negative and are fastidious in their nutritional requirements. Growth is enhanced considerably by microaerobic conditions (5% CO2).

If yeast and mould contamination in the food sample is of concern (for example in dry sausages), it is recommended to add sorbic acid to the medium in order to enhance inhibition. By addition of 0.1mg of clindamycin and 10mg of ciprofloxacin per litre, the selective enumeration of Lactobacillus acidophilus in dairy products (according to the standards ISO 20128) can be achieved. MRS (ISO) Agar can be acidified to pH 5.4 for the enumeration of Lactobacillus bulgaris in yoghurt.

Technique

  1. Prepare the MRS (ISO) Medium by suspending 33.35g per 500ml of distilled water, and heat to dissolve completely. Sterilize by autoclaving at 121°C for 15 minutes. Cool to 50°C, mix well and pour into sterile Petri dishes.
  2. Add the food sample to a diluent, as specified by ISO 6887-1, and process in a `Stomacher’ for 30 seconds, or alternatively weigh the sample into the diluent and leave for 30 minutes shaking periodically
  3. Inoculate 0.1ml of the prepared sample onto the prepared medium surface and spread
  4. Incubate the plates in a microaerophilic atmosphere (5% CO2) at 30 ± 1°C and examine after 3 days (72 hours)
  5. Report as number of colonies per gram of food

Storage conditions and Shelf life

Store the dehydrated medium at 10–30°C and use before the expiry date on the label.
Store the prepared plates at 2–8°C.

Appearance

Dehydrated medium: Dark straw coloured powder
Prepared medium: Orange/brown coloured gel

Quality control


Organism:

Expected results

Lactobacillus gasseri
ATCC® 19992 *

0.5–2mm pale straw colonies

Lactobacilles sake ATCC® 15521

0.5–2mm pale straw colonies

Streptococcus lactis
ATCC® 19435

0.5–2mm cream colonies

Pediococcus pentosaceus
ATCC® 33316

Pinpoint–3mm cream colonies

Bacillus cereus
ATCC® 11778 *

No growth

Escherichia coli
ATCC® 8739 *

No growth

Escherichia coli
ATCC® 25922*

No growth

* This organism is available as a Culti-Loop®

Precautions

Sterilization of this medium in some autoclaves, at 121°C for 15 minutes, may cause the medium in some cases to fall outside the specified pH limit of 5.7 ± 0.1. Adjustment of the medium in these rare cases is recommended using acetic acid or sodium hydroxide.

THIS PRODUCT MAY PACK DOWN ON PROLONGED STORAGE BUT THE QUANTITY WILL NOT BE AFFECTED. THE FREE FLOWING STATE MAY BE RESTORED BY THOROUGHLY SHAKING THE CONTAINER BEFORE OPENING IT.

References

  1. de Man J. C., Rogosa M. and Sharpe M. Elisabeth (1960) Appl. Bact. 23. 130-135.
  2. Briggs M. (1953) J. Dairy Res. 20. 36-40.
 
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