MRS AGAR - Biokar Diagnostics

MRS AGAR
INTENDED USE
MRS Agar is used for the growth and enumeration of cultures of Lactobacillus in dairy and other food
products and in animal feeding stuffs. The medium can be used to culture slowly-growing lactobacilli
such as Lactobacillus brevis and Lactobacillus fermentum. Acidified to low pH, it can be used to
enumerate Lactobacillus bulgaricus in yogurts. Following the NF / ISO standard 15214 for the
enumeration of mesophilic lactic acid bacteria, MRS agar should be used at pH 5.7. If yeast & mold
contamination in the sample being tested is of concern (for example in dry sausages), it is useful to add
sorbic acid to the medium in order to enhance inhibition. By addition of 0.1 mg of Clindamicine and 10
mg of Ciprofloxacine per liter, a medium known as MRS/CLC/CIP Agar can be obtained ; this medium
is used for the selective enumeration of Lactobacillus acidophilus in dairy products, according to the
standards ISO 20128 / IDF 192.
For the isolation and enumeration lactic acid bacteria in animal feeding stuffs, the pH of the MRS Agar
should be adjusted following the NF EN standards.
HISTORY
De Man, Rogosa and Sharpe developed a formulation in 1960 for a medium specifically designed for
the culture of lactobacilli in dairy products, without the need to add tomato juice (an ingredient of highly
variable composition).
PRINCIPLES
- The different peptones, glucose, magnesium and manganese salts supply the nutritive elements
required for the growth of lactobacilli.
- Tween 80 is composed of a mixture of oleic esters and is a source of fatty acids essential for the
growth of these bacteria.
- Dipotassium phosphate stabilizes the pH during bacterial growth.
- Ammonium citrate and sodium acetate inhibit the development of most contaminants, including
streptococci and molds.
1/5
Biokar Diagnostics – Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – F60002 Beauvais Cedex – France
Tél : + 33 (0)3 44 14 33 33 – Fax : + 33 (0)3 44 14 33 34 – www.biokar-diagnostics.com
PREPARATION
- Suspend 70.3 g of dehydrated medium (BK089) in 1 liter of distilled or deionized water.
- Slowly bring to boiling, stirring slowly with constant agitation until complete dissolution. Incomplete
agar melting during preparation will invariably lead to significant inconsistency in the gel strength of
the solidified agar, after sterilization and cooling.
- Adjust the pH, depending on the product being analyzed :
ƒ
ƒ
For yogurts, it is recommended to use acetic acid in order to reach a final pH of
5.4 ± 0.1.
For enumeration of mesophilic lactic acid bacteria, it is not necessary to adjust the pH of
the medium.
ƒ
For animal feeding stuffs, it is recommended to use a sodium hydroxyde solution to reach a
final pH of 6.5 ± 0.2 (NF EN 15785), 6.2 ± 0.2 (NF EN 15786 and NF EN 15787).
- Dispense in tubes or flasks.
- Sterilize in an autoclave at 121°C for 15 minutes.
NOTE 1 :
The acid pH of the medium increases its heat sensitivity ; a proper respect of sterilization procedures is
essential.
NOTE 2 :
When using the ready-to-use medium BM089 at pH 5.7 (or if the medium has been prepared in
advance from the dehydrated form), melt the agar for the minimum amount of time in order to obtain
complete liquefaction. Do not repeat this operation more than once.
INSTRUCTIONS FOR USE
- Cool and maintain the medium at 44-47°C.
- Add, if necessary, the appropriate antibiotic solutions (i.e. Clindamicine and/or Ciprofloxacine). In
this context, the medium must be inoculated via streaking, after drying poured Petri dishes.
- Transfer 1 mL of the product to analyze and its serial tenfold dilutions to sterile Petri dishes.
- Pour in 15 mL of medium.
- Homogenize by swirling.
- Let solidify on a cold surface.
- Place the inoculated plates in the conditions dictated by the protocol being applied (i.e. 5% CO2).
- Incubate at 30 or 37°C for 2 to 3 days, depending on the protocol.
RESULTS
Enumerate colonies for each plate containing a maximum of 300 colonies. As there is a possibility that
other non lactic bacteria may develop, verify under the microscope that the cells are Gram-positive,
non-sporulated bacilli.
2/5
Biokar Diagnostics – Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – F60002 Beauvais Cedex – France
Tél : + 33 (0)3 44 14 33 33 – Fax : + 33 (0)3 44 14 33 34 – www.biokar-diagnostics.com
TYPICAL COMPOSITION
(can be adjusted to obtain optimal performance)
For 1 liter of medium :
- Polypeptone ...................................................................................10.0 g
- Meat extract ...................................................................................10.0 g
- Yeast extract ....................................................................................5.0 g
- Glucose ..........................................................................................20.0 g
- Tween 80 .......................................................................................1.08 g
- Dipotassium phosphate ...................................................................2.0 g
- Sodium acetate ................................................................................5.0 g
- Ammonium citrate ............................................................................2.0 g
- Magnesium sulfate ...........................................................................0.2 g
- Manganese sulfate.........................................................................0.05 g
- Bacteriological agar .......................................................................15.0 g
pH of the ready-to-use medium at 25°C : 5.7 ± 0.1
QUALITY CONTROL
- Dehydrated medium : cream powder, slightly clumped and brittle.
- Prepared medium : amber agar.
- Typical culture response after 72 hours of incubation at 30°C in 5% CO2 :
Growth
(Productivity Ratio : PR)
Microorganisms
Lactobacillus casei subsp. rhamnosus
Lactobacillus plantarum
Lactobacillus sake
Escherichia coli
Bacillus cereus
ATCC® 7469
ATCC 8014
CIP 103139T
ATCC 25922
ATCC 11778
PR ≥ 70%
PR ≥ 70%
PR ≥ 70%
inhibited, score 0
inhibited, score 0
STORAGE / SHELF LIFE
Dehydrated medium : 2-20°C.
- The expiration date is indicated on the label.
Prepared medium (benchmark value*) :
- Media in vials : 6 months at 2-8°C.
Code
PACKAGING
Ready-to-melt medium in vials (pH 5.7) :
- 10 x 200 mL
BM08908
Dehydrated medium :
- 500 g bottle
BK089HA
3/5
Biokar Diagnostics – Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – F60002 Beauvais Cedex – France
Tél : + 33 (0)3 44 14 33 33 – Fax : + 33 (0)3 44 14 33 34 – www.biokar-diagnostics.com
PHOTO SUPPORT :
Product Reference : BK089HA, BM08908
Media used for :
Lactobacillus
rhamnosus
casei
Enumeration of lactic acid bacteria.
subsp.
Typical aspect
White colonies of uniform size.
MRS Agar
Ref : BM08908
Incubation 72 hours at 30°C (in 5% CO2)
Characteristics : White colonies of uniform size. Colonies found on the surface of the agar or on the
very bottom will have a larger size due to the absence of agar restricting growth.
4/5
Biokar Diagnostics – Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – F60002 Beauvais Cedex – France
Tél : + 33 (0)3 44 14 33 33 – Fax : + 33 (0)3 44 14 33 34 – www.biokar-diagnostics.com
BIBLIOGRAPHY
De Man, J.C., Rogosa, M., and Sharpe, M.E. 1960. A medium for the cultivation of lactobacilli. J. App. Bacteriol., 23, (1): 130135.
Journal Officiel du 4 janvier 1978. Méthode officielle d'analyse pour le dénombrement de la flore spécifique du yaourt ou
yoghourt. (arrêté du 25 Novembre 1977).
NF V 04-503. Septembre 1988. Viandes et produits à base de viande. Dénombrement des Bactéries lactiques.
FIL-IDF 149A. Juillet 1997. Levains lactiques de cultures de bactéries lactiques. Norme de composition.
NF ISO 15214 (V 08-030). Septembre 1998. Microbiologie des aliments. Méthode horizontale pour le dénombrement des
bactéries lactiques mésophiles. Technique par comptage des colonies à 30 °C.
ISO 7889 / IDF 117. Février 2003. Yaourt. Dénombrement des micro-organismes caractéristiques. Technique de comptage
des colonies à 37 °C.
XP CEN ISO/TS 11133-2 (V 08-104-2). Janvier 2004. Microbiologie des aliments. Guide pour la préparation et la production
des milieux de culture. Partie 2 : Guide général pour les essais de performance des milieux de culture.
ISO 20128 / IDF 192. Mai 2006. Produits laitiers. Dénombrement de Lactobacillus acidophilus présomptifs sur un milieu
sélectif. Technique de comptage des colonies à 37 °C.
NF EN 15785 (V 18-238). Décembre 2009. Aliments des animaux. Isolement et dénombrement du Bifidobacterium spp.
NF EN 15786 (V 18-230). Décembre 2009. Aliments des animaux. Isolement et dénombrement du Pediococcus spp.
NF EN 15787 (V 18-231). Décembre 2009. Aliments des animaux. Isolement et dénombrement du Lactobacillus spp.
*Benchmark value refers to the expected shelf life when prepared under standard laboratory conditions following manufacturer’s instructions. It is provided as a
guide only and no warranty, implied or otherwise is associated with this information.
The information provided on the package take precedence over the formulations or instructions described in this document.
The information and specifications contained in this technical data sheet date from 2010-01-13.
They are susceptible to modification at any time, without warning.
Code document : BK089/A/2003-10 : 9.
5/5
Biokar Diagnostics – Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – F60002 Beauvais Cedex – France
Tél : + 33 (0)3 44 14 33 33 – Fax : + 33 (0)3 44 14 33 34 – www.biokar-diagnostics.com