Culture Media

Lactobacilli MRS Broth and Agar Composition, Preparation, Principle

MRS Broth was developed by deMan, and co. in order to facilitate the most abundant growth of lactobacilli derived from feces, oral...

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Sourav Bio
This article writter by Sourav Bio on December 09, 2021

Writer and Founder of Microbiologynote.com. I am from India and my main purpose is to provide you a strong understanding of Microbiology.

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Lactobacilli MRS Broth and Agar Composition, Preparation, Principle
Lactobacilli MRS Broth and Agar Composition, Preparation, Principle

MRS Broth was developed by deMan, and co. in order to facilitate the most abundant growth of lactobacilli derived from feces, oral and other samples. Lactobacilli MRS Broth is a better media for lactobacilli. It allows for healthy growth and is especially suitable for several very discerning strains that grow poorly in other media.

Composition of Lactobacilli MRS Broth

IngredientsGms / L
Proteose peptone10.0
Beef extract10.0
Yeast extract5.0
Dextrose20.0
Polysorbate 801.0
Ammonium citrate2.0
Sodium acetate5.0
Magnesium sulphate0.1
Manganese sulphate0.05
Dipotassium phosphate2.0

Final pH ( at 25°C) 6.5±0.2

Composition of Lactobacilli MRS Agar

IngredientsGms / L
Proteose peptone10.0
Beef extract10.0
Yeast extract5.0
Dextrose20.0
Polysorbate 801.0
Ammonium citrate2.0
Sodium acetate5.0
Magnesium sulphate0.1
Manganese sulphate0.05
Dipotassium phosphate2.0
Agar15g

Principle of Lactobacilli MRS Broth

Lactobacilli MRS Medium, first created in the year 1960 by de Man, Rogosa & Sharpe and Sharpe, is based on the formula, but with slight modifications. The MRS broth is rich in nitrogen, carbon, as well as vitamins that aid in development of lactobacilli as well as other microorganisms. Proteose Peptone and beef extract are carbonaceous and nitrogenous substances. The extract of yeast provides vitamin B complex, while dextrose is an energy source and carbohydrate. Polysorbate 80 acts as an surfactant that facilitates the absorption in nutrients of lactobacilli providing fatty acids needed to regulate the metabolism of Lactobacilli. It is a specific medium that makes use of sodium ammonium citrate and ammonium acetate to stop overgrowth caused by contamination of organisms. The sodium acetate inhibits the growth of many competitive bacteria (although certain Lactobacilli like Leuconostoc and Pediococcus can expand). Ammonium citrate blocks Streptococci, molds and other microorganisms. Magnesium sulfate as well as manganese sulf are ions that aid in metabolism. Growth is considered to be a positive outcome. A Durham tube could be added to distinguish Lactobacillus species and Leuconostoc species.

Intended Use

Lactobacilli MRS Agar, as well as Lactobacilli MRS Broth have been suggested to be used in the isolation, enumeration , and cultivation of Lactobacillus species.

Preparation of Lactobacilli MRS Broth

  1. Take a suspension of 55.15 grams into 1000 milliliters of distillate water.
  2. It is possible to heat the oven to melt your medium to the point of complete dissolution.
  3. Distribute into bottles, tubes or flasks if desired.
  4. Sterilize with autoclaving with 15 lbs pressurization (121degC) during 15 minutes.

Procedure

Direct Counts

  1. To determine the direct count of lactobacilli, add 15-20 mL of sterile, molten (45-50degC) Lactobacilli MRS Agar into sterilized Petri dishes with 1 mL volumes of dilute test sample.
  2. Inoculate all the media by turning the plates in one direction, and afterward in the reverse direction.
  3. Let the medium set on an even surface over 5 to 10 minutes.
  4. Additionally plates with Lactobacilli MRS Agar could be used to obtain direct detection of bacteria with the use of the streak inoculation method.
  5. Incubate agar plates in 35degC for 3 days or at 30degC for five days in an atmosphere that is aerobic and supplemented by carbon dioxide.

Broth Enrichment

  1. Samples can be directly inoculated directly into Lactobacilli MRS broth.
  2. Incubate the broth tubes at 35degC for three days, or at 30degC over 5 days, under an aerobic environment.
  3. Subculture growth occurs in broth tubes in appropriate solid media.

Result Interpretation of Lactobacilli MRS Broth

Lactobacilli appear as white, large colonies found within or over Lactobacilli MRS Agar, or as Turbidity within Lactobacilli MRS Broth. The growth can be subcultured onto the appropriate medium to be used in other procedures. Check the appropriate reference for suggestions on the culture of Lactobacillus Spp.

  • Positive result: Growth, turbidity (Lactobacillus gasseri)
  • Negative result: No growth (Uninoculated medium)

Storage and Shelf Life

The dehydrated medium between 2 and 8 degrees Celsius. Make use of the product prior to the expiry date printed indicated on the label. After opening, the product must be stored dry after sealing the bottle tightly in order to prevent the formation of lumps as a result of the highly hygroscopic nature the product. Unproper storage can result in the formation of lumps. Keep the product in a dry, ventilated place safe from extreme temperatures and ignition sources. The container should be sealed tightly following use. It is recommended that the product be utilized within the expiry date.

Quality Control

Lactobacilli MRS Agar

  • Dehydrated Appearance: Light tan, free-flowing, homogeneous.
  • Solution: 7.0% solution, soluble in purified water upon boiling. Solution is medium amber, clear to slightly opalescent.
  • Prepared Appearance: medium amber extremely slightly to somewhat transparent.
  • Reaction of 7.0% Solution at 25°C: pH 6.5 ± 0.2

Lactobacilli MRS Broth

  • Appearance of Dehydrated: Tan, homogeneous, appears damp.
  • Solution 5.5 percent solution which is soluble in purified drinking water upon boiling. Solutions are medium-amber to transparent to somewhat transparent. It is opalescent to very slightly.
  • Prepared Appearance: medium amber translucent to slightly opaque.
  • Reaction of 5.5% Solution at 25degC: pH 6.5 +- 0.2

Following organisms can be commonly utilized for quality control testing in Anaerobe Systems.

Organism TestedResultsTime
Lactobacillus vaginalisGrowth24 – 48 hrs
Lactobacillus acidophilusGrowth24 – 48 hrs
Lactobacillus crispatusGrowth24 – 48 hrs
Lactobacillus jenseniiGrowth24 – 48 hrs
Lactobacillus fermentumGrowth24 – 48 hrs

Physical Properties of MRS Broth

  • Appearance Color: From yellow to cream. fluid powder
  • Color and clarity of the prepared medium: Medium amber colored transparent to slightly transparent solution in tubes
  • Reaction: A reaction that occurs in 5.51 percent w/v of aqueous solution at 25°C. pH: 6.5+-0.2
  • pH: 6.30-6.70
  • Culture Response: The cultural aspects observed following an incubation period of 35-37°C for 18-24 hours or more. (with 5% CO2)Uses of Lactobacilli MRS Broth
  • MRS Broth is a medium to cultivate and count of Lactobacillus Spp.
  • It aids in the development of all Lactobacilli found in the oral cavity dairy products, food products as well as feces and various other sources.
  • It can be used to determine if the organism produces gas when it ferments glucose. Certain Lactobacillus as well as Leuconostoc spp. create gas.

Limitations of Lactobacilli MRS Broth

  • It is suggested that immunological, biochemical, mass spectrometry, or molecular tests be conducted on the colonies of pure culture to ensure complete identification.
  • Other organisms than lactobacilli could be able to grow in this environment.
  • Due to the different nutrition requirements, certain species may do not grow well or do not thrive in this medium.
  • Lactobacilli MRS Broth has not been designed to be used to diagnose the presence of a disease or other ailments in humans.
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Writer and Founder of Microbiologynote.com. I am from India and my main purpose is to provide you a strong understanding of Microbiology.

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