Lactobacilli MRS Broth- Composition, Principle, Preparation, Results, Uses

Lactobacilli are a group of gram-positive, rod-shaped bacteria that belong to the lactic acid bacteria (LAB) family. They are widely distributed in nature and can be found in various environments, such as the human and animal gastrointestinal tract, oral cavity, vagina, fermented foods, and dairy products. Lactobacilli play important roles in food preservation, fermentation, probiotics, and health benefits.

Lactobacilli MRS Broth is a complex medium that contains various ingredients to support the growth and metabolism of lactobacilli and other lactic acid bacteria. The composition of Lactobacilli MRS Broth per liter of distilled water is as follows:

• Proteose peptone: 10 g
• Beef extract: 8 g
• Yeast extract: 4 g
• Dextrose: 20 g
• Polysorbate 80: 1 g
• Sodium acetate: 5 g
• Dipotassium hydrogen phosphate: 2 g
• Triammonium citrate: 2 g
• Magnesium sulfate: 0.2 g
• Manganese sulfate: 0.05 g

The final pH of the medium is adjusted to 6.5 ± 0.2 at 25°C.

Each ingredient has a specific role in the medium:

• Proteose peptone and beef extract provide nitrogenous and carbonaceous compounds, such as amino acids, peptides, nucleotides, vitamins, and minerals, that are essential for the growth and maintenance of lactobacilli.
• Yeast extract provides vitamin B complex, which is involved in various enzymatic reactions and coenzyme synthesis in lactobacilli.
• Dextrose serves as a carbohydrate and energy source for lactobacilli. It is also the substrate for glucose fermentation, which produces lactic acid and gas as end products.
• Polysorbate 80 is a surfactant that facilitates the uptake of nutrients by lactobacilli. It also supplies fatty acids that are required for the membrane synthesis and lipid metabolism of lactobacilli.
• Sodium acetate is a selective agent that suppresses the growth of many competing bacteria, such as enteric bacteria, staphylococci, streptococci, and bacilli. It also lowers the pH of the medium by forming acetic acid, which inhibits the growth of alkaline-tolerant bacteria.
• Dipotassium hydrogen phosphate acts as a buffer that maintains the pH of the medium within the optimal range for lactobacilli. It also provides phosphorus, which is a component of nucleic acids, phospholipids, and ATP in lactobacilli.
• Triammonium citrate is another selective agent that inhibits the growth of streptococci, molds, and many other microorganisms. It also provides nitrogen and citrate, which are used in the tricarboxylic acid cycle and other metabolic pathways in lactobacilli.
• Magnesium sulfate and manganese sulfate provide magnesium and manganese ions, respectively, that are used as cofactors for various enzymes in lactobacilli.

The composition of Lactobacilli MRS Broth is designed to provide optimal conditions for the cultivation and enumeration of lactobacilli from various sources, such as oral cavity, dairy products, foods, feces, and other sources. It supports the luxuriant growth of all lactobacilli species and some other lactic acid bacteria, such as Leuconostoc and Pediococcus. However, it may also allow the growth of some non-lactobacilli organisms that can tolerate the selective agents or have similar nutritional requirements as lactobacilli. Therefore, it is recommended that further identification tests be performed on the isolated colonies to confirm their identity.

Lactobacilli MRS broth is a selective and differential medium that supports the growth of lactobacilli and other lactic acid bacteria, while inhibiting the growth of most other microorganisms. The principle of this medium is based on the following factors:

• The medium contains various sources of carbon, nitrogen, and vitamins that are essential for the metabolism of lactobacilli. These include proteose peptone, beef extract, yeast extract, dextrose, and polysorbate 80. Polysorbate 80 also acts as a surfactant, facilitating the uptake of nutrients by lactobacilli and supplying fatty acids required for their growth.
• The medium contains sodium acetate and ammonium citrate as selective agents that suppress the growth of many competing bacteria. Sodium acetate creates an acidic environment that is unfavorable for most bacteria, except for lactobacilli and some other lactic acid bacteria, such as Leuconostoc and Pediococcus. Ammonium citrate inhibits the growth of streptococci, molds, and many other microorganisms that cannot utilize citrate as a carbon source.
• The medium contains magnesium sulfate and manganese sulfate as cofactors that are involved in various enzymatic reactions of lactobacilli. These minerals also enhance the formation of lactic acid by lactobacilli, which lowers the pH of the medium and further inhibits the growth of non-lactic acid bacteria.
• The medium can be used to differentiate lactobacilli from Leuconostoc based on their ability to produce gas from glucose fermentation. A Durham tube can be added to the medium to trap any gas bubbles that are formed by the bacteria. Lactobacilli do not produce gas, while Leuconostoc do.

The principle of Lactobacilli MRS broth is to provide optimal conditions for the growth and identification of lactobacilli and other lactic acid bacteria from various sources. The medium is selective for lactobacilli due to its acidic pH and the presence of sodium acetate and ammonium citrate. The medium is differential for lactobacilli and Leuconostoc based on their gas production from glucose fermentation. The medium can be used to cultivate and enumerate lactobacilli from oral cavity, dairy products, foods, feces, and other sources.

Lactobacilli MRS broth is a commercially available dehydrated medium that can be easily prepared in the laboratory. The following steps are involved in the preparation of Lactobacilli MRS broth:

1. Suspend 55.15 grams of the dehydrated medium in 1000 ml of distilled water. Mix well to dissolve the medium completely. You may need to heat the solution slightly to facilitate the dissolution.
2. Distribute the medium in tubes, bottles or flasks as desired. If you want to use Durham tubes to detect gas production, add them to the tubes before distributing the medium.
3. Sterilize the medium by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Make sure that the caps or stoppers are loosely fitted to allow steam to escape.
4. After autoclaving, cool the medium to room temperature and store it in a dark and dry place until use. Do not use the medium if it shows signs of deterioration, such as discoloration, turbidity or contamination.

To inoculate the medium, use a sterile loop or needle to transfer a small amount of the test organism from a pure culture to the surface of the medium. Incubate the inoculated medium at 35°C for 24-48 hours under anaerobic conditions. You can use an anaerobic jar or chamber to create an oxygen-free environment for the growth of lactobacilli. Alternatively, you can add a reducing agent, such as cysteine hydrochloride, to the medium before sterilization to lower the oxidation-reduction potential and inhibit aerobic growth.

After incubation, examine the medium for growth and gas production. Growth is indicated by turbidity or cloudiness of the medium. Gas production is indicated by displacement of the liquid in the Durham tube or formation of bubbles in the medium. Compare your results with those of uninoculated controls and positive and negative controls (if available). Record your observations and interpretations in a table or a report.

Here is an example table for recording your results:

Lactobacilli MRS Broth is a differential medium that allows the detection of gas production from glucose fermentation by lactobacilli and other organisms. Gas production is indicated by the displacement of the medium or the formation of bubbles in a Durham tube, if present. The medium also supports the growth of lactobacilli, which appear as white to cream-colored colonies on the surface or throughout the broth.

The following table summarizes the expected results for some common lactobacilli and other organisms on Lactobacilli MRS Broth:

(+) Positive result; (-) Negative result

A positive result for growth indicates that the organism is able to utilize the nutrients and conditions provided by the medium. A negative result for growth indicates that the organism is inhibited by the selective agents or requires different nutrients or conditions for growth. A positive result for gas production indicates that the organism is able to ferment glucose and produce carbon dioxide and/or hydrogen as by-products. A negative result for gas production indicates that the organism either does not ferment glucose or does not produce gas from glucose fermentation.

It is important to note that some lactobacilli may produce acid but not gas from glucose fermentation, and some may produce gas only under certain conditions. Therefore, gas production alone is not sufficient to identify lactobacilli. Other tests, such as Gram staining, catalase reaction, carbohydrate fermentation, and molecular methods, should be performed to confirm the identity of lactobacilli and differentiate them from other organisms.

Lactobacilli MRS Broth is a widely used medium for the cultivation and enumeration of Lactobacillus spp. and other lactic acid bacteria. It has several applications in different fields, such as:

• Food microbiology: Lactobacilli MRS Broth can be used to isolate and identify lactobacilli from various food products, such as cheese, yogurt, fermented vegetables, meat, and beverages. It can also be used to monitor the quality and safety of these products by detecting the presence of spoilage or pathogenic microorganisms that may grow along with lactobacilli.
• Probiotics research: Lactobacilli MRS Broth can be used to select and characterize probiotic strains of lactobacilli that have beneficial effects on human health. It can also be used to study the interactions between lactobacilli and other gut microbiota, as well as their effects on the host immune system and metabolism.
• Industrial biotechnology: Lactobacilli MRS Broth can be used to optimize the production of lactic acid and other metabolites by lactobacilli in fermentation processes. It can also be used to screen for novel enzymes or genes from lactobacilli that have potential applications in biocatalysis, bioconversion, or biosynthesis.
• Oral microbiology: Lactobacilli MRS Broth can be used to isolate and identify lactobacilli from oral samples, such as saliva, plaque, or caries. It can also be used to assess the role of lactobacilli in oral health and disease, such as dental caries, periodontitis, or halitosis.

Lactobacilli MRS Broth is a widely used medium for the cultivation and enumeration of Lactobacillus spp. However, it has some limitations that should be considered when using it for microbiological purposes. Some of these limitations are:

• It is not a selective medium for Lactobacillus spp. alone. Other organisms, such as Leuconostoc, Pediococcus, Enterococcus, and Streptococcus, may also grow in this medium. Therefore, it is recommended that further tests, such as biochemical, immunological, molecular, or mass spectrometry, be performed on the isolated colonies to confirm their identity and exclude possible contaminants.
• It may not support the growth of all Lactobacillus spp. Some strains may have different nutritional requirements or environmental preferences that are not met by this medium. For example, some Lactobacillus spp. may require higher or lower pH, higher or lower temperature, or additional supplements to grow optimally. Therefore, it is advisable to use other media or methods in conjunction with this medium to ensure the recovery of all Lactobacillus spp. from a given sample.
• It may not differentiate between gas-producing and non-gas-producing Lactobacillus spp. Although a Durham tube can be added to the medium to detect gas production during glucose fermentation, some Lactobacillus spp. may produce gas only under certain conditions or in small amounts that are not visible in the tube. Moreover, some non-Lactobacillus spp., such as Leuconostoc spp., may also produce gas in this medium. Therefore, it is suggested that other tests, such as carbohydrate fermentation profiles or enzyme assays, be used to distinguish between gas-producing and non-gas-producing Lactobacillus spp.
• It may not reflect the physiological state or viability of Lactobacillus spp. in the sample. The growth of Lactobacillus spp. in this medium depends on several factors, such as inoculum size, incubation time, oxygen availability, and pH stability. These factors may vary depending on the source and handling of the sample. Therefore, it is possible that some Lactobacillus spp. may be present but not grow in this medium due to stress or injury caused by environmental changes or processing treatments. Alternatively, some Lactobacillus spp. may grow in this medium but not be viable or active in the sample due to dormancy or senescence induced by unfavorable conditions or aging. Therefore, it is recommended that other methods, such as direct microscopic counts, viability assays, or metabolic activity measurements, be used to assess the physiological state or viability of Lactobacillus spp. in the sample.

In conclusion, Lactobacilli MRS Broth is a useful medium for the cultivation and enumeration of Lactobacillus spp., but it has some limitations that should be taken into account when interpreting the results. It is advisable to use complementary methods and tests to confirm the identity, diversity, and activity of Lactobacillus spp. in a given sample.

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