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Robertson’s Cooked Meat Medium (RCM Medium) Preparation, Composition.

Cooked Meat Medium was originally developed by Robertson to cultivate specific anaerobes isolated from wounds. The current formulation is a modified version, sometimes...

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This article writter by MN Editors on November 11, 2021

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Robertson’s Cooked Meat Medium (RCM Medium) Preparation, Composition.
Robertson’s Cooked Meat Medium (RCM Medium) Preparation, Composition.

Robertson’s Cooked Meat Medium (RCM Medium)

  • Cooked Meat Medium was originally developed by Robertson to cultivate specific anaerobes isolated from wounds.
  • The current formulation is a modified version, sometimes referred to by the name of Chopped Meat Medium, which allows for the growth of a variety of spores and non-spore-forming strict anaerobes.
  • It is able to stimulate the growth of bacteria starting with very little inocula, and to sustain the viability of these cultures over a longer periods of time. The mixed cultures of bacteria thrive inside Cooked Meat Medium without displacing those with slower growth.
  • The growth products are not able to rapidly destroy inoculated cells, which is why it’s a good medium to store aerobic and anaerobic species.
  • It is used to cultivate as well as maintenance Clostridia and to determine the proteolytic activity of anaerobes.
  • It’s also referred to in the form of cooked broth (CMB) because it is made up of chunks of fat-free minced cooked heart meat from ox as well as nutritious broth.
  • It is responsible for the development of both spore-forming as well as non-spore-forming essential anaerobes, and it also distinguishes between saccharolytic and putrefactive species.
  • The oxygen content in media for culture can be decreased by a variety of substances, such as glucose, thioglycollate or thioglycollate cooked meat pieces ascorbic acid and cysteine.
  • Thioglycollate broth that contains nutrient soup and one percent thioglycollate also utilized to grow anaerobes.

Principle of Robertson’s Cooked Meat Medium (RCM Medium)

Cooked M-Medium is a source of HMH Peptone B, They contains amino acids as well as other nutrients. Also, it contains glutathione, a substance that reduces the growth of anaerobes that are obligate. The sulfhydryl chains are responsible for reducing the effect, are more readily available in protein denatured, and so cooked meat is included in the cooking medium.

Dextrose permits rapid and massive growth of anaerobic bacteria over short amounts of time. It also results in a faster identification of the most important anaerobes. The rate of growth in this medium is visible by the appearance of bubbles or turbidity creation by certain organisms. Disintegration and discoloration of meat particles are signs of proteolysis.

For the best results, the medium must be utilized on the day that it was prepared otherwise, it needs to be cooked or steam for a couple of minutes, allowed to cool , without agitation before being inoculated. Inoculation should be performed at the base of the tube, in the meat particles in anaerobic culture. Aerobes thrive on top, whereas anaerobic species are found deeper in the medium. To separate Clostridium from food items, utilize a stomacher for the preparation of 10 percent suspension of food item in Peptone Water (M028) diluent.

Create dilutions and plates for suspensions and dilutions for Willis as well as Hobbs Medium Base (M1375 ), Tryptose Sulphite Cycloserine (T.C.S.) Agar Base (M837). Put a metronidazole disc onto the inoculum. Keep it incubating anaerobically for a few hours at 37 degrees Celsius.

To determine the number of clostridia present, fill the plates with concentrations onto Perfringens Agar Base (O.P.S.P.) (M579). Make duplicate plates and incubate them both aerobically and anaerobically in order to differentiate between clostridia as well as other organisms. Add a portion of the suspension into two tubes of cooked Medium. One tube should be heated for 10 minutes at 80degC and incubate the same way as before. The growth of Clostridium is visible by gas bubbles or turbidity. This medium may be further checked for the evidence of Clostridium.

Composition of Robertson’s Cooked Meat Medium (RCM Medium)

Cooked Meat Medium250.0 gm
Peptic Digest of Animal Tissue17.5 gm
Dextrose5.0 gm
Sodium Chloride5.0 gm
Yeast Extract5.0 gm
Iron Filings10.0 gm
Hemin10.0 ml
Vitamin K10.0 ml

Final pH 6.8 +/- 0.3 at 25ºC.

* Modified and/or added according to the need to meet requirements for performance

  • Cooked Meat Medium: In the cooked medium, meat particles function as a detoxifying and reducing substance, thus preventing harmful by-products created in the organism that reproduces. Since reducing substances are accessible in protein denatured which is why it is cooked prior to being used for the medium.
  • Iron filings: Reducing substance. The iron filings and the muscle tissue facilitate the expansion of strict anaerobes.
  • Nutritional supplements: Nutritional needs of the majority of bacteria can be met through peptic digests of tissues from animals, yeast extract and dextrose. Hemin as well as vitamin K can be included to increase the development of anaerobic microorganisms. The amino acids as well as other nutrients are also provided by the muscles protein found in the granules of heart tissue.

Preparation of the RCM Medium

  1. Robertson’s cooked meat medium best prepared using dehydrated granules that are ready to use readily available from the majority of retailers of cultural media.
  2. With a small tube, or scoop that has been marked to hold 1g of granules disperse the medium in 1g amounts into screw-cap bottles and tubes.
  3. Add 10ml of distilled water, mix and let it soak for 5 minutes.
  4. Sterilize the medium using autoclaving (with caps loosening) at 121°C during 15 minutes. After cooling, secure the caps of the bottles. Then, date the medium and assign the medium an identification number for the batch.
  5. Place your medium somewhere cool and, dark location, ensuring that you screw the bottles cops securely screwed.

Quality Control of Robertson’s Cooked Meat Medium

Positive controls

  • Clostridium histolyticum ATCC 19401: Luxuriant growth; proteolysis
  • Clostridium perfringens ATCC 13124: Luxuriant growth; saccharolysis ( redness of the medium), proteolysis

Negative control

  • Uninoculated medium: No change

Inoculation on Robertson’s Cooked Meat Medium

  • Based on the specimen, the meat is inoculated with either a swab Pasteur pipette or wire loop. When using a swab,, this should be put in on the inside of the vessel.
  • For the cultivation of strict anaerobes the medium should be used as fresh, or after placing (with the top of the bottle loosened) in a pot of water that is boiling for about 10 minutes to remove oxygen dissolved in it or the bath in a bath of water at 80 degrees Celsius for 30 mins to ensure that it is oxygen-free.
  • Let the medium cool to room temperature prior to inoculating it. On the surface, CMB medium can be covered with a thin layer of liquid paraffin that is sterile.

Interpretation of Results

Robertson’s Cooked Meat Medium result
Cooked meat media with Clostridium perfringens. Tubes a & b showing 24 hrs anaerobic bacterial growth after inoculation with samples. Tube c showing cooked meat medium before inoculation. | Source: https://www.researchgate.net/figure/Cooked-meat-media-with-Clostridium-perfringens-Tubes-a-b-showing-24-hrs-anaerobic_fig1_319651311
  • No turbidity: No growth
  • Turbidity: Presence of growth

Note: In order to avoid growth reports, the samples is a need to blindly subculture into blood agar , and then being incubated anaerobically and aerobically.

Clostridium species are divided into two main groups due to their actions in the media.

  • Saccharolytic group: There is rapid production of gas and acid but there is no digestion of the meat. The cultures may exhibit a pungent smell, and proteins that are reddened.
  • Proteolytic group: Proteolysis triggers the decomposition of meat, resulting in the creation of sulfur that smells foul compounds as well as blackening. But, certain saccharolytic strains also release Hydrogen Sulfide, which causes blackening, but in a lesser extent.

Aerobic and anaerobic growth

The medium tube is then placed in the incubator with the cap open without seal needed. Aerobes develop at the top while the more anaerobic species are found deeper within the medium.

Characteristics of the culture observed following incubation at 35 to 37 degrees Celsius for 40 to 48 hours, using an inoculum of 50-100 CFU.

OrganismGrowth
Clostridium botulinumluxuriant
Clostridium perfringensluxuriant
Clostridium sporogenesluxuriant
Enterococcus faecalis ATCCluxuriant
Streptococcus pneumoniaeluxuriant

Precautions/Limitations

  • For the best outcomes, the medium should be applied on the day that it is made or else it must be cooked or steam for a few minutes before being allowed to cool , without agitation before being inoculated.
  • The inoculation should be done close to the top of the tube, in the meat particles in anaerobic strains.

Uses of Robertson’s cooked meat medium

  • Cultivation of microaerophilic, aerobic and anaerobic microorganisms specifically Clostridium species. It promotes the development of both spore-forming as well as non-spore-forming, obligate anaerobes.
  • It’s a great enrichment broth for the cultivation of microorganisms that are derived from a tiny inoculum.
  • Researchers have also discovered that cooking meat medium is able to preserve the viability of the organisms for an extended period of time, and can be useful in keeping anaerobic stock organisms.
  • The Food and Drug Administration recommends it for recension and the identification of Clostridium perfringens in food items.

Precautions of Robertson’s Cooked Meat (RCM) Medium

  • To ensure the longevity of culture stocks, keep your growth tubes at room temperatures following the initial incubation period at 35°C and subculture it at least every 4 to 6 months.
  • Conditions for storage and shelf life of the medium Storage conditions: Store the dehydrated medium at temperatures of 10-30 degrees Celsius and use it by expiry date is noted on the label. keeping the medium at room temperature in the dark, with tightly sealed caps for up to 6 month.
  • It is a great substrate for the primary development and growth of anaerobic and aerobic organisms.
  • The color and clarity of the medium prepared are amber-colored, transparent to slightly opalescent supernatant on insoluble grains.
  • RCM is used to cultivation of microaerophilic, aerobic and anaerobic microorganisms specifically Clostridia and it helps to promote the growth of both non-spore-forming and spore-forming obligate anaerobes.
  • FDA has advised RCM to count and identify of Clostridium fringens isolated from Foods.
  • The exceptional properties of recovery in RCM means that mixed cultures typically result from inoculation of samples.
  • The medium’s blackening is not a possibility in the case of acid pH.
  • Carbohydrate fermentation can inhibit proteolysis.
  • The amount of oxygen in culture media can be reduced through a variety of reduction agents, such as glucose glutathione from cooked meat pieces, cysteine and ascorbic acid.

References

  • http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=CM0081&c=UK&lang=EN
  • https://himedialabs.com/TD/M149.pdf
  • https://www.bd.com/europe/regulatory/Assets/IFU/US/L007448(10)(0706).pdf
  • https://www.microxpress.in/product.php?type=A&c_id=9&id=322
  • http://www.tulipgroup.com/MicroExpress/Accumix/PackInsert/Dehydrated%20Culture%20Media/C%20Files/Cooked_Meat_Medium_Robertson_Cooked_Meat_Medium.pdf
  • https://universe84a.com/robertsons-cooked-meat-rcm-medium/
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