Introduction of Robertson’s Cooked Meat (RCM) Medium

Robertson’s Cooked Meat (RCM) Medium is the recommended medium for the cultivation of anaerobic as well as aerobic bacteria. It is especially for pathogenic Clostridium species from clinical, food, and water samples. It can also be used as a maintenance medium for stock cultures of anaerobes as well as aerobes. RCM is also known as cooked meat broth (CMB) since it contains pieces of fat-free minced cooked meat of ox heart and nutrient broth.

Principle of Robertson’s Cooked Meat (RCM) Medium

HMH peptone B: It is a component of cooked meat and it provides amino acids and other nutrients. Glutathione, a reducing substance that permits the growth of obligate anaerobes is also found in it. The sulfhydryl groups, which impart a reducing effect, are more available in denatured protein and hence cooked meat is added to the medium. Glucose (dextrose) of the medium allows rapid and heavy growth of anaerobic bacteria in a short time and leads to more rapid identification of important anaerobes. Sodium chloride (NaCl) allows for adjustments in osmotic pressure.

Growth in this medium (RCM) is indicated by turbidity or bubble formation by some organisms. Proteolysis causes the decomposition of the meat with the formation of foul-smelling sulfur compounds and blackening. However, some saccharolytic strains also produce hydrogen sulfide which will cause blackening but to a lesser degree. Saccharolytic anaerobes may cause rapid production of acid and gas but no digestion of the meat. Cultures may have a slightly sour smell, with reddened protein. For best results, the medium should be used on the day it is prepared, otherwise, it should be boiled or steamed for a few minutes and allowed to cool without agitation, and then inoculated. Inoculation should be made near the bottom of the tube in the meat particles for anaerobic cultures. Aerobes grow at the top of the medium while more anaerobic species grow deeper in the medium.

Composition of RCM Medium

Ingredients	Gms / Litre
HMH peptone B	98.0
Proteose peptone	20.0
Dextrose (Glucose)	2.0
Sodium chloride	5.0
Final pH (at 25°C)	7.2±0.2

Preparation of Robertson’s Cooked Meat (RCM) Medium

Suspend 12.5 grams RCM in 100 ml purified/distilled water in a flask or suspend 1.25 grams in 10 ml distilled water in a test tube. Mix thoroughly it and allow it to stand for 15 minutes until all the particles are thoroughly wetted. Using test tubes needs a tube rack or an autoclavable beaker. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.

Test Requirements RCM Medium

• Test sample (water/food/clinical specimens)
• RCM Medium
• Inoculating loop ( optional depending on the nature of specimens)
• Bunsen burner
• BOD Incubator
• Water bath/Steamer (optional)
• Negative control (NC) and positive control (PC)-Control strains

Test Procedure of RCM Medium

Anaerobic Culture: The freshly prepared medium is preferred which is inoculated as soon as it has cooled to approximately 35°C. RCM tubes that are not used on the day of preparation should be placed in a boiling water bath or steamer for about 15 minutes to pull out dissolved oxygen. They should be allowed to cool without agitation and then inoculated. Inoculate near the bottom of the tube in the meat particles. Incubate for up to 7 days For Aerobic organisms at 35°C with loosened caps while in the case of Anaerobic organisms use freshly reduced medium and incubate for up to 21 days at 35°C. Examine daily for changes in the medium. Make films and subcultures at intervals

Result and Interpretation of RCM Medium

No turbidity	No growth
Turbidity	Presence of growth
Saccharolytic group	There is rapid production of acid and gas but no digestion of the meat. Cultures may have a slightly sour smell, with reddened protein.
Proteolytic group	Proteolysis causes the decomposition of the meat with the formation of foul-smelling sulfur compounds and blackening. However, some saccharolytic strains also produce Hydrogen sulfide which will cause blackening but to a lesser degree.
Aerobic and anaerobic growth	The tube of medium is incubated with the cap loose and no seal is required. Aerobes grow at the top whilst more anaerobic species grow deeper in the medium.
	Cultural characteristics were observed after incubation at 35-37°C for 40-48 hours taking inoculum size 50-100 CFU-
Clostridium botulinum ATCC 25763	Luxuriant growth
Clostridium tetani	Luxuriant growth, proteolysis (blacking of the medium)
Clostridium sporogenes ATCC 11437	Luxuriant growth
Enterococcus faecalis ATCC 29212	Luxuriant growth
Streptococcus pneuminiae ATCC 6303	Luxuriant growth
Positive controls	Expected results
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

Note: To say no growth reporting, there should be blindly subcultured into blood agar and finally incubated aerobically and anaerobically.

Keynotes on RCM Medium

1. For the maintenance of stock cultures, hold the growth tube at room temperature after the initial incubation at 35°C and subculture it every 4-6 months.
2. Storage conditions and Shelf life of the medium: Store the dehydrated medium at 10-30°C and use it before the expiry date on the label while storing the prepared medium at room temperature, in the dark with tightened caps for up to 6 months.
3. It is an excellent medium for the primary growth and maintenance of aerobic and anaerobic organisms.
4. The color and Clarity of the prepared medium are amber-colored, clear to slightly opalescent supernatant over insoluble granules.
5. RCM is used for the cultivation of aerobic, microaerophilic, and anaerobic microorganisms, especially Clostridia, and it supports the growth of both spore-forming and non-spore-forming obligate anaerobes.
6. FDA has recommended RCM for the enumeration and identification of Clostridium perfringens from Foods.
7.  The excellent recovery properties of RCM mean that mixed cultures commonly result from sample inoculation.
8. Blackening of the medium will not occur if the pH is acid.
9. Carbohydrate fermentation may inhibit proteolysis.
10. Oxygen in culture media can be reduced by various reducing agents such as glucose, thioglycolate, glutathione of cooked meat pieces, cysteine, and ascorbic acid.
11. Clostridium species may be divided into two main groups by their action on the medium.

(i) Saccharolytic group: There is rapid production of acid and gas but no digestion of the meat. Cultures may have a slightly sour smell, with reddened protein.

(ii) Proteolytic group: Proteolysis causes the decomposition of the meat with the formation of foul-smelling sulfur compounds and blackening. However, some saccharolytic strains also produce Hydrogen sulfide which will cause blackening but to a lesser degree.

Gallery of RCM

Specimen inoculated RCM having bacterial growth

Bacterial growth in RCM

Robertson’s Cooked Meat with Proteolytic anaerobe (black)

Robertson’s Cooked Meat (RCM) Medium Showing Left-Saccharolytic anaerobes(red) and Proteolytic anaerobes (black) whereas Right-Uninoculated RCM

Fungal growth over RCM

Fungal growth on RCM-left-Aspergillus niger, right-Aspergillus flavus

Bacterial and fungal growth on RCM

Further Readings

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