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REINFORCED CLOSTRIDIAL AGAR (RCM AGAR)

Code: CM0151

A solid medium for the cultivation and enumeration of anaerobes, especially Clostridium species.

* Adjusted as required to meet performance standards

Directions
Suspend 52.5g in 1 litre of distilled water. Bring to the boil to dissolve completely. Sterilise by autoclaving at 121°C for 15 minutes.

Description
Reinforced Clostridial Agar is a solid version of Oxoid Reinforced Clostridial Medium CM0149, suitable for the cultivation and enumeration of clostridia and other anaerobes, lactobacilli, and many other species of bacteria. Reinforced Clostridial Agar does not differ significantly in performance from the pork-starch-pea agar of Anderson¹ for the count of anaerobes. It is employed for the estimation of clostridia in food - see below (also Barnes et al.² and Angelotti³). Attenborough and Scarr⁴ employed RCM Agar, in conjunction with Membrane Filters, for the count of Clostridium thermosaccharolyticum in sugar.

Reinforced Clostridial Agar is also frequently employed for the investigation of intestinal flora: Perry et al.⁵ for investigation of bovine rumen streptococci; Williams Smith & Crabb⁶ used the Oxoid medium with added sodium chloride or blood for counts on human or animal faeces; Barnes & Goldberg⁷ employed the Oxoid medium with added chlortetracycline hydrochloride or sodium azide and ethyl violet, for the examination of poultry faeces; the Oxoid medium was also used by Goldberg et al.⁸ for the examination of poultry faecal samples. Williams Smith⁹ employed Oxoid Reinforced Clostridial Agar with added blood for the `total’ and Lactobacillus count of human and animal faeces; with added blood and neomycin for determination of Bacteroides, anaerobic Gram-negative cocci, `total’ streptococci, sporeformers, yeasts, and `actino’ types.

Sneath¹⁰ used Oxoid RCM Agar and other media for the anaerobic count of micro-organisms from soil samples up to approximately 300 years old. Gregory et al.¹¹ also employed the Oxoid medium for the estimation of anaerobes in moulding hay.

Technique
Barnes and Ingram^12,13described the use of RCM Agar for the total viable count of clostridia employing their black glass rod technique. In this method the diluted sample is added to plugged test tubes containing about 9 ml of RCM Agar held at 48°C. The tubes are quickly rotated to mix the contents, and a sterile black glass rod inserted into each tube before the agar sets. They are then sealed with about 1.5cm of RCM Agar containing 1/20,000 methylene blue. Growth is very rapid in this medium and it is necessary to count the colonies before gas production disrupts the agar. The authors suggest that the tubes should be incubated overnight at 25°C and then at 35°C for several hours. The colonies are clearly visible against the black background.

Oxoid RCM Agar may also be used for enumerating anaerobes using the Miller-Prickett technique (Miller et al.¹⁴). The Miller-Prickett tube is a flattened test tube 15 x 2.5 x 1.3 cm.

Mossel et al.¹⁵, although working with other media, suggested the following procedure which may be used with RCM Agar:
1. Transfer, in triplicate, 1 ml of serial decimal dilutions of the food under investigation into sterilised, plugged Miller- Prickett tubes. Cool the freshly prepared medium to approximately 50°C and, without shaking, add about 15ml to each tube.
2. Seal immediately with melted sterile paraffin, and allow to set in a water bath at about 15°C.
3. Incubate for 1 to 10 days at a temperature between 30°C and 55°C, depending on the type of clostridia expected.
4. Run at least one blank to detect contamination occurring during the procedure.
5. Count colonies.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared medium at 2-8°C.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Light straw to straw coloured gel

Quality control

* This organism is available as a Culti-Loop®

Precautions
Further identification tests must be carried out on organisms isolated from this medium.

References
1. Anderson A. A. (1951) J. Bact. 62. 425-430.
2. Barnes Ella M., Despaul J. E. and Ingram M. (1963) J. Appl. Bact. 26. 415-427.
3. Angelotti R., Hall H. E., Foter M. J. and Lewis K. H. (1962) Appl. Microbiol. 10. 193-199.
4. Attenborough Sheila J. and Scarr M. Pamela (1957) J. Appl. Bact. 20. 460-466.
5. Perry K. D., Wilson M. K., Newland L. G. M. and Briggs C. A. E. (1955) J. Appl. Bact. 18. 436-442.
6. Williams Smith H. and Crabb W. E. (1961) J. Path. Bact. 82. 53-66.
7. Barnes Ella M. and Goldberg H. S. (1962) J. Appl. Bact. 25. 94-106.
8. Goldberg H. S., Barnes Ella M. and Charles A. B. (1964) J. Bact. 87. 737-742.
9. Williams Smith H. (1961) J. Appl. Bact. 24. 235-241.
10. Sneath P. H. A. (1962) Nature 195. 643-646.
11. Gregory P. H., Lacey M. E., Festenstein G. N. and Skinner F. A. (1963) J. Gen. Microbiol. 33. 147-174.
12. Barnes Ella M. and Ingram M. (1956) J. Appl. Bact. 19. 117-128.
13. Ingram M. and Barnes Ella M. (1956) Lab. Practice 5. 145.
14. Miller N. J., Garrett O. W. and Prickett P. S. (1939) Food Res. 4. 447-451.
15. Mossel D. A. A., De Bruin A. S., Diepen H. M. J., van Vendrig C. M. A. and Zoutwelle G. (1956) J. Appl. Bact. 19. 142-154.