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Culture Media Supplements

CLOSTRIDIUM DIFFICILE AGAR BASE

Code: CM0601

for the isolation of Clostridium difficile when used with Culture Media Supplements SR0096 or SR0173

Typical Formula*

gm/litre

Proteose peptone

40.0

Disodium hydrogen phosphate

5.0

Potassium dihydrogen phosphate

1.0

Magnesium sulphate

0.1

Sodium chloride

2.0

Fructose

6.0

Agar

15.0

pH 7.4 ± 0.2 @ 25°C

 
*Adjusted as required to meet performance standards
CLOSTRIDIUM DIFFICILE SELECTIVE SUPPLEMENT

Code: SR0096

Vial contents (each vial is sufficient for 500mls of medium)
per vial
per litre

D-cycloserine

125.0mg

250.0mg

Cefoxitin

4.0mg

8.0mg

Directions
Suspend 34.5g in 500ml of distilled water and bring gently to the boil to dissolve completely. Sterilise by autoclaving at 121°C for 15 minutes. Allow to cool to 50°C and add aseptically the contents of 1 vial of Oxoid Clostridium Difficile Supplement (SR0096) reconstituted as directed, together with 7% (v/v) Defibrinated Horse Blood (SR0050). Sheep Blood (SR0051) may be used in place of Horse Blood but some strains of the organism will show a slightly reduced growth recovery. Mix well and pour into sterile Petri dishes.

Description
Clostridium difficile was first isolated in 1935 by Hall and O’Toole1 who proposed the name `difficile’ because it was very difficult to isolate. In 1940 Snyder2 isolated Clostridium difficile from infants aged 10 weeks to 1 year. No further isolations were reported until 1960, when the organism was cultured by McBee3 from the intestinal contents of a seal, and in 1962 Smith and King4 reported its presence in human infections.
Toxicogenic isolates of Clostridium. difficile have been demonstrated to be a major cause of antibiotic-associated ileo-caecitis in laboratory animals5 and pseudomembranous colitis in man6,7. Keighley8 found Clostridium difficile was associated with colitis and diarrhoea without pseudomembranous changes after antibiotic therapy following gastrointestinal operations.

Hafiz and Oakley9 devised a medium for the selective isolation of Clostridium difficile based on the observation that the organism has a high tolerance to cresol, which it produces during its growth, and used Reinforced Clostridial Medium (CM0151) plus 0.2% phenol or p-cresol.
George et al10 in a study of selective media for the routine isolation of Clostridium difficile from faecal specimens found this medium was inhibitory compared with growth on blood agar. They recommended the use of a fructose containing nutrient medium plus egg yolk, with D-cycloserine and cefoxitin as selective agents for the isolation of Clostridium difficile.

The combination of Oxoid Clostridium difficile Agar Base plus the Culture Media Supplement (SR0096) is based on the formulation proposed by George et al.10

The selective agents D-cycloserine (500µg/ml) and cefoxitin (16µg/ml) inhibit growth of the majority of Enterobacteriaceae, as well as Streptococcus faecalis, staphylococci, Gram-negative non-sporing anaerobic bacilli and Clostridia species. (except Clostridium difficile) which may be found in large numbers in faecal samples.

Levett11, noting reports12,13 that some strains of Clostridium difficile had low minimum inhibitory concentrations to both cycloserine and cefoxitin, reduced the antibiotic concentrations to 125µg per ml cycloserine and 4µg per ml cefoxitin and combined this with alcohol shock14 to compensate for the reduction in selectivity. Clostridium difficile was isolated from all of the 33 faecal specimens plated on to CCFA Medium containing cycloserine and cefoxitin at 250µg per ml and 8µg per ml respectively, but from only 25/33 specimens plated on to medium containing 500µg per ml cycloserine and 16µg per ml cefoxitin. The specimen should be treated with alcohol before inoculation (see technique).

It can be expected that medium containing the lower concentration of antibiotics will yield a greater growth of contaminating organisms if antibiotics are used alone, but Levett reported that there was no difference in the growth of contaminating organisms on plates containing either concentration of antibiotics following alcohol shock treatment of the specimen.

Phillips and Rogers15 have described a simple modification to the medium in which the ability of Clostridium difficile to produce p-cresol from p-hydroxyphenyl acetic acid is used for the rapid presumptive identification by gas chromatographic detection of the p-cresol. Addition of 7% horse blood to the agar base increases the recovery of Clostridium difficile and produces larger colonies compared with Egg Yolk Emulsion used by George et al.10

Technique

  1. Lightly inoculate the medium with the faecal sample spreading part of the original inoculum in order to obtain well separated colonies.
  2. Incubate plates at 35°C for 18-24 hours in a conventional anaerobic gas jar. The use of the Oxoid Anaerobic Jar (HP0011) with an Anaerobic Gas Generating Kit (BR0038) is strongly recommended. Alternatively use Anaerogen (AN0025 or AN0035). Anaerogen does not require the addition of water or a catalyst.
  3. Colonies of Clostridium difficile after 48 hours incubation are 4-6mm diameter irregular, raised opaque, grey-white.

Technique for Alcohol Shock Treatment (if required)

  1. Mix equal parts of industrial methylated spirit or absolute alcohol and the faecal specimen.
  2. Homogenise using a vortex mixer.
  3. Leave at room temperature for 1 hour.
  4. Inoculate on to Clostridium difficile Selective Agar and incubate anaerobically.

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 no longer than 5-7 days.

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

Quality control

Positive control:

Expected results

Clostridium difficile NCTC 11204

Good growth; grey-white coloured colonies 

Negative controls:

 
Staphylococcus aureus ATCC® 25923*No growth

Escherichia coli ATCC® 25922*

No growth

* This organism is available as a Culti-Loop®

Precautions
Colonies of Clostridium difficile from faecal cultures are smaller when egg yolk is used in place of horse blood.

The Oxoid formula does not contain the neutral red indicator proposed by George et al.10 because it is designed for use with horse blood. On this medium the typical colour of the colony of Clostridium difficile will not appear however there will be a fluorescent reaction.

Typical Gram stain morphology of Clostridium difficile may not be evident in colonies picked from this medium because of the antibiotics present. Subculture to blood agar to obtain characteristic morphology10.

CLOSTRIDIUM DIFFICILE MOXALACTAM NORFLOXACIN (CDMN) SELECTIVE SUPPLEMENT

Code: SR0173

Vial contents (each vial is sufficient for 500 ml of medium)

per vial

per litre

Cysteine hydrochloride

250.0mg

500.0mg

Norfloxacin

6.0mg

12.0mg

Moxalactam

16.0mg

32.0mg

Directions
Aseptically add 2ml of sterile distilled water to a vial and mix gently to dissolve the supplement completely. Avoid frothing. Add to 500ml of Clostridium difficile Agar Base, prepared as directed and cooled to 50°C. Add 7% v/v of Defibrinated Horse Blood (SR0050). Mix well and pour into Petri dishes.

Description
Clostridium difficile CDMN medium is an alternative selective medium based on a formula described by Aspinall et al.16 for the isolation of Clostridium difficile from faeces. It has been found to be significantly more productive than CCFA medium. Inclusion of cysteine hydrochloride speeds the growth rate of Clostridium difficile. CDMN medium was reported to isolate 20% more Clostridium difficile strains than CCFA and the use of norfloxacin and moxalactam as selective agents reduces the number of contaminating micro-organisms by 30% when compared to CCF16.

Pre-treatment of specimens with alcohol is not necessary with this medium but its use will further enhance selectivity. See Clostridium difficile Selective Supplement (SR0096) for the technique.

Storage conditions and Shelf life
CDMN supplement (SR0173) should be stored at 2-8°C in the dark.

Positive control:

Expected results

Clostridium difficile NCTC 11204

Good growth; grey-white coloured colonies

Negative controls:

 
Escherichia coli ATCC® 25922*No growth

Clostridium perfringens ATCC® 13124

No growth


References
1. Hall I. and O’Toole E. (1935) Am. J. Dis. Child. 49. 390.
2. Snyder M. L. (1940) J. Infect. Dis. 66. 1.
3. McBee R. H. (1960) J. Bact. 79. 311.
4. Smith L. D. S. and King E. O. (1962) J. Bact. 84. 65.
5. Bartlett J. G., Onderdonk A. B., Cisneros R. L. and Kasper D. L. (1977) J. Infect. Dis. 136. 701-705.
6. Bartlett J. G., Chang T. W., Gurwith M., Gorbach S. L. and Onderdonk A. B. (1978) N. Engl. J. Med. 298. 531-534.
7. George W. L., Sutter V. L., Goldstein E. C. J., Ludwig S. L. and Finegold S. M. (1978) Lancet. i. 802-803.
8. Keighley M. R. B., Burdon D. W., Alexander Williams J. et al (1978) Lancet ii. 1165-1167.
9. Hafiz S. and Oakley C. L. (1976) J. Med. Microbiol. 9. 129-136.
10. George W. L., Sutter V. L., Citron D. and Finegold S. M. (1979) J. Clin. Microbiol. 9. 214-219.
11. Levett (1985) J. Clin. Pathol. 38. 233-234.
12. George W. L., Kirby B. D., Sutter V. L. and Finegold S. M. in Schlessinger D. Editor Microbiology 1979 Washington D.C. American Society for Microbiology, 2670271.
13. Dzink J. and Bartlett J. G. (1980) Antimicrob. Ag. Chemother. 17. 695-698.
14. Borriello S. P. and Honour H. (1981) J. Clin. Pathol. 34. 1124-1127.
15. Philips K. D. and Rogers P. A. (1981) J. Clin. Pathol. 34. 643-644.
16. Aspinall S.T. and Hutchinson D.N. (1992) J. Clin. Pathol. 45. 812-814.

 
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