CLOSTRIDIUM DIFFICILE AGAR BASE
CAT Nº: 1447
For the isolation of Clostridium difficile when used with supplements
FORMULA IN g/l
Peptone Proteose
40.00
Magnesium Sulfate
0.10
Fructose
6.00
Sodium Chloride
2.00
Disodium Hydrogen Phosphate
5.00
Bacteriological Agar
15.00
Potassium Dihydrogen Phosphate
1.00
Final pH 7.4± 0.2 at 25ºC
PREPARATION
Suspend 34.5 grams of the medium in 500ml of distilled water. Mix well and dissolve by heating with frequent agitation.
Boil for one minute until complete dissolution. Sterilize in autoclave at 121ºC for 15 minutes. Cool to 45-50ºC and
aseptically add one vial of Clostridium Difficile Supplement (Cat. 6061), previously reconstituted in 5 ml of sterile distilled
water and 7% sterile defibrinated horse blood. Homogenize gently and dispense into Petri dishes. Be careful to avoid
bubble formation when adding the blood and rotate the flask or bottle slowly to create a homogeneous solution. The
prepared medium should be stored at 8-15°C. The color of the prepared medium with the blood added is brown. Sheep
blood may be used instead of horse blood, but some strains of the organisms will show a slightly reduced growth
recovery.
The dehydrated medium should be homogeneous, free flowing and beige in color. If there are any physical changes,
discard the medium.
Clostridium Difficile Supplement (Cat. 6061)
(Composition: one vial for 500 ml)
D-Cycloserine……………………………………….125.0 mg
Cefoxitin………………………………………………….4.0 mg
USES
CLOSTRIDIUM DIFFICILE AGAR BASE when used with supplement is a selective medium for the isolation of Clostridium
difficile from fecal specimens.
Clostridium difficile was first isolated from meconium and infant feces in 1935 by Hall and O’Toole, who proposed the
name “difficile” because it was very difficult to isolate. Keighley associated Clostridium difficile with colitis and diarrhea
without pseudomembranous changes after antibiotic therapy following gastrointestinal operations.
Peptone Proteose provides nitrogen, vitamins, minerals and amino acids essential for growth. Fructose is the
fermentable carbohydrate used to enhance recovery and growth of C. difficile. Potassium dihydrogen phosphate and
Disodium hydrogen phosphate act as a buffer system. Magnesium Sulfate is a magnesium ion required in a large
variation of enzymatic reactions, including DNA replication. Sodium chloride supplies essential electrolytes for transport
and osmotic balance. Horse blood provides essential growth factors in Clostridium Difficile Agar. Bacteriologycal Agar is
the solidifying agent.
The combination of Clostridium Difficile Agar with the supplement is based on the formulation proposed by George W. L,
Sutter V.L., Goldstein E.C.J., Ludwig S.L. and Finegold S.M. The selective agents D-Cycloserine, and Cefoxitin inhibit the
growth of most Enterobacteriaceae, as well as Enterococcus faecalis, staphylococci, gram-negative, non-sporing
anaerobic bacilli, and Clostridia species (except Clostridium difficile), which may be found in large quantities in fecal
samples.
1
LABORATORIOS CONDA, S.A.
www.condalab.com
Incubate at 35 - 37ºC and observe after 18-48 hours in anaerobic conditions. After 48 hours Clostridium difficile colonies
grow circular, raised, opaque grey, sometimes with irregular borders, and 4-6 mm in diameter.
MICROBIOLOGICAL TEST
The following results were obtained in the performance of the medium from type cultures, with Clostridium Difficile
Supplement (Cat. 6061) and 7% defibrinated Horse Blood added, after incubation at a temperature of 35- 37ºC and
observed after 18-48 hours.
Microorganisms
Growth
Clostridium difficile NCTC 11204
Staphylococcus aureus ATCC 25923
Escherichia coli ATCC 25922
Good
Inhibited
Inhibited
BIBLIOGRAPHY
George W. L., Sutter V.L., Goldstein E.C.J., Ludwig S.L and Finegold S.M (1978) Lancet. i. 802-803
Hall I. and O’Toole E. (1935) am. J. Dis. Child. 49. 390.
Keighley M.R.B, Burdon D.W., Alexander Williams J. et al (1978) Lancet ii. 1165-1167.
STORAGE
25ºC
Once opened keep powdered medium closed to avoid hydration.
2ºC
2
LABORATORIOS CONDA, S.A.
www.condalab.com