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Dehydrated Culture Media

ROSE-BENGAL CHLORAMPHENICOL AGAR

Code: CM0549

for the selective enumeration of moulds and yeasts from foods

Typical Formula*

gm/litre

Mycological peptone

5.0

Glucose

10.0

Dipotassium phosphate

1.0

Magnesium sulphate

0.5

Rose-Bengal

0.05

Agar

15.5

pH 7.2 ± 0.2 @ 25°C

 
* Adjusted as required to meet performance standards

CHLORAMPHENICOL SUPPLEMENT

Code: SR0078

Vial contents

SR0078E
(1 vial per 500ml medium)

SR0078H
(1 vial per 2 litres medium)

per litre

Chloramphenicol

50mg

200mg

100mg

Directions
Suspend 16.0g per 500ml (32.0g/l) of distilled water and bring to the boil to dissolve completely. Reconstitute one vial SR0078E per 500ml medium or one vial SR0078H per 2 litres medium, as directed. Add the vial contents to Rose Bengal Chloramphenicol Agar Base (CM0549) and mix gently. Autoclave at 121°C for 5 minutes. Cool to 50°C, mix gently and pour into Petri dishes.

Description
Rose-Bengal Chloramphenicol Agar is a selective medium for the enumeration of yeasts and moulds from a wide variety of foodstuffs. The medium has a neutral pH and chloramphenicol is used as a selective agent to suppress the growth of bacteria. Several investigators have noted advantages in the use of media at neutral pH containing antibiotics1,2.

Rose-Bengal is taken up by mould and yeast colonies thereby assisting enumeration of small colonies3. Rose-Bengal also controls the size and height of mould colonies, such as Neurospora and Rhizopus spp. Over-growth of slow growing strains by more luxuriant species is thus prevented and plate counting is assisted.

The choice of a suitable medium for enumeration of yeasts and moulds is greatly dependent on the nature of the foodstuffs under investigation and the organisms that occur on them4. Rose-Bengal Chloramphenicol Agar is recommended for fresh proteinaceous foods whose associated flora consists mainly of Gram negative rod-shaped bacteria although it should be noted that chloramphenicol alone may not be sufficient to inhibit the bacterial background. Because of the stability of chloramphenicol, Rose-Bengal Chloramphenicol Agar is also suitable when higher and prolonged incubation temperatures around 35°C are required.

Technique
Add 1ml aliquots of a suitable series of decimal dilutions to empty 9cm Petri dishes. Two dishes are used for each dilution. Then add to each dish approximately 15ml of medium cooled to 50°C. Mix gently, turning the plates three times clockwise and three times counter clockwise.
Allow the medium to gel then turn the petri dishes upside down and incubate them for 5 days at 22 ± 2°C.

Inspect the dishes and count the colonies on those that contain an estimated 50-100 colonies. Calculate the number of yeasts or moulds per 1g or 1ml by multiplying the number of colonies by the dilution factor. Consult the appropiate references for further information5,6,7.

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 away from light.
NB: Rose-Bengal photo-oxidises to form toxic compounds. Store plates of the medium in the dark and avoid exposure to light8.

Appearance
Dehydrated medium: Pink, free-flowing powder
Prepared medium: Pink gel

Quality control

Positive controls:

Expected results

Saccharomyces cerevisiae ATCC® 9763 *

Good growth; pink coloured colonies

Aspergillus niger ATCC® 16404*

White mycelia, black spores

Negative controls:

 

Escherichia coli ATCC® 25922 *

No growth

Enterococcus faecalis ATCC® 29212 * No growth
* This organism is available as a Culti-Loop®

Precautions
It is essential to store plates of media containing Rose-Bengal in the dark to prevent toxic photo-oxidation of the dye. See above.
Identify moulds and yeasts by morphological appearance and microscopic examination. Colonies of bacteria and yeasts can be confused.

References
1. Mossel D. A. A., Visser M. and Mengerink W. H. J. (1962) Lab. Pract. 11. 109-112.
2. Koburger J. A. (1968) Bact. Proc. 13. A73.
3. Jarvis B. (1973) J. Appl. Bact. 36. 723-727.
4. Mossel D A. A., Vega C. L. and Put H. M. C. (1975) J. Appl. Bact. 39. 15-22.
5. American Public Health Association (1976) Compendium of Methods for the Microbiological Examination of Foods. APHA Inc. Washington DC.
6. American Public Health Association (1978) Standard Methods for the Examination of Dairy Products. 14th Edn. APHA Inc. Washington DC.
7. American Public Health Association (1981) Standard Methods for the Examination of Water and Wastewater. 15th Edn. APHA Inc. Washington DC.
8. Kramer C. L. and Pady S. M. (1961) Kansas Academy of Science Vol. 64 No. 2 1961. Inhibition of growth of Fungi on Rose-Bengal media by light.

 
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