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Material Safety Data Sheet

Organisms

Organisms in the Clinical sector this product works with:

Dehydrated Culture Media

Sector: Clinical

CHOLERA MEDIUM TCBS

Code: CM0333

A selective isolation medium for pathogenic vibrios.

Typical Formula*

gm/litre

Yeast extract

5.0

Bacteriological peptone

10.0

Sodium thiosulphate

10.0

Sodium citrate

10.0

Ox Bile

8.0

Sucrose

20.0

Sodium chloride

10.0

Ferric citrate

1.0

Bromothymol blue

0.04

Thymol blue

0.04

Agar

14.0

pH 8.6 ± 0.2 @ 25°C

* Adjusted as required to meet performance standards 

Directions
Suspend 88g in 1 litre of distilled water. Boil to dissolve the medium completely. DO NOT AUTOCLAVE.
Pour plates without further heating and dry before use.

Description
Kobayashi, Enomoto, Sakazaki and Kuwahara1 developed TCBS media from the selective isolation agar of Nakanishi2.

The Oxoid TCBS medium conforms to the formulation of Kobayashi et al., except that it contains specially processed ox bile, free from the defects noted by Nakanishi and Kobayashi.


The complexity of the composition of this medium means that uniformity of growth is a difficult standard to maintain. Several investigations have shown variation between batches of TCBS Medium made by different companies3,4,5,6.

Quality control by the manufacturers of this medium is especially important because satisfactory inhibition of normal gut flora and lack of inhibition of certain Vibrio species is very critical. West et al.7 showed that Oxoid TCBS Medium came closest to their criteria for a satisfactory product.
WHO has established a minimum acceptable guideline for the recovery of Vibrio species on TCBS Medium8.

The Oxoid medium is suitable for the growth of Vibrio cholerae, Vibrio parahaemolyticus, and most other Vibrios9.

Most of the Enterobacteriaceae encountered in faeces are totally suppressed for at least 24 hours. Slight growth of Proteus species and Enterococcus faecalis may occur but the colonies are easily distinguished from vibrio colonies.


Oxoid TCBS Medium is complete and requires no additives or aseptic additions of blood. It therefore shows a considerable advantage over Lauryl Sulphate Tellurite Agar which requires further additions after sterilisation. Apart from this convenience factor, it also possesses superior growth characteristics for Vibrio species, compared with tellurite media. Whilst inhibiting non-vibrios, it promotes rapid growth of pathogenic vibrios after overnight incubation at 35°C. For the isolation of other vibrios from environmental samples, incubation at lower temperatures, 20-30°C is needed.

Colonial appearance of organisms on TCBS medium
24 hours incubation at 35°C.

Organisms

Colonies

Vibrio cholera and El Tor type

Yellow, flat, 2-3mm diameter

Vibrio parahaemolyticus

Blue-green, 3-5mm diameter

Vibrio alginolyticus

Yellow, 3-5mm diameter

Vibrio metschnikovii 10

Yellow, 3-4mm diameter

Vibrio fluvialis 11

Yellow, 2-3mm diameter

Vibrio vulnificus 12

Blue-green, 2-3mm diameter

Vibrio mimicus 13

Blue-green, 2-3mm diameter

Enterococcus species

Yellow, 1mm diameter

Proteus species

Yellow-green, 1mm diameter

Pseudomonas species

Blue-green, 1mm diameter

Some strains of Aeromonas hydrophila grow producing yellow colonies but Plesimonas shigelloides does not usually grow well on TCBS.

Technique
Streak the faeces or a subculture from an enrichment medium, e.g. Alkaline Peptone Water, across the surface of Oxoid TCBS Cholera Medium. Incubate plates for 18-24 hours at 35°C for clinical specimens or lower temperature for environmental samples.

Cultures grown on TCBS should be examined quickly after removal from an incubator as the yellow colonies of Vibrio cultures e.g. Vibrio cholerae, may revert to a green colour when left at room temperature9.

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/green coloured, free-flowing powder
Prepared medium: Green coloured gel

Quality control

Positive controls:

Expected results

Vibrio furnissii NCTC 11218 (a non-pathogenic strain6)

Good growth; yellow colonies

Vibrio parahaemolyticus NCTC  10885

Good growth; green colonies

Negative control:

 

Escherichia coli ATCC® 25922 *

No growth

* This organism is available as a Culti-Loop®

Precautions
The identification of the various Vibrio species on TCBS Medium is presumptive and further tests are required for confirmation.
Yellow colonies on TCBS Medium will give unsatisfactory oxidase reactions.
Colonies taken from TCBS Medium are `sticky’ and react poorly in slide agglutination tests. Subculture to nutrient agar is required before slide agglutination tests can be carried out.
Some strains of Vibrio vulnificus produce better recovery at 30°C.

References
1. Kobayashi T., Enomoto S., Sakazaki R. and Kuwahara S. (1963) Jap. J. Bacteriol. 18. 10-11, 387-311.
2. Nakanishi Y. (1963) Modern Media 9. 246.
3. McCormack W. M., DeWitt W. E., Bailey P. E., Morris G. K., Socharjono P. and Gangarosa E. J. (1974) J. Inf. Dis. 129. 497-500.
4. Morris G. K., Merson M. H., Huq I., Kibrya A. K. M. G. and Black R. (1979) J. Clin. Microbiol. 9. 79-83.
5. Nicholls K. M., Lee J. V. and Donovan T. J. J. Appl. Bact. 41. 265-269.
6. Taylor J. A. and Barrow G. I. (1981) J. Clin. Path. 34. 208-212
7. West P. A., Russek E., Brayton P. R. and Colwell P. R. (1982) J. Clin. Microbiol. 16. 1110-1116.
8. WHO Scientific Working Group (1980) Bull. WHO 58. 353-374.
9. Furniss A. L., Lee J. V. and Donovan T. J. (1978) The Vibrios. PHLS Monograph No. 11.
10. Lee J. V., Donovan T. J. and Furniss A. L. (1978) Int. J. Sys. Bact. 28. 99-111.
11. Lee J. V., Shread P., Furniss A. L. and Bryant T. N. (1981) J. Appl. Bact. 50. 73-94.
12. Farmer J. J. 111 (1979) The Lancet. 2. 903.
13. Davis B. R., Fanning G. R., Madden J. M., Steigerwall A. G., Bashford H. B., Smith H. L. and Brenner D. J. (1981) J. Clin. Microbiol. 14. 631-639.

 
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