CRITERION™ TCBS (THIOSULFATE CITRATE BILE SALTS SUCROSE) AGAR

Cat. no. C7040 CRITERION™ TCBS Agar 176gm
Cat. no. C7041 CRITERION™ TCBS Agar 500gm
Cat. no. C7042 CRITERION™ TCBS Agar 2kg
Cat. no. C7043 CRITERION™ TCBS Agar 10kg
Cat. no. C7044 CRITERION™ TCBS Agar 50kg

INTENDED USE

Hardy Diagnostics CRITERION™ TCBS (Thiosulfate Citrate Bile Salts Sucrose) Agar is recommended for the selective isolation and cultivation of Vibrio spp. from clinical specimens.

This dehydrated culture medium is a raw material intended to be used in the making of prepared media products, which will require further processing, additional ingredients, or supplements.

SUMMARY

TCBS Agar is prepared according to the formula developed by Kobayashi, et al.(6) It is highly selective for the isolation of V. cholerae and V. parahaemolyticus, in addition to other Vibrio spp. TCBS has a very high pH (8.5-9.5) which suppresses growth of intestinal flora other than Vibrio spp.(5) The medium consists of plant and animal proteins, a mixture of bile salts, one percent sodium chloride, sodium thiosulfate, ferric citrate, sucrose, and yeast extract. The bile salts inhibit growth of gram-positive microorganisms; one percent sodium chloride is incorporated into the medium to provide optimum growth and metabolic activity of halophilic Vibrio spp.; sodium thiosulfate provides a source of sulfur and also acts in combination with ferric citrate to detect the production of hydrogen sulfide; sucrose serves as the fermentable carbohydrate that, with the help of bromothymol blue and thymol blue indicators, allows for the differentiation of those Vibrio spp. which utilize sucrose.

V. cholerae and its biotype Eltor ferment sucrose, which results in a pH shift and production of yellow-brown colonies. According to Fishbein, et al., V. parahaemolyticus will produce light bluish colonies.(7) Certain strains of Proteus and enterococci may grow and produce small, yellow colonies that are easily distinguished.

Vibrio species that are considered medically important can be divided into two groups, V. cholerae and the non-cholera Vibrio spp.(5) They are as follows:

V. cholerae :
V. cholerae serogroup O1
V. cholerae serogroup non O1
V. cincinnatiensis
V. damsela
V. fluvialis
V. furnissii
V. hollisae
V. metschnikovii
V. mimicus
V. parahaemolyticus
V. vulnificus (lactose-fermenter)
Non-cholera Vibrio spp.:
V. alginolyticus
V. carchariae

Vibrio cholerae is the causative agent of cholera. Other Vibrio species have been associated with gastroenteritis and extraintestinal infections, especially of the ear, soft tissue, and blood. Life-threatening septicemia has been linked to V. vulnificus . Most Vibrio infections are associated with seawater contact. Symptoms are often similar to more common inland microbial agents.

FORMULA

Gram weight per liter: 88.0gm/L
Sucrose 20.0gm
Agar 14.0gm
Sodium Chloride 10.0gm
Dipeptone 10.0gm
Sodium Citrate 10.0gm
Sodium Thiosulfate 10.0gm
Oxbile (Oxgall) 5.0gm
Yeast Extract 5.0gm
Sodium Cholate 3.0gm
Ferric Citrate 1.0gm
Bromothymol Blue 0.04gm
Thymol Blue 0.04gm

Final pH 8.6 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Store the sealed bottle(s) containing dehydrated culture medium at 2-30ºC. Dehydrated culture medium is very hygroscopic. Keep lid tightly sealed. Protect dehydrated culture media from moisture and light. The dehydrated culture media should be discarded if it is not free-flowing or if the color has changed from its original light tan with greenish tinge.

Store the prepared culture media at 2-8ºC.

PRECAUTIONS

METHOD OF PREPARATION FOR DEHYDRATED CULTURE MEDIA

1. Suspend 89.0gm of the dehydrated culture media in 1 liter of distilled or deionized water.

2. Heat to boiling and mix to dissolve completely.

3. Heat Sensitive. Do not autoclave.

4. Cool to 45-50ºC. and dispense into sterile petri dishes or as desired.

PROCEDURE AND INTERPRETATION OF RESULTS

For information on procedures and interpretation of results, consult listed references or refer to the prepared media Instructions for Use (IFU) for Cat. No. G55.

LIMITATIONS

TCBS Agar may not support good growth of some Vibrio spp. (e.g., V. hollisae and V. metschnikovii). The identification of the various Vibrio spp. on TCBS Agar is presumptive and further tests are required for confirmation.

Sucrose-fermenting Proteus species produce yellow colonies which resemble those of Vibrio spp.

It recommended that a non-selective media be used in conjunction with selective media for optimum recovery of pathogenic organisms.

Cultures grown on TCBS Agar should be examined immediately after removal from the incubator as yellow colonies of vibrios, e.g., V. cholerae may revert to a green color when left at room temperature.(8)

Most Vibrio spp. and/or colonies that appear yellow on TCBS Agar will produce unsatisfactory oxidase reactions.

If slide agglutination tests are to be carried out, organisms must be subcultured to nutrient agar. Colonies taken from TCBS Agar react poorly in slide agglutination tests due to their 'sticky' nature.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as autoclaves, incinerators, and incubators, etc., are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Vibrio parahaemolyticus
ATCC ® 17802
A 18-24hr 35°'C Aerobic Growth; blue-green centered colonies
Escherichia coli
ATCC ® 25922
B 18-24hr 35°C Aerobic Partial to compete inhibition; small, clear colonies
Proteus mirabilis
ATCC ® 12453
B 18-24hr 35°C Aerobic Partial to complete inhibition; small, clear to yellow colonies

User Quality Control

PHYSICAL APPEARANCE

CRITERION™ TCBS Agar powder should appear homogeneous, free-flowing, and light tan with a greenish tinge in color. The prepared media should appear slightly opalescent with no precipitate, and dark green in color.

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

3. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

5. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

6. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

7. U.S. Food and Drug Administration. Bacteriological Analytical Manual. AOAC, Arlington, VA.
http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm2006949.htm.

8. Kobayashi, T., et al. 1963. Jap. J. Bacteriol.; 18:387.

9. Applied Microbiology; 20:176, 1970.

10. Furniss, A.L., et al. 1978. PHLS Monograph; No. 11.


ATCC is a registered trademark of the American Type Culture Collection.

062116gr