TCBS (THIOSULFATE CITRATE BILE SALTS SUCROSE) AGAR

Cat. no. G55 TCBS Agar, 15x100mm Plate, 18ml 10 plates/bag

INTENDED USE

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

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

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

Ingredients per liter of deionized water:*

Sucrose 20.0gm
Dipeptone 10.0gm
Sodium Citrate 10.0gm
Sodium Thiosulfate 10.0gm
Sodium Chloride 10.0gm
Yeast Extract 5.0gm
Oxbile (Oxgall) 5.0gm
Sodium Cholate 3.0gm
Ferric Citrate 1.0gm
Bromothymol Blue 0.04gm
Thymol Blue 0.04gm
Agar 15.0gm

Final pH 8.6 +/- 0.2 at 25 degrees C.

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8 degrees C. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

The expiration date applies to the product in its intact packaging when stored as directed.

This product has the following shelf life from the date of manufacture:

90 Days: G55 TCBS Agar Plate

Refer to the keyword "Storage", in the Hardy Diagnostics software program HUGO™, for more information on storing culture media.

PRECAUTIONS

This product is for in vitro diagnostic use only and is to be used only by adequately trained and qualified laboratory personnel. Observe approved biohazard precautions and aseptic techniques. All laboratory specimens should be considered infectious and handled according to "standard precautions". The "Guideline for Isolation Precautions" is available from the Centers of Disease Control and Prevention at www.cdc.gov/ncidod/dhqp/gl_isolation.html .

For additional information regarding specific precautions for the prevention of the transmission of all infectious agents from laboratory instruments and materials, and for recommendations for the management of exposure to infectious disease, refer to CLSI document M-29: Protection of Laboratory Workers from Occupationally Acquired Infections: Approved Guideline.

Sterilize all biohazard waste before disposal.

Refer to the keyword "Precautions", in the Hardy Diagnostics software program HUGO™, for more information regarding general precautions when using culture media.

Refer to the keyword "MSDS", in the Hardy Diagnostics software program HUGO™, for more information on handling potentially hazardous material.

PROCEDURE

Specimen Collection: Infectious material should be submitted directly to the laboratory within two to three hours of collection. Specimens should be protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport medium and refrigerated until inoculation. Consult listed references for information on specimen collection. (1-5)

Method of Use: Media should be warmed to room temperature prior to inoculation. The specimen, or sampling from a well mixed transport medium, should be inoculated directly onto the TCBS Agar and streaked so as to obtain isolated colonies. Incubate aerobically at 35 degrees C. for 18-24 hours. Examine for characteristic colonial morphology. Cultures grown on TCBS should be examined immediately after removal from the incubator as yellow colonies of Vibrio spp. (e.g., V. cholerae ) may revert to a green color when left at room temperature. (8) Consult listed reference for additional information regarding typical colonial morphology. (2,3,5)

INTERPRETATION OF RESULTS

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.

Typical colonial morphology on TCBS Agar:
V. cholerae Yellow colonies.
V. parahaemolyticus Blue to green centered colonies.
V. alginolyticus , V. fluvialis , V. furnissii Yellow colonies.
V. mimicus , V. damsela Green colonies.
V. vulnificus Green (85%) or yellow (15%).
V. hollisae Green (very poor growth).
V. metschnikovii Yellow (reduced growth).
Proteus /Enterococci Partial to complete inhibition. If growth, small,
yellow to translucent colonies.
Pseudomonas / Aeromonas Partial to complete inhibition. If growth, blue colonies.
Escherichia coli Partial to complete inhibition. If growth, translucent colonies.

LIMITATIONS

It is recommended that biochemical and/or serological tests be performed on colonies from pure culture for complete identification.

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.

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

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

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.

Refer to the keyword "Limitations", in the Hardy Diagnostics software program HUGO™, for more information regarding general limitations on culture media.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

The following organisms are routinely used for testing at Hardy Diagnostics:

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Vibrio parahaemolyticus
ATCC ® 17802
B 18-24hr 35 o C Aerobic Growth; blue-green centered colonies
Escherichia coli
ATCC ® 25922
B 18-24hr 35°C Aerobic Partial to complete 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

Check for signs of contamination and deterioration. Users of commercially prepared media may be required to perform quality control testing with at least one known organism to demonstrate growth or a positive reaction; and at least one organism to demonstrate inhibition or a negative reaction (where applicable). Refer to the following keywords, in the Hardy Diagnostics software program HUGO™, for more information on QC: "Introduction to QC", "QC of Finished Product", and "The CLSI (NCCLS) Standard and Recommendations for User QC of Media". Also see listed references for more information. (1-5)

* Refer to the keyword "Inoculation Procedures", in the Hardy Diagnostics software program HUGO™, for a description of inoculation procedures.

PHYSICAL APPEARANCE

TCBS Agar should appear slightly opalescent with no precipitate, and dark green in color.

V. parahaemolyticus on TCBS Agar

Vibrio parahaemolyticus (ATCC ® 17802) colonies growing on TCBS Agar (Cat. no. G55). Incubated aerobically for 24 hours at 35 deg. C.

E. coli inhibited on TCBS Agar

Escherichia coli (ATCC ® 25922) growth inhibited on TCBS Agar (Cat. no. G55). Incubated aerobically for 24 hours at 35 deg. C.

REFERENCES

1. Anderson, N.L., et al. 2005. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Murray, P.R., et al. 2011. Manual of Clinical Microbiology, 10th ed. American Society for Microbiology, Washington, D.C.

3. Forbes, B.A., et al. 2007. Bailey and Scott's Diagnostic Microbiology, 12th ed. 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. 2006. Color Atlas and Textbook of Diagnostic Microbiology, 6th ed. J.B. Lippincott Company, Philadelphia, PA.

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

7. Applied Microbiology ; 20:176, 1970.

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


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

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