TREHALOSE FERMENTATION BROTH

Cat. no. Y205 Trehalose Fermentation Broth with Durham Tube,
13x100mm Tube, 5ml
20 tubes/box

INTENDED USE

Hardy Diagnostics Trehalose Fermentation Broth with Durham Tube is recommended for use in the determination of trehalose fermentation reactions of yeasts. When incubated at 42ºC., Trehalose Fermentation Broth aids in the presumptive identification of Candida glabrata.(8)

SUMMARY

Trehalose Fermentation Broth is a modification of the assimilation Trehalose Broth developed by Stockman and Roberts and Trehalose Fermentation Broth developed by Land, et al.(6,8) The medium employs the use of trehalose as the carbohydrate source and bromcresol green as the pH indicator.

Due to the increased frequency of severe infection by Candida glabrata in immunosuppressed patients, clinical laboratories are in need of a rapid presumptive identification system for this medically important yeast.(7)

The use of Trehalose Fermentation Broth as an aid in the presumptive identification of Candida glabrata is based on the ability of the organism to ferment trehalose in 24-48 hours, when incubated at 42ºC. Trehalose fermentation is detected by both the development of acid (yellow color in the medium) and gas (the appearance of gas bubbles in the inverted durham tube). In a study conducted by Land, et al., it was discovered that among the yeast taxa that are frequently isolated and considered clinically important, 96% of C. glabrata and 5% of C. tropicalis were able to ferment trehalose at 42ºC.(8) In a study by Fenn, et al., Hardy Diagnostics Trehalose Fermentation Broth was found to have a sensitivity of 96% and a specificity of 100%.(9)

When HardyCHROM™ Candida is used as the primary plating medium, only colonies that morphologically resemble C. glabrata should be inoculated to Trehalose Fermentation Broth. C. glabrata produces smooth, light pink colonies on HardyCHROM™ Candida while C. tropicalis appear smooth and dark rose to wine. C. krusei appear rough, spreading, and white in color. C. albicans present as smooth, creamy, green colonies. The potential for misidentifying C. tropicalis as C. glabrata, however, can be reduced if HardyCHROM™ Candida (Cat. no. G301) is used as the primary medium.

Trehalose Fermentation Broth can also be inoculated with suspect isolates taken from Blood Agar (Cat. no. A10), Chocolate Agar (Cat. no. E14), Columbia CNA (Cat. no. A50), Corn Meal Agar with Tween® 80 (Cat. no. W10), or SabDex Agar, Emmons (Cat. no. W20). When taken from these media, isolates should be tested for trehalose fermentation only if the following criteria are met:

1) produce stunted growth on Blood Agar
2) appear as small, oval yeast on wet mount preparations
3) fail to produce germ tubes
4) fail to produce pseudohyphae on Corn Meal Agar with Tween® 80


Hardy Diagnostics recommends the use of HardyCHROM™ Candida as the primary plating medium, in conjunction with Trehalose Fermentation Broth with Durham Tube, to allow for a simplified, more rapid presumptive identification of Candida glabrata.

FORMULA

Ingredients per liter of deionized water:*

Trehalose 40.0gm
Yeast Nitrogen Base 10.0gm
Bromcresol Green 20.0mg

Final pH 5.5 +/- 0.2 at 25ºC.

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. 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 and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: This product is not intended for primary isolation of patient specimens. This product is used in conjunction with other biochemical tests to identify cultures of isolated organisms.

Method:
1. Obtain isolated colonies which are 24-48 hours old.
Note: Isolates can be taken from; Blood Agar, Chocolate Agar, Columbia CNA, Corn Meal Agar with Tween® 80,
SabDex Agar, Emmons, or HardyCHROM™ Candida.

2. Inoculate Trehalose Fermentation Broth with one well isolated colony.
Note: If air bubbles are present in the tubed media prior to inoculation, the tube should be inverted until the air is
released from the durham tube.

3. Apply 1ml of Sterile Mineral Oil (Cat. no. Z80), melted paraffin, or vaspar to the inoculated broth.

4. Tighten the cap on tubes and incubate at 42ºC. (incubator or heat block) for 24-48 hours.

5. Observe for color change and gas bubbles in durham tube at 24 and 48 hours.

INTERPRETATION OF RESULTS

A positive trehalose fermentation test is denoted by the development of a bright yellow color in the medium and the presence of gas bubbles within the inverted durham tube.

A negative test is failure of the organism to produce both a yellow color change and gas.

LIMITATIONS

If an excessively heavy inoculum is used, a false-positive reaction may occur due to endogenous carbohydrates present in the yeast.

If air bubbles are present in the tubed media prior to inoculation, the tube should be inverted until the air is released from the durham tube. Failure to remove air bubbles prior to inoculation may result in false-positive results.

Failure to incubate Trehalose Fermentation Broth at 42ºC. with oil, paraffin, or vaspar overlay for 24-48 hours may lead to erroneous results.

Approximately 5% of C. tropicalis are able to ferment trehalose at 42ºC.(8)

If using HardyCHROM™ Candida as the primary medium, it is important to note that a smooth, dark rose to wine colony is presumptively identified as C. tropicalis.

Rare strains of Saccharomyces cerevisiae may ferment trehalose at 42ºC., however, they can be differentiated from C. glabrata on the basis of size and the inability of C. glabrata to form pseudohyphae on Corn Meal Agar with Tween® 80.(8)

Colonies taken from media other than HardyCHROM™ Candida must appear as small, oval yeast on wet mount preparations, produce stunted growth on Blood Agar, fail to produce germ tubes, lack pseudohyphae on Corn Meal Agar with Tween® 80, and ferment trehalose in order to be presumptively identified asC. glabrata.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, Sterile Mineral Oil (Cat. no. Z80), paraffin, vaspar, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Candida glabrata
ATCC ® 66032
E 24-48hr 42°C Aerobic Positive; yellow medium with gas bubbles in durham tube
Candida albicans
ATCC ® 60193
E 24-48hr 42°C Aerobic Negative; blue medium with no gas bubbles in durham tube

User Quality Control

PHYSICAL APPEARANCE

Trehalose Fermentation Broth should appear clear, and royal blue in color.

C. glabrata growing in Trehalose Fermentation Broth

Candida glabrata (ATCC ® 66032) growing in Trehalose Fermentation Broth (Cat. no. Y205). The yellow color change and presence of gas in the durham tube within 24-48 hours was indicative as positive trehalose fermentation. Incubated aerobically for 24 hours at 42ºC.

C. albicans growing in Trehalose Fermentation Broth

Candida albicans (ATCC ® 60193) growing in Trehalose Fermentation Broth (Cat. no. Y205). The absence of a yellow color change and gas in the durham tube within 24-48 hours was indicative as negative for trehalose fermentation. Incubated aerobically for 48 hours at 42ºC.

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

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

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

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

6. Stockman, L. and Roberts, G. 1985. Abstracts of Annual Meeting of American Society of Microbiology, P. 377 (F-80), Washington, D.C.

7. Kwon-Chung, K.J. and Bennett, J.E. 1992. Medical Mycology, Lea & Febiger.

8. Land, G., et al. 1996. Journal of Clinical Microbiology; Vol. 34, No. 9:2300-2303.

9. Fenn, et al. 1999. Comparison of Four Methodologies for Rapid and Cost-Effective Identification of Candida glabrata. Journal of Clinical Microbiology; Vol. 37, No. 10:3387-3389.


ATCC is a registered trademark of the American Type Culture Collection.
Tween is a registered trademark of ICI Americas, Inc.

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