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TRYPTONE BILE X-GLUCURONIDE (TBX) MEDIUM (7692)

Intended Use
Tryptone Bile X-Glucuronide (TBX) Medium is used in the isolation and identification of E. coli in food.
Product Summary and Explanation
1
Tryptone Bile X-Glucuronide (TBX) Medium is a modification of Tryptone Bile Agar. Tryptone Bile Agar was
2,
developed to improve the detection of E. coli in foods. TBX Medium is enhanced by the addition of a
chromogenic agent, X-glucuronide, detecting glucuronidase activity. The presence of the enzyme -Dglucuronidase differentiates most E. coli spp. from other coliforms, and is the same enzyme used in the MUG
3
reaction. X-glucuronide reacts slightly differently and when released into the medium is insoluble,
accumulating within the cell.
Principles of the Procedure
Tryptone provides nitrogen, vitamins, and amino acids in TBX Medium. Bile Salts are the selective agent
against Gram-positive bacteria, particularly bacilli and fecal streptococci. E. coli absorbs the chromogenic
substrate, 5-bromo-4-chloro-3-indolyl- -D-glucuronide, X-glucuronide. The enzyme -glucuronidase splits the
bond between chrompohore 5-bromo-4-chloro-3-indolyle and -D-glucuronide. The released chromophore is
colored and accumulates within the cells. E. coli colonies are colored blue. Agar is the solidifying agent.
Formula / Liter
Tryptone .................................................................................. 20 g
Bile Salts ................................................................................ 1.5 g
X-glucuronide ..................................................................... 0.075 g
Agar ....................................................................................... *15 g
*9 - 18 according to gel strength

Final pH: 7.2 0.2 at 25C


Formula may be adjusted and/or supplemented as required to meet performance specifications.

Precautions
1. For Laboratory Use.
2. IRRITANT. Irritating to eyes, respiratory system, and skin.
Directions
1. Suspend 36.6 g of the medium in one liter of purified water.
2. Heat with frequent agitation and boil for one minute to completely dissolve the medium.
3. Autoclave at 121C for 15 minutes.
Quality Control Specifications
Dehydrated Appearance: Powder is homogeneous, free flowing, and light beige.
Prepared Appearance: Prepared medium is trace to moderately hazy and colorless to light beige.
Expected Cultural Response: Cultural response on Tryptone Bile X-Glucuronide (TBX) Medium at 35
2C and examined for growth at 18 24 hours incubation.
Microorganism
Enterobacter aerogenes ATCC 13048

Growth
growth

Reaction
white

Escherichia coli ATCC

25922

growth

blue

Escherichia coli ATCC

35218

growth

blue

Escherichia coli ATCC

11775

growth

blue

growth

white

growth

white

growth

white

growth

white

Escherichia coli O157:H7 ATCC


Klebsiella pneumoniae ATCC
Salmonella choleraesuis ATCC
Shigella flexneri ATCC

12022

35150

13883
13076

The organisms listed are the minimum that should be used for quality control testing.
PI 7692, Rev 02, August 2009

Test Procedure
The pour plate or surface spread plate method can be used to inoculate the prepared medium:
Pour Plate Method
1. Pipette 1 mL of a homogenate or appropriate dilution into a sterile Petri dish.
2. Add 15 mL of the medium, cooled to 45 - 50C, mix gently.
3. For the recovery of sub-lethally injured E. coli, incubate plates at 37C or 30C for 4 hours.
4. Continue incubation at 44C for additional 18 20 hours.
Surface Plating Technique
1. Pipette 0.1 mL of a homogenate or appropriate dilution onto a prepared plate of TBX Medium and
aseptically distribute the sample evenly across the plate surface.
2. For the recovery of sub-lethally injured E. coli, incubate plates at 37C or 30C for 4 hours.
3. Continue incubation at 44C for additional 18 20 hours
Results
E. coli colonies are blue (X- -D-glucuronide reaction).
Three to four percent of -Glucuronidase-negative E. coli strains (E. coli O157) form white colonies, or they
cannot grow at elevated temperatures of 44C (E. coli O157:H7).
Storage
Store sealed bottle containing the dehydrated medium at 2 - 30C. Once opened and recapped, place
container in a low humidity environment at the same storage temperature. Protect from moisture and light by
keeping container tightly closed.
Expiration
Refer to expiration date stamped on the container. The dehydrated medium should be discarded if not free
flowing, or if the appearance has changed from the original color. Expiry applies to medium in its intact
container when stored as directed.
Limitation of the Procedure
Due to varying nutritional requirements, some strains may be encountered that grow poorly or fail to grow.
Packaging
Tryptone Bile X-Glucuronide (TBX) Medium

Code No.

7692A
7692B
7692C

500 g
2 kg
10 kg

References
1.
2.
3.

Anderson, J. M., and A. C. Baird-Parker. 1975. J. Appl. Bact. 39:111-117.


Gross, R. J. and B. Rowe. 1985. J. Hyg. Camb. 95:513-550.
Feng, P.C. S. and P. A. Hartmann. 1982. Appl. Environ. Microbiol. 43:1320-1329.

Technical Information
Contact Acumedia Manufacturers, Inc. for Technical Service or questions involving dehydrated culture media preparation or
performance at (517)372-9200 or fax us at (517)372-2006.

PI 7692 Rev 02, August 2009

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