TRYPTONE BILE AGAR

Code: CM0595

A rapid and direct plate method for the enumeration of Escherichia coli in food.

Directions
Suspend 36.5g in 1 litre of distilled water and bring gently to the boil to dissolve completely. Sterilise by autoclaving at 121°C for 15 minutes. Cool to 50°C and pour 12-15ml of the medium into sterile Petri dishes.

Description
Tryptone Bile Agar has been developed according to the formulation of Anderson and Baird-Parker¹ for the detection and enumeration of Escherichia coli in foods.

It has several advantages over older methods:
1. It is faster.
2. It is less variable.
3. It gives better recovery from frozen samples.
4. It detects anaerogenic and poor lactose-fermenting strains.

The Direct Plating Method (DPM) described by Anderson and Baird-Parker is a modification of that described by Delaney et al.². This method, developed for the detection and enumeration of Escherichia coli in water and food samples, utilises the ability of Escherichia coli to produce indole from tryptophan at 44°C when grown on a cellulose acetate membrane on plates of Tryptone Bile Agar.

The authors concluded that the formation of indole was a more reliable characteristic for both enterotoxigenic and non-enterotoxigenic strains of Escherichia coli than lactose fermentation. Ewing³ found that only 90% of Escherichia strains produce acid from lactose within two days, whereas 99% of strains produce indole.

The International Commission on Microbiological Specifications for Foods (CMSF)⁴ compared the Most Probable Number (MPN) and the Anderson-Baird-Parker Direct Plating Method (DPM) and concluded that the DPM was preferable to the MPN method of enumeration of Escherichia coli in raw meats, because of less variability, better recovery from frozen samples, greater rapidity and the smaller quantity of medium needed.

The Direct Plating Method will enumerate both anaerogenic and late lactose-fermenting strains of Escherichia coli which would be missed by the MPN method. According to Ewing³ these organisms comprise as many as 10% of Escherichia strains.

Holbrook et al.⁵ have further modified the Direct Plating Method for detection and enumeration of sublethally damaged cells of Escherichia coli in frozen, dried, heat processed or acid foods. In this modification the inoculum is applied to a cellulose acetate membrane on Minerals Modified Glutamate Agar and incubated for 4 hours at 37°C. The resuscitation step permits the repair of stressed cells before the transfer of the membrane to a Tryptone Bile Agar plate.

It has been shown that the presence of high levels of fermentable carbohydrates will inhibit the synthesis of tryptophanase⁶ and thereby stop indole formation. Holbrook et al. have demonstrated that the resuscitation step reduces the high concentration of sugar present in the inoculum to a level which does not interfere with the production of indole by Escherichia coli when grown on Tryptone Bile Agar. The resuscitation step should always be carried out when testing dairy or other products containing high concentrations of sugars.

The indole reagent described by Vracko and Sherris⁷ was found to be the most suitable, giving the most distinct reaction and reproducibility. The reagent, 5% p-dimethylaminobenzaldehyde in 1N hydrochloric acid is easy to prepare and will not deteriorate when kept for three months in the dark at room temperature.

All indole positive strains give well defined pink colonies when `stained’ using the indole reagent; colonies that do not produce indole are straw coloured.

The growth of indole positive organisms other than Escherichia coli is inhibited by the selective action of the bile salts and the elevated incubation temperature.

The `stained’ membranes may be `fixed’ by drying in direct sunlight or under a low pressure fluorescent ultra violet lamp with a `Woods’ type filter. When dried the intensity of the staining reaction is improved, and such membranes may be stored for reference.

Technique
Direct Plating Method
1. Prepare plates of Tryptone Bile Agar and dry the surface.
2. Place a cellulose acetate filter membrane (85mm diameter, 0.45µ  pore size), which need not be sterilised, on the surface of the medium. Gently flatten with a sterile spreader to remove trapped air.
3. Prepare the food sample by diluting 1 in 5 or 1 in 10 with 0.1% (w/v) sterile Peptone Water CM0009 and homogenise in a `Stomacher’ or a laboratory blender⁸.
4. Pipette 0.5 or 1.0ml of the homogenate on to the membrane and spread over the surface with a sterile glass spreader.
5. Allow the homogenate to soak in and incubate plates stacked, not more than three high, with lids uppermost, for 18-24 hours in a water jacketed incubator at 44 ± 1°C.
6. Remove the plates from the incubator and pipette 1-2ml of the indole reagent into each labelled lid.
7. Lift the membrane with a pair of forceps from the plate and lower on to the reagent.
8. Place the stained membranes in direct sunlight or under a low pressure uv lamp for 5-10 minutes. Indole positive colonies are stained pink.
9. Multiply the number of pink colonies by the dilution factor and express the result as the number of Escherichia coli per gram of food.
10. The `stained’ membrane may be `fixed’ by prolonged drying in direct sunlight or under a uv lamp, and kept for reference.

Resuscitation Procedure
1. Preparation of Minerals Modified Glutamate Agar plates.
Make up 1 litre of Minerals Modified Glutamate Medium CM0607 and add 12g of Agar No.1 LP0011. Bring gently to the boil until dissolved completely and sterilise by autoclaving at 116°C for 10 minutes. Cool to 50°C and pour 12-15ml of the medium into sterile dishes.
2. Place a cellulose acetate filter membrane on to the well dried surface of a plate of Minerals Modified Glutamate Agar. Gently flatten with a sterile spreader to remove trapped air.
3. Prepare the food sample by diluting 1 in 5 or 1 in 10 with 0.1% (w/v) Peptone Water CM0009 and homogenise in a `Stomacher’ or a laboratory blender.
4. Pipette 0.5 or 1.0ml of the homogenate on to the membrane and spread completely over the surface with a sterile glass spreader.
5. Allow the homogenate to soak in, and incubate the plates with the lids uppermost in piles of not more than three for 4
hours at 35°C.
6. Transfer the membrane filter from the plate using sterile forceps and gently lower on to the dried surface of a Tryptone Bile Agar plate.
7. Incubate the plates as described for the Direct Plating Method, strain, and count the number of pink indole positive colonies.
If required the unstained plates may be placed in the refrigerator overnight and the indole test carried out the following morning.

Indole Reagent
5% p-dimethylaminobenzaldehyde in 1N hydrochloric acid.

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 plates at 2-8°C.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Light straw coloured gel

Quality control

* This organism is available as a Culti-Loop®

References
1. Anderson J. M. and Baird-Parker A. C. (1975) J. Appl. Bact. 39. 111-117.
2. Delaney J. E., McCarthy J. A. and Grasso R. J. (1962) Wat. Sewage Works. 109. 289.
3. Ewing W. H. (1972) COC Atlanta, US Dept. of Health, Education & Welfare.
4. International Commission on Microbiological Specifications for Foods (1979) Can. J. Microbiol. 25. 1321-1327.
5. Holbrook R., Anderson J. M. and Baird-Parker A. C. (1980) Food Technol. in Aust. 32. 78-83.
6. Clarke P. H. and Cowen S. T. (1952) J. Gen. Microbiol. 6. 187-197.
7. Vracko R. and Sherris J. C. (1963) Amer. J. Clin. Path. 39. 429-432.
8. Sharpe A. N. and Jackson A. K. (1972) Appl. Microbiol. 24. 175-178.