SOP: Rinse Method for Bottles
GIFSQ — Global Institute for Food Safety & Quality
Principle
Bottles are rinsed internally with a known small volume of sterile Ringer solution; plate counts from the rinse, incubated at two temperatures, give the average colony count per bottle, and inoculation into double strength MacConkey’s broth tests for coliform bacilli. Testing all bottles travelling abreast through a washing machine shows whether one set of jets or carriers is out of alignment.
Apparatus required
- Sterile rubber corks
- Sterile pipettes
- Petri dishes
- Test tubes and bottles (4 oz) with Durham’s tubes
- Incubator (37°C and 22°C)
Reagents
a. Sterile Ringer solution — To 1,000 ml of distilled water add 2.15 g of NaCl (AR), 0.075 g of potassium chloride (AR), 0.12 g of anhydrous calcium chloride (AR) and 0.5 g of sodium thiosulphate. Autoclave for 20 min at 15 psig. Use the solution as diluent at one-quarter of its original strength; it is ionically balanced.
b. Yeast extract agar (routine work) — The chapter’s ingredient table is partly illegible in the available copy: the legible routine-work figures are peptone 5 g, agar 10 g and water 1,000 ml. Heat and dissolve the ingredients, adjust the pH to 7.3 and filter if necessary. Bottle or tube the medium and autoclave at 10 psig for 10 min. Check the complete recipe against the printed book.
c. MacConkey’s double strength broth — To 1,000 ml of water add 10 g of sodium tauroglycocholate, 10 g of NaCl and 40 g of peptone; prepare as the single strength broth. Add 40 g of lactose and 20 ml of 1% neutral red solution. Dispense 10 ml into each 6 × 0.75 in. test tube and 50 ml into each 4 oz bottle, with Durham’s tubes in each. Adding 10 ml of sample to a tube (or 50 ml to a bottle) brings the medium down to single strength. (Bromocresol purple at 1.5% may replace neutral red at 0.04%.)
Sample preparation
Where bottles are taken after a washing machine in which a row of bottles travels abreast, examine every bottle in one such row — this shows whether any set of jets or carriers is out of line. Never examine fewer than six bottles. Cap or stopper the bottles at once.
Procedure — Rinse examination of bottles
To each bottle, add 20 ml of sterile Ringer solution
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Close with sterile rubber corks
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Roll the bottles on their sides so that the whole of the internal surface is rinsed
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Leave them on their sides and roll at intervals of 30 min
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Pipette 5 ml from each bottle into each of two Petri dishes
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Add 15–20 ml of yeast extract agar, mix, and incubate one plate from each bottle at 37°C and one at 22°C for 48 hr
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Pipette 5 ml from each bottle into 10 ml of double strength MacConkey’s broth and incubate at 37°C for 48 hr for the coliform test
Calculation
Count per bottle = (Mean of the 37°C and 22°C plate counts) × 4
Where,
- Mean of the 37°C and 22°C plate counts = average of the colony counts from the two plates of the bottle
- 4 = factor converting the 5-ml plated portion to the 20 ml of rinse used per bottle
Take the mean of the 37°C and 22°C plate counts and multiply by 4. This is the count per bottle. (The source prints “the average colony count per container” at this point — carried here as “per bottle”, the evident meaning in the bottle procedure; check the printed book.) Find the average of the counts, omitting any figure which is 25 times greater than others — this indicates a possible fault in that particular line.
Interpretation
No colony-count standards have been laid down for beverage bottles. For milk bottles, the 1947 Classification of the UK Ministry of Agriculture and Fisheries treats an average colony count of 200 or less as satisfactory, 200 to 600 as fairly satisfactory and over 600 as unsatisfactory. Coliform bacilli must not, however, be present in 5 ml of the rinse.
Precautions
- Examine not less than six bottles in any case.
- Close the bottles with sterile rubber corks and keep them on their sides, rolling at intervals of 30 min, so that the whole internal surface is rinsed.
References
- De Man, J.C., Rogosa & Sharpe, J. Appl. Bact. 23, 130 (1960).
- Knox, R. & J. Walker, J. Hyg. Camb., 45, 187 (1947).
- (Author name illegible in the available copy — printed as “Gos) C.H.”), Microbiological Methods, 2nd edn., Butterworth & Co. Ltd., London, p. 347 (year printed as 1997).