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International Starch Institute
Science Park Aarhus, Denmark
ISI 30-2e Determination of Heat Resistant Spore Count

1. Scope The method is applicable to granular starch in native or modified form.

 

LT 18 Dec 1964
Rev.: LT 30 Aug 1999
2. Principle The sample is serial diluted, incubated on peptone substrate, pasteurised and incubated

 

3. Apparatus 3.1 Electrical heated incubator thermostatically controlled
3.2 Pipette 2000 -3000 microlitre with disposable tips or glass pipette
3.3 Pipette 500 microlitre with disposable tips or glass pipette
3.4 Laboratory balance
3.5 Flask with wide opening, 100 ml.
3.5 Substrate tubes 160 mm x 16 mm with cap use sterile disposable
3.7 Autoclave Sterilise at 120 +/- 2 oC 20 min.
3.8 Bunsen burner

 

Work antiseptically throughout
4. Media 4.1 Peptone milk.

Preparation of
peptone milk

10 g peptone is dissolved in litre fresh skimmed milk. Use heating as necessary. Cool and add up to 1 litre with skimmed milk. Add 10 ml 1% solution of bromcresolpurpur in alcohol. Dissolve 0.8 g cysteinhydrochloride in a small quantity of water and neutralise the solution to pH 7.2 - 7.3 with 2 N KOH. Add the cysteinhydrochloride solution to the peptone milk.

 

BACTO peptone or equivalent
5. Procedure Pour peptone milk (4.1) approx. 10 ml in tubes (3.5), cover and sterilise  in autoclave at 115 oC for 20 min. Sterile substrate keeps for 1-2 weeks

Dilution I

Weigh aseptically 10 g (W) starch sample to the nearest 100 mg  into a sterile 100 ml flask (3.5). Dilute to 4-1 by adding three times as much sterile water. Close flask and shake vigorously. The starch must be kept evenly suspended throughout

 Dilution II

Dilute to 4-2 by adding 3 ml dilution I to tube (3.5) with 9 ml sterile water (A5)

Dilution III

Dilute to 4-3 by adding 3 ml dilution II to tube (3.5) with 9 ml sterile water (B5)

Inoculation

Inoculate three tubes (3.5) of sterile substrate  with 2 ml of each of the three dilutions (I, II and III) and inoculate also three tubes (3.5) of sterile substrate with 0.5 ml of dilution III (to make dilution 4-4 ) - making up a total of 12 inoculated substrate tubes.
Tubes 1 2 3 4 5
A O O O O Water
B O O O O Water
C O O O O  
Dilution 4-1 4-2 4-3 4-4  

Pasteurising

Pasteurise inoculated substrate tubes at 80 oC for 20 min. in a water bath.

 Incubation

Cool and incubate at 30 oC for 4 days..

Counting

A substrate tube is positive if growth can be observed after 4 days of incubation. Number of heat resistant spores are read from the following table:
Number of positive tubes Spores per g
1 <1
2 1
3 2
4 4
5 6
6 9
7 15
8 25
9 42
10 73
11 136
12 >136


6. Calculation Report Heat Resistant Spore Count per gram of sample without decimals

 

7. Note Sterilise utensils in autoclave at 120 +/-2 0C 20 min or in hot air 160 +/-5 0C 2 hours or 140 +/-5 oC 3 hours.

 

Properly wrapped
8. Reference Research Centre Roskilde, Lab. book page 114.

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Keywords: laboratory method determination count heat resistant spore spores starch