ISI 30-2e Determination of Heat Resistant Spore Count

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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 ½ 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.