ISI 31-1e Determination of Total Viable Count


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

 

 KJSV Sep 1964
Rev.: LT 14 Jan 1998
2. Principle The sample is serial diluted, incubated and counted on agar plates

 
3. Apparatus 3.1 Electrical heated incubator thermostatically controlled
3.2 Petri dishes, approx. 9 cm in diameter use sterile disposable
3.3 Pipette 1000 microlitre with disposable tips or glass pipette
3.4 Pipette 100 microlitre with disposable tips or glass pipette
3.5 Laboratory balance
3.6 Pyrex flask with screw cap, 100 ml.
3.7 Substrate tubes 160 mm x 16 mm with cap
3.8 Autoclave Sterilise at 120 +/- 2 oC 20 min.
3.9 Drigalski spatula
3.10 Bunsen burner

 

 Work antiseptically throughout
4. Media 4.1 Plate count agar. Melt and fill flasks (3.6). Sterilise in autoclave (3.8) Gibco's Standard Methods Agar.
4.2 Physiological saline, 0.9% NaCl. Fill 9 ml into tubes (3.7). Sterilise in autoclave (3.8) isotonic solution
4.3 Physiological saline, 0.9% NaCl. Fill 90 ml into flasks (3.6). Sterilise in autoclave (3.8)

 
5. Procedure Melt agar (4.1) and pour approx. 5 ml in sterile Petri dishes (3.2) or buy sterile plates ready to use

Dilution 101

 Weigh 10 g (W) sample to the nearest 100 mg on (3.5) into a sterile flask (3.6). Add half of saline of a flask (4.3). Close flask and shake vigorously. Add residual saline of flask (4.3). Suspend sample painstaking

 Dilution 102

 Add 1 ml dilution with (3.3) to tube (4.2). Mix.

Dilution 10x

 Continue serial dilution until a plate count of 30 - 300 is expected.

Inoculation

 Plating: Transfer x ml dilution to each of three agar plates and distribute evenly with Drigalski spatula. Mark dishes. Also plate a blank with saline only
Plate 1 ml of dilution 101 and mark dishes D = 1. Plate 0.1 ml of dilution 101 and mark dishes D = 2. Plate 0.1 ml of subsequent serial dilutions and mark dishes D = 3 etc. Plate dilutions in expectation of 30-300 colonies only

 Incubation

 Incubate agar plates upside down at 30 oC for 3 days.

Counting

 Count colonies (T) on plates with 30-300 colonies

 
6. Calculation Report Total Viable Count with one decimal times the factor of ten as the average counts of three plates:

Count per g of sample = T x 10D / W

 

 Example:
1.7x104 counts/g (ISI 31-1e)
7. Note Alternatively add x ml dilution to empty Petri dishes. Pour melted agar of 45 oC on top. Distribute by tilting.
Sterilise utensils in autocalve 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 NMKL 86

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