Enumeration of Viable Bacillus Spores

Scope

This method applies to liquid and solid Bacillus samples.

Principle

The viable spore count per gram (or per millilitre) of Bacillus is determined by the spore-concentration method — a tenfold serial dilution followed by spread-plate counting.

Instruments and equipment

• Electronic balance0.0001 g
• Pressure steam sterilizer (autoclave)—
• Constant-temperature incubator—
• Ultrasonic cell disruptor — JY92-IIN (Ningbo Scientz Biotechnology)650 W · 60%
• Magnetic stirrer — JOANLAB0–1500 rpm
• Constant-temperature water bath0–100 °C
• Vortex mixer—
• Capped glass bottles250 · 500 mL
• Sterilizable centrifuge tubes7 mL
• Graduated cylinders100 · 1000 mL
• Micropipettes100 µL · 1000 µL · 5 mL
• Beaker2000 mL
• Petri dishesΦ90 mm
• Glass spreader—

Media and diluent

Diluent

Sodium chloride 18 g, Tween 80 2 mL, purified water 2000 mL. Heat until fully dissolved, dispense into five 500 mL Erlenmeyer flasks and sterilize at 121 °C for 30 min.

NA medium

Peptone 10.0 g, beef extract powder 3.0 g, sodium chloride 5.0 g, agar powder 15.0 g, purified water 1000 mL. Dissolve all components in water and mix. Adjust to pH 7.0–7.2 with dilute sodium hydroxide solution, heat to dissolve, and make up to 1000 mL with water. Dispense into three 500 mL Erlenmeyer flasks and autoclave at 121 °C for 30 min.

Plate preparation

In a laminar-flow (clean) bench, pour the sterilized NA medium into Petri dishes, about 15 mL per dish. Let the plates set flat and reserve for use.

Pretreatment and activation

5.1Liquid samples

Accurately weigh 10.0 g of sample into a capped glass bottle containing 90.0 g of diluent and shake vigorously for 1 min. For liquid products such as fermentation broth, sonicate in a conventional ultrasonic bath (45 W) for 15 min.

5.2Solid samples

Weigh a defined amount of sample (see Appendix Table 1) into a 250 mL capped glass bottle and add 100 g of diluent. Soak for 20 min, then stir on a magnetic stirrer at 1000 rpm for 30 min. Sonicate the suspension with the ultrasonic cell disruptor: insert the probe 1.5–2 mm below the liquid surface, set the power to 60%, sonicate for 4 min, ice-water bath for 1 min, then sonicate again for 4 min and ice-water bath for 1 min. After processing, keep the suspension stirring at 1000 rpm, ready for use.

Sample dilution

Take n test tubes and transfer the processed suspension, performing tenfold serial dilution up to tube n–1; mix well. Place tube n–1 in an 80 °C water bath — 10 min for solid samples, 20 min for liquid samples. After the water bath, immediately cool the sample in cold water to room temperature, then carry out a further tenfold dilution to tube n; mix and reserve. Here n is the number of tubes (see Appendix Table 1).

Spread plating and incubation

Select 4 uncontaminated NA plates free of water droplets and label each with a number, date and dilution. Transfer 0.1 mL of the sample dilution from tube n (prepared in Section 6) onto each NA plate and spread it radially from the centre outward with a sterile spreader. Lay the plates flat on the bench for 20–30 min so the liquid absorbs into the medium, then invert them and incubate at 37 ± 2 °C for 18–24 h. Count the colonies on the plates.

Counting

After incubation, count the Bacillus colonies on the plates. Plates with fewer than 30 or more than 300 colonies are not counted.

8.1Liquid samples

The viable Bacillus spore count N (cfu/mL) is calculated by Formula (1):

8.2Solid samples

The viable Bacillus spore count N (cfu/g) is calculated by Formula (2):

Determination of contamination rate

9.1Principle

NA medium is used to detect bacterial contaminants and PSA medium to detect fungal contaminants. Based on colony morphology and microscopic examination, typical Bacillus can be distinguished; the contaminant colonies are then counted. The percentage of contaminants in the total colony count is the contamination rate of the sample.

9.2Instruments and equipment

As in Section 3 of this procedure.

9.3Reagents and solutions

PSA medium. Weigh 200 g of peeled potato and cut into small pieces. Boil in 1000 mL of tap water for 30 min, then filter through four layers of gauze to collect the juice. Add 20.0 g sucrose and 15.0 g agar, heat to dissolve, and make up to 1000 mL with water. Dispense into three 500 mL Erlenmeyer flasks and sterilize at 118 °C for 30 min, then add chloramphenicol at 0.1‰, mix well, pour plates and reserve. NA medium, diluent and plate preparation are as in Section 4.

9.4Procedure

9.4.1Sample pretreatment — as in Section 5.

9.4.2Sample dilution — as in Section 6.

9.4.3Determination — Select 2 uncontaminated NA plates and 2 PSA plates free of water droplets and label them with a number, date and dilution. Perform tenfold serial dilution of the suspension from tube n–2 (of Section 9.4.2), mix, take 0.1 mL and add it to the PSA and NA plates respectively, then spread evenly with a sterile spreader. Lay the plates flat for 20–30 min so the liquid absorbs into the medium, then invert and incubate at 30 ± 2 °C — NA plates for 2 days and PSA plates for 5 days — and count.

9.5Calculation

9.5.1For liquid samples, the contaminant count X (cfu/mL) is calculated by Formula (3):

9.5.2For solid samples, the contaminant count X (cfu/g) is calculated by Formula (4):

9.5.3The contamination rate Y is calculated by Formula (5):

Related documents

• Applicable Bacillus standard documents.
• Media and diluent preparation records.
• Spore-count raw data records.

Change record

This document was revised in May 2026.

Appendix Table 1