Bacillus Viable Spore Count

Scope

This method applies to liquid and solid Bacillus samples.

Test Principle

The spore concentration method is used — after a ten-fold serial dilution series, spread-plate counting determines the number of viable spores per gram (or milliliter) of Bacillus sample.

Instruments & Equipment

• Electronic balance0.0001 g
• Autoclave (steam sterilizer)—
• 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—
• Lidded glass bottles250 · 500 mL
• Sterilizable centrifuge tubes7 mL
• Graduated cylinder100 · 1000 mL
• Pipettes100 µL · 1000 µL · 5 mL
• Beaker2000 mL
• Petri dishesΦ90 mm
• Glass spreader—

Media & 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 minutes.

NA Medium

Peptone 10.0 g, beef extract 3.0 g, sodium chloride 5.0 g, agar powder 15.0 g, purified water 1000 mL. Dissolve and mix the ingredients in water, adjust to pH 7.0–7.2 with dilute sodium hydroxide solution, dissolve by heating, and bring to 1000 mL with water. Dispense into three 500 mL Erlenmeyer flasks and autoclave at 121°C for 30 minutes.

Plate Preparation

In a laminar flow cabinet, pour the sterilized NA medium into petri dishes, approximately 15 mL per dish. Allow the plates to set flat before use.

Pretreatment & Activation

5.1Liquid Samples

Accurately weigh 10.0 g of sample into a lidded glass bottle containing 90.0 g of diluent and shake vigorously for 1 minute. For liquid products such as fermentation broth, ultrasonic treatment for 15 minutes in a standard ultrasonic cleaner (45 W) may be used.

5.2Solid Samples

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

Sample Dilution

Take n test tubes and transfer the treated suspension, performing a ten-fold serial dilution up to tube n–1; mix thoroughly. Place tube n–1 in an 80°C water bath — 10 minutes for solid samples, 20 minutes for liquid samples. Immediately after the water bath, cool the sample to room temperature with cold water, then perform one more ten-fold dilution to tube n; mix and set aside. Here n is the number of tubes (see Appendix Table 1).

Plating & Incubation

Select 4 NA plates free of contamination and surface moisture, and label each with an ID, date, and dilution level. Transfer 0.1 mL of the tube-n dilution prepared in Section 6 onto each NA plate, and spread evenly outward from the center with a sterile spreader in a radial pattern. Leave the plates flat on the bench for 20–30 minutes to allow the liquid to fully absorb into the medium, then invert and incubate at 37±2°C for 18–24 hours before observing and counting colonies on the plates.

Counting

After incubation, count the Bacillus colonies on each plate. Plates with fewer than 30 or more than 300 colonies are excluded from the count.

8.1Liquid Samples

The viable Bacillus spore count N (CFU/mL) in liquid samples is calculated using Equation (1):

8.2Solid Samples

The viable Bacillus spore count N (CFU/g) in solid samples is calculated using Equation (2):

Contamination Rate Determination

9.1Principle

NA medium is used to detect bacterial contamination, and PSA medium to detect fungal contamination. Typical Bacillus colonies are distinguished based on colony morphology and microscopic examination; contaminant colonies are then counted. The percentage of contaminant colonies out of the total colony count is the sample's contamination rate.

9.2Instruments & Equipment

Same as Section 3 of this method.

9.3Reagents & Solutions

PSA Medium. Weigh 200 g of peeled potato, cut into small pieces, add 1000 mL of tap water, and boil for 30 minutes; filter the liquid through four layers of gauze. Add 20.0 g sucrose and 15.0 g agar, dissolve by heating, and bring to 1000 mL with water. Dispense into three 500 mL Erlenmeyer flasks, sterilize at 118°C for 30 minutes, add chloramphenicol to 0.1‰, mix, and pour into plates. NA medium, diluent, and plate preparation follow the same method as Section 4.

9.4Procedure

9.4.1Sample pretreatment — same as Section 5.

9.4.2Sample dilution — same as Section 6.

9.4.3Determination — select 2 NA plates and 2 PSA plates free of contamination and surface moisture, and label each with an ID, date, and dilution level. Perform a ten-fold serial dilution of the tube-n–2 suspension from Section 9.4.2, mix, and transfer 0.1 mL onto each PSA and NA plate, spreading evenly with a sterile spreader. Leave flat for 20–30 minutes to allow the liquid to absorb into the medium, then invert and incubate at 30±2°C — 2 days for NA plates, 5 days for PSA plates — before counting.

9.5Calculation

9.5.1The contaminant count X (CFU/mL) in liquid samples is calculated using Equation (3):

9.5.2The contaminant count X (CFU/g) in solid samples is calculated using Equation (4):

9.5.3The contamination rate Y is calculated using Equation (5):

Related Documents

• Applicable Bacillus-related standard documents.
• Media and diluent preparation records.
• Raw spore count data records.

Revision History

This document was revised in May 2026.

Appendix Table 1