GB/T 13092-2025Enumeration of molds count in feeds (English PDF)

5.2 Sodium hydroxide solution (1 mol/L). Weigh

20.0 g of sodium hydroxide; dissolve in water; cool to room temperature; make constant volume to 500 mL with water; and mix well.

5.3 Hydrochloric acid solution (1 mol/L). Pipette 42 mL of hydrochloric acid; dilute with water; make constant volume to 500 mL with water; and mix well.

5.4 Phosphate buffer stock solution. Weigh

34.0 g of potassium dihydrogen phosphate; dissolve in 500 mL of water; adjust the pH to

7.2 with approximately 175 mL of sodium hydroxide solution (5.2); dilute to 1000 mL with water; mix well; and store at 2°C~8°C.

5.5 Sterile phosphate buffer solution. Take

1.25 mL of phosphate buffer stock solution (5.4); dilute with water to 1000 mL; and mix well. Dispense into suitable containers; and autoclave at 121 °C for 15 min.

5.6 Sterile physiological saline. Weigh

8.5 g of sodium chloride; dissolve in water and make constant volume to 1000 mL; and mix well. Dispense into suitable containers; and autoclave at 121 °C for 15 min.

5.7 Dichloran Rose Bengal Chloramphenicol Agar (DRBC) medium. Prepare according to Appendix A; or purchase commercially available medium.

5.8 Sterile liquid paraffin. Take an appropriate amount of liquid paraffin and autoclave at 121 °C for 20 min.

5.9 Sterile Tween

80.Take an appropriate amount of Tween 80 and autoclave at 121 °C for 20 min.

5.10 Sterile homogenizing cup.

5.11 Sterile homogenizing bag.

5.12 Sterile petri dish. 90mm in diameter.

6.2 Balance. Accuracy of

0.1 g,

0.01 g, and

0.1 mg.

6.3 Homogenizer. Rotation speed 8000 r/min ~ 10000 r/min.

6.4 Beating homogenizer.

6.5 Thermostat. 42 °C ± 2 °C.

6.6 Vortex mixer.

6.7 pH meter. Accuracy ± 0.02, or precision pH test paper.

6.8 Magnifier or colony counter.

6.9 Oscillator. Reciprocating type.

9.1.1 Preparation of specimen solution

9.1.1.1 Specimen (excluding oily type) Using sterile operation, take 25 g (mL) of specimen and homogenize it according to one of the following three methods to prepare a

1.10 specimen homogenate.

--- Place it in a sterile homogenizing cup (5.10) containing 225 mL of sterile phosphate buffer solution (5.5) or sterile physiological saline (5.6); and homogenize at 8000 r/min to 10000 r/min for 1 min to 2 min;

--- Place it in a sterile homogenizing bag (5.11) containing 225 mL of sterile phosphate buffer solution (5.5) or sterile physiological saline (5.6); and homogenize using a tapping homogenizer for 1 min to 2 min;

--- Place it in a sterile glass conical flask containing 225 mL of sterile phosphate buffer solution (5.5) or sterile physiological saline (5.6); and oscillate on an oscillator for 30 min.

9.1.1.2 Oily specimen Using sterile operation, take 25 g (mL) of the specimen and place it in a sterile Erlenmeyer flask. First, add

12.5 mL of sterile liquid paraffin (5.8); then add 25 mL of sterile Tween 80 (5.9). Oscillate and mix for 10 min in a 42 °C±2 °C thermostat. Add 187.5 mL of sterile phosphate buffer solution (5.5) or sterile physiological saline (5.6) at 42 °C±2 °C. Emulsify in a 42 °C±2 °C thermostat for 1 min to 2 min to prepare a

1.10 specimen homogenate.

9.1.2.1 Using a 1mL sterile pipette or micropipette, pipette 1mL of a

1.10 specimen homogenate (

9.1.1.1 or 9.1.1.2) and slowly inject it along the tube wall into a sterile test tube containing 9mL of sterile phosphate buffer solution (5.5) or sterile physiological saline (5.6). Vortex for 30s to prepare a

1.100 specimen homogenate.

9.1.2.2 Following the operating procedure in 9.1.2.1, perform 10-fold incremental dilutions; changing the sterile pipette or micropipette tip after each incremental dilution to prepare a 1.10n specimen homogenate.