GB/T 13093-2023Determination of bacterial count in feeds (English PDF)

5.4 Phosphate buffer stock solution: Weigh

34.0 g potassium dihydrogen phosphate and dissolve it in 500 mL water. Adjust the pH to

7.2 with about 175 mL of 1 mol/L sodium hydroxide solution, dilute to 1000 mL with water, mix well, and store at 2 °C~8 °C.

5.5 Sterile phosphate buffer: Take

1.25 mL of phosphate buffer stock solution (5.4), dilute to 1000 mL with water, dispense into suitable containers or 9 mL per tube, and sterilize by autoclaving at 121 °C for 15 min.

5.6 Sterile physiological saline: Weigh

8.5 g of sodium chloride, dissolve it in water, and make up to 1000 mL. Dispense into suitable containers or 9 mL per tube and sterilize by autoclaving at 121 °C for 15 min.

5.7 Plate count agar medium: Prepare according to Appendix A.

5.8 Sterile liquid paraffin: Take liquid paraffin and sterilize it by autoclaving at 121 °C for 20 min.

5.9 Sterile Tween 80: Take Tween 80 and sterilize it by autoclaving at 121 °C for 20 min.

8.1.2.2 Follow the procedure in

8.1.2.1 to make 10-fold incremental dilutions. The sterile pipette or micropipette tip shall be replaced after each incremental dilution.

8.1.2.3 According to the estimation of the contamination status of the sample, select 2 ~3 sample homogenates of appropriate serial dilutions, and pipette 1 mL of sample homogenate of each dilution into a sterile plate; make two plates for each dilution. At the same time, pipette 1 mL of sterile phosphate buffer or sterile physiological saline into a sterile plate as a blank control.

8.2 Cultivation After the dilution is transferred to the plate, promptly pour 15 mL~20 mL of the culture medium cooled to 46 °C~50 °C (can be placed in a 48 °C±2 °C constant temperature device) into the plate, and carefully rotate the plate to fully mix the sample and the culture medium. The time from diluting the sample to pouring the culture medium shall not exceed 30 min. If it is estimated that the sample may diffusely grow on the surface of the culture medium, after the culture medium is completely solidified, a thin layer of plate count agar medium (about 4 mL) cooled to 48 °C±2 °C can be covered on the solidified agar surface. After the agar solidifies, invert the plate horizontally and culture it in a 36 °C±1 °C constant temperature incubator for 48 h±2 h.

8.3 Bacterial colony counting

8.3.1 When counting bacterial colonies, observation can be made with the naked eye. If necessary, a magnifying glass or colony counter can be used to check to prevent omissions. Bacterial counts are expressed in colony-forming units (CFU).

8.3.2 Select plates with colony counts between 30 CFU ~ 300 CFU and no spreading colony growth for counting. For plates with less than 30 CFU, record the specific colony count; for plates with more than 300 CFU, record as TNTC (too numerous to count).

8.3.3 When one of the two plates has large flake-like colonies growing, the plate without large flake-like colonies shall be selected for colony counting; if the flake-like colonies are less than half of the plate, and the colonies on the other half are evenly distributed, the half of the plate with evenly distributed colonies shall be selected for colony counting, and the count result multiplied by 2 represents the colony number on this plate.

8.3.4 If there are chain-like growths without clear boundaries between colonies on the plate, each single chain shall be counted as one colony.

8.3.5 If there is colony growth on the blank control, the test result will be invalid.

9.1 Calculation of bacterial count

9.1.1 If the number of colonies on only one dilution plate is within the appropriate counting range, calculate the average value of the colony counts on the two plates and then multiply the average value by the corresponding dilution multiple to obtain the bacterial count per gram or per milliliter of the sample. See Example 1 in Appendix B.

9.1.2 If the colony counts on two serial dilution plates are within the appropriate counting range, calculate it according to formula (1): where: N -- The bacterial count in the sample; C -- The number of colonies on the plates (the plates on which the number of colonies is in the appropriate range); n1 -- The number of plates of the first dilution (low dilution multiple); n2 -- The number of plates of the second dilution (high dilution multiple); d -- Dilution factor (first dilution). The calculated result is the bacterial count per gram or per milliliter of the sample, see Example 2 in Appendix B.

9.1.3 If the colony counts on all dilution plates are greater than 300 CFU, count the plate with the highest dilution and record the other plates as TNTC. The result is calculated by multiplying the average colony count by the highest dilution multiple, see Example 3 in Appendix B.

9.1.4 If the colony counts on the plates of all dilutions are less than 30 CFU, the calculation shall be based on the average colony count of the lowest dilution multiplied by the dilution multiple, see Example 4 in Appendix B.

9.1.5 If no bacteria grow on any dilution plate, calculate by multiplying the lowest dilution multiple by less than 1, see Example 5 in Appendix B.