1.0 Direction for use
Step 1. Sample preparation
Prepare the 1:10 solution by placing 25mL (g) of sample into 225mL of sterile saline, then prepare 1:100 solution by sucking 1mL of solution (1:10) with sterile pipette into 9mL sterile saline. Prepare tenfold increasing serial dilution of sample. If necessary, use 1mol/L NaOH or 1mol/L HCL to adjust the PH value to 6.6-7.2.
Step 2 Inoculation
Select 2 or 3 diluted solutions for ordinary food, while for the liquid solution which has little bacteria, directly suck original sample to do the test. Place bacteria colony count plate on the sterile experiment table; uncover the film on the surface. Suck 1mL of solution with sterile pipette, and then drip it slowly and evenly onto the count plate. Then cover the film gently and stand it for about 10s until the medium solidify. Inoculate two count plates for each diluted solution. Do a blank control for each test.
Step 3 Culture
Superimpose the count plates together and place them back to the ziplock bag and seal it well. Then put it in the incubator with transparent side up. The quantity of superimposed count plates should not be more than 12. For ordinary food, the temperature of culture should be 36±1 °C and culture time should be 15~24hs. While for aquatic food, the temperature of culture should be 30±1 °C, and culture time should be 48hs.
Bacteria colony shows red spot on the count plate. Select the count plates of which the colony quantity is between 30CFU and 300CFU to do the counting.
3.0. Counting method & report
3.1 If there is just one diluted sample suitable for counting, then count the average number of the colonies on these count plates, and then multiply it by the corresponding dilution multiple. Report it as estimated CFU/mL or CFU/g.
3.2 If the colony numbers on count plates from two serial dilutions are both between 30CFU and 300CFU, calculate the average number of colonies as per below formula:
N in the formula means the bacteria number of the sample.
∑C means the total bacteria number of these two serial dilutions.
n1 means the quantity of count plates of which the dilution multiple is lower between these two
n2 means the quantity of count plates of which the dilution multiple is higher between these two
d means the higher dilution ratio between two different dilution ratios.
(Higher dilution ration
Low dilution ration
The quantity of colonies
∑C 232+224+33+35 544
N = —————— = ———————— = ———— = 24727
(n1+0.1n2)d (2+0.1×2)×10-2 0.022
Round the above result to 25000 (or 2.5×104).
3.3 If the colony numbers on count plates of every dilution ratio are more than 300CFU, count the average number of bacteria colonies on count plates of the maximum dilution, and then multiply it by maximum dilution multiple.
3.4 If the colony numbers on count plates of every dilution ratio are less than 30CFU, count the average number of bacteria colonies on count plates of the minimum dilution, then multiply it by minimum dilution multiple.
3.5 If no colony was found on the count plates from all dilutions of sample (including original liquid sample), report the count in terms of less than one multiplied minimum dilution multiple.
3.6 If the average numbers of bacteria colonies from all the dilutions are not between 30CFU and 300CFU, some of them are less than 30CFU or some of them are more than 300CFU, count the average number of bacteria colonies which is closest to 30CFU or 300CFU, and then multiply it by the corresponding dilution multiple.
3.7 Report of the bacteria quantity
3.7.1 If the colony counts less than 100, round it as per rounding-off rule and use two effective digits to report it in terms of scientific notation.
3.7.2 If the colony counts more than 100 (incl. 100), round the third digit as per rounding-off rule, and use the first two digits to report it in terms of scientific notation.
3.7.3 If there are too numerous to count, report as TNTC.
3.7.4 If there are some bacteria colonies growing on the blank control count plates, the test is invalid.
3.7.5 Use CFU/g as report unit for solid samples, and use CFU/mL as report unit for liquid samples.
4. Surface sampling method
Suck 1mL of sterile phosphate buffer solution or sterile saline onto count plate, and stand it for 10s to let the medium solidify. Uncover the upper film, and make the central filter part of the count plate contact with the surface of the sample. Then press it by hand, cover the film, and put it in the incubator for cultivation.
5. Additional explanatory note
5.1 The coincidence rate of detecting result achieved by using bacteria colony count plate and by traditional plate counting method is higher than 80%.
5.2 Prepare and sterilize phosphate buffer:
Stock solution: weigh 34.0g monopotassium phosphate (KH2PO4), and dissolve it in 500mL of pure water or distilled water, and then use about 175mL of 1 mol/L sodium hydroxide (NaOH) to adjust the PH value to 7.2. Dilute it to 1000mL with distilled water and then store it in the refrigerator.
Diluent: Suck 1.25mL of stock solution and dilute it to 1000mL with distilled water. Subpackage them by suitable container. Autoclave them at 121℃ for 15mins or put them in the disinfection cabinet.
5.3 Prepare and sterilize normal saline: Weigh 8.5 g sodium chloride (NaCl) and dissolve it by 1000mL of distilled water or pure water. Subpackage it by suitable container. Autoclave them at 121℃ for 15mins or put them in the disinfection cabinet.
5.4 Dispose the used count plates according to biology safety waste treatment rule, because there are live bacteria on them.