1、 Principle and Scope of Application
Load lactose bile salt selective culture medium and TTC chromogenic reagent onto paper, and those that can grow and ferment lactose to produce acid on the paper after cultivation are considered to be positive for coliform bacteria. Record the number of coliform bacteria positive papers at each dilution, and find the corresponding coliform bacteria number based on the coliform bacteria MPN table. The paper method for testing food coliform bacteria corresponds to the nine tube method in the national standard method, simplifying the previous few steps of the experiment into one step and reducing the time from 78 hours to less than 24 hours. This eliminates a lot of auxiliary work such as preparing culture media, disinfecting, and cleaning culture vessels, and allows for sampling and testing at any time by opening the packaging. Execution standard: National Food Safety Standard - Microbial Inspection of Food - MPN Counting Method for Coliform Bacteria (GB 4789.3).
2、 Operation method
1. Sample processing: Weigh 25g (or 25mL) of the sample according to aseptic procedures and place it in a sampling bottle or homogenization cup containing 225mL of sterile phosphate buffer diluent (or physiological saline). Shake thoroughly to make a 1:10 sample homogenate. The pH value of the sample solution should be between 6.5 and 7.5, and adjusted with 1mol/L sodium hydroxide or 1mol/L hydrochloric acid if necessary. Use a 1mL sterilized pipette to draw a 1:10 sample homogenate, inject it into a test tube containing 9mL of diluent, shake the test tube to make a 1:100 sample homogenate. Based on the estimation of sample contamination, follow the above steps to prepare a 10 fold series of diluted sample homogenates at once. For each incremental dilution, replace a 1mL sterile pipette or tip. The entire process from preparing the sample homogenate to completing the sample inoculation should not exceed 15 minutes.
2. Vaccination
2.1. Select two dilutions for inoculation. Ordinary foods generally use two dilutions of 1:10 and 1:100, while beverages use two dilutions of the original solution and 1:10. Each serving of this product consists of three large pieces of paper (10mL each, with two stacked in the bag as one large piece) and six small pieces of paper (1mL each).
2.2. Use a sterilized pipette to draw 10mL of 1:10 sample mixture and add it to a plastic bag containing large paper sheets (equivalent to 1g of sample). After suction, place it flat and repeat three times. Then use a 1mL sterilized pipette to draw 1mL of the same dilution of the sample mixture and add it to a plastic bag containing small paper sheets (equivalent to 0.1g of the sample), and repeat three times. Take another 1mL sterilized pipette and add 1mL of 1:100 sample mixture to a plastic bag containing small paper sheets (equivalent to 0.01g of sample), repeat three times.
3. Cultivation: Place the inoculated paper sheets (stackable) in a 36 ℃± 1 ℃ incubator and incubate for 15-24 hours.
3、 Report on the Most Probable Number (MPN) of Coliform Bacteria
1. If the paper turns yellow or shows red spots on a yellow background, it is a positive paper for Escherichia coli. The paper remains unchanged in purple blue color or presents red spots on a purple blue background, but there is no yellow around it, all of which are negative for coliform bacteria.
2. Record the number of positive paper strips for each dilution of coliform bacteria, and find the corresponding coliform bacteria number according to the "MPN Table for Coliform Bacteria in Food and Beverage".
3. If the first gradient of vaccination is the original solution, the results found on the table should be divided by 10.
4、 Precautions
The unit used in the new national standard has been changed to the most probable number of coliforms per gram (or milliliter) of sample, and the standard inoculation dose is 0.333g (mL). The MPN table is also designed based on this. The standard inoculation amount for using this product is 3.33g (mL), and inoculating 10mL of 1:10 sample homogenate on a large paper is equivalent to 1g of sample. Therefore, the numbers in the table should be reduced by 10 times accordingly. The inoculation amount of the beverage sample is 33.3mL, and 10mL of the sample solution should be inoculated on a large paper. The numbers in the table should be reduced by 100 times.
5、 Save conditions
This product needs to be stored in a refrigerator at 4 ℃ to 10 ℃ with a shelf life of one year. After opening the aluminum foil bag, any unused paper should be put back into the aluminum foil bag, sealed, and placed in the refrigerator. It should be used up within one month. Condensation may occur in high humidity environments, it is best to warm the entire package back to room temperature before opening.

Click to view: Food and Beverage Coliform (MPN) Retrieval Table