1. Principle & application scope

Listeria detecting board is a kind of disposable culture medium containing standard nutrient medium, selective reagent, soluble bibulous gel and chromogenic substrate (X-GLU) of β-D-glucosidase. Listeria detecting board is suitable for the detection of Listeria in food and environment. This product can detect Listeria, including Listeria monocytogenes, Listeria innocua and Listeria welshi, but it cannot distinguish these organisms from one another.

2. Using method

2.1 For food sample:

2.1.1 Sample preparation: Prepare the 1:10 solution by placing 25g (mL) of sample into 225mL of sterile phosphate buffer solution or sterile saline. According to the pollution level of sample, prepare tenfold increasing serial dilution of sample if necessary. Select 2 or 3 diluted solutions to inoculate.

2.2 For environmental sample:

2.2.1 Environmental sampling: use sterile swab stick or other sampling equipment to collect environmental sample. Moisten the sampling equipment with humectants like sterile water, sterile saline, sterile phosphate buffer solution or buffered peptone water. The volume of the humectants should be less than 10mL.

2.2.2 Sample preparation: add 10mL of sterile saline or phosphate buffer solution, and then shake them evenly. According to the pollution level of sample, prepare tenfold increasing serial dilution of sample if necessary. Select 2 or 3 diluted solutions to inoculate.  

2.3 Inoculation

Lift the adhesive label of the upper film of Listeria detecting board; use a sterile pipette to suck 1mL of sample solution and drip it to the filter paper evenly, then cover the upper film and shake the detecting board horizontally. Stand it for about 10s until the sample solution is absorbed into the filter paper. Inoculate two detecting boards for each different diluted solution.

2.4 Culture

Superimpose the detecting boards and place them upside down in the incubator at 36℃±1℃ for 18-24 hours.

3. Results

Listeria species show bluish green on the detecting board. If there are not any colonies or the colonies on the detecting board show other colors, it is a negative result.

4. Counting method

4.1 Sample counting

4.1.1 Select the detecting boards of which the colony quantity is between 30 and 300 CFU to do the counting.

4.1.2 If there is only one diluted sample suitable for counting, calculate the average number of colonies on two detecting boards, and then multiply it by corresponding dilution multiple.

4.1.3 If the colony numbers on detecting boards from two serial dilutions are both between 30CFU and 300CFU, then calculate the number of colonies in sample by below formula (1): 

        ∑C

N= ———————— •••••••••••••••••••（1） 

    （n₁+0.1n₂）d

In the formula: 

N means the Listeria quantity in sample. 

∑C means the total number of Listeria.

n₁ means the quantity of detecting boards of which the dilution multiple is lower between two serial dilutions.

n2 means the quantity of detecting boards of which the dilution multiple is higher between two serial dilutions.

d means the higher dilution ratio.

For example:

    (245+247+31+30)

N= ————————— = 2514          

    (2+0.1×2)×10^-1

The result is 2.5×10³CFU/g(mL)

4.1.4 If no colony was found on the detecting boards from all dilutions of sample, report the count as less than one multiply the minimum dilution multiple. 

4.1.5 Estimate low value: If the colony numbers on two detecting boards of original sample (water and liquid beverage) or initial dilute suspension are less than 30CFU, then calculate the average number of colonies on two detecting boards by below formula (2): 

       ∑C

NE = ——— •••••••••••••••••••（2）

       nd      

NE means estimated value of the colony quantity in per milliliter or gram sample.

∑C means total number of colonies on two detecting boards.

n means the quantity of detecting boards.

d means the dilution ratio of original suspension or inoculated suspension.

4.1.6 Estimate high value: If the colony numbers on detecting boards from all the dilutions are more than 300CFU, use the detecting boards having a count closest to 300CFU. Calculate the average number of colonies. Other detecting boards can be recorded as TNTC (too numerous to count). The formula (3) is:

      ∑C 

NE = ——— •••••••••••••••••••（3）

      nd

NE means the estimated value of the colony quantity in per milliliter or gram sample.

∑C means total number of colonies on two deteting boards.

n means the quantity of detecting boards.

d means the corresponding dilution ratio of the sample suspension.

5. Report

Use CFU/g as report unit for solid sample, and use CFU/mL as report unit for liquid sample.

6. Additional explanatory note

Dispose the used detecting boards according to the biology safety waste treatment rule, because there are live bacteria on them.