Pour plate technique

The standard procedure for pour plating, described below, has a detection limit of 10 CFU/g for solid products or 1 CFU/ml for liquid products. This procedure may be adapted, if necessary, to achieve detection limit of 1 CFU/g for solid products. The main applications for the pour plating technique are total aerobic mesophilic counts,  Enterobacteriaceae counts, enterococci counts and lactic acid bacteria counts. There are some limitations to this technique, the main one being the need to melt the culture medium before use. Some media supplemented with heat-sensitive components after sterilization may not be reheated to melt the agar before use.

1- Material required for the analyses

-    Material for preparing the sample and serial dilutions .

-   The culture medium recommended for the test to be carried out, described in specific chapters.

- Sterile, empty 20 × 100 mm Petri dishes.

-  Laboratory incubator set to the temperature specified by the test to be performed.

2- Procedure

Properly identify the tubes and plates that will be inoculated by labeling them with the sample code, the dilution, and the standard abbreviation of the culture medium. Melt the culture media in a boiling water bath, main-taining the boiling for only the time necessary to soften and liquefy the agar. Cool immediately in cold water and keep at a temperature of 44 to 46°C until the time of use )in a temperature-controlled water bath or incubator).

a)  Preparation of the samples and serial dilutions

b) Inoculation: In general, inoculation is done for several tests at the same time. For each test that is being conducted, select three adequate dilutions  of the samples (see the notes below) and inoculate 1 ml of each dilution in separate, sterile and empty Petri dish, opening the plates only enough

to insert the pipette. Work in a laminar flow cabinet or in the proximity of the flame of a Bunsen burner. Deposit the inoculum off-centre in the Petri dish, since this will later on facilitate mixing with the culture medium. Position the pipette at an angle of about 45° touching the bottom of the plate. Use a different pipette for each dilution, with a maximum holding capacity of 10 ml. The uncertainty of the volume measurement must not exceed 5%. Observe carefully whether the plate used actually corresponds to the sample and dilution that are being inoculated. Change the position of the plates as they are being inoculated, to avoid the risk of inoculating the same plate more than one time, or to leave a plate un-inoculated.

 Note b.1)   Select the dilutions as a function of the estimated contamination level of the sample, so as to obtain plates containing 25 to 250 colonies. If the expected contamination level of the inoculum falls in range from 2.500 to 25.000 CFU/g or ml, for example, the recommended dilutions are 10⁻¹, 10⁻² and 10^−3, which correspond to 0.1, 0.01 and 0.001 g or ml of sample. If the contamination level is expected to exceed this range, higher dilutions must be inoculated. If the contamination is expected to be below this range, it is possible to start inoculating 1 ml of the sample without any dilution for liquid products. In the case of solid products it is not possible to inoculate samples without dilution, but it is possible to inoculate up to 2 ml of the initial dilution in one and the same plate, or a greater volume, distributed or divided over several plates (2 ml/plate.( If it is not possible to previously estimate the level of contamination of the sample, it is recommended to inoculate more than three dilutions, made from the initial dilution.

 Note b.2)   For the analysis of certain foods, the recommended initial dilution is greater than 1:10. If the counts in these products are expected to be low, the inoculated volume of the initial dilution should be increased, in the same way as described in Note b.1. When using this technique, the inoculation of 0.1 g of solid product or 1 ml of liquid product should be maintained, if possible.

 Note b.3)   To increase the accuracy of the counts, it is recommended not to use pipettes with a holding capacity greater than 2 ml to dispense volumes of 1 ml. It is also possible to inoculate two plates with the same dilution (duplicate) .

 Note b.4)  does not require the inoculation of three dilutions of the sample, nor that of two plates for each dilution. It establishes two successive dilutions, without duplicate, or with a duplicate if only one dilution is to be used. However, the way to calculate the results is somewhat different .

c)  Addition of the culture medium: For each test that is being conducted, withdraw the culture medium from the water bath or incubator at 44–46°C and, if the flask is wet, dry with a paper towel taking care to avoid spattering onto the plates at the moment of plating. Avoid agitation and abrupt movements to prevent the formation of bubbles. Pour 12 to 15 ml of the medium into the inoculated plates, observing whether the identification of the plates corresponds to the culture medium used. Mix the medium with the inoculum, moving the plates gently on a flat surface, in movements forming the number eight or in circular movements, 8–10 times clockwise and 8–10 times counter-clockwise. The plates should be moved about with utmost care, to avoid droplets of medium from spattering onto the rims or lids of the plates. To facilitate this step of the operation, prefer using high plates (20 × 100 mm). The plates can be stacked one on top of the other during the addition of the growth medium and homogenization with the inoculum, but they must subsequently be evenly distributed over the cold surface of a bench, to accelerate cooling and solidification of the medium.

 Note c.1)   When several tests are being conducted simultaneously, the activities and teamwork should be organized and programmed so as satisfy the following conditions, established by the Compendium (Swanson et al., 2001): the time between depositing the inoculum in a plate and the addition of culture medium should not exceed 10 minutes, to prevent the inoculum from drying out and to adhere to the plates. Mixing the culture medium with the inoculum should be done immediately after adding the medium, in order to avoid the risk of solidification of the agar. Furthermore, the Compendium recommends that the complete procedure, from the preparation of the first dilution until finishing the inoculation of all the culture media, should not take longer than 20 minutes.

Note c.2)  For foods containing or consisting of particles (meals and flours, for example) the distinction between colonies and particles may be difficult in the first dilution. To avoid this problem, TTC (2,3,5 triphenyltetrazolium chloride) may be added to the culture medium since most bacteria form red colonies in the presence of TTC. For each 100 ml of medium, add 0.5 ml of a 1% aqueous TTC solution, previously sterilized by filtration.

d) Incubation: Wait until solidification of the culture medium is completed, invert the plates (if required by the method being used) and incubate at the conditions of temperature, time and atmosphere specified for each test. The culture medium should reach the incubation temperature within a maximum time interval of two hours. Avoid excessive stacking of the plates and do not place an excessive number of plates in each incubator, to ensure even distribution of the temperature. Within the first 48 h of incubation, the plates may not lose more than 15% of their weight caused by drying out. Excessive moisture is also undesirable, since it increases the risk of spreading. Depending on the temperature, humidity control of the incubator may be necessary.

e)  Counting the colonies and calculating the results.

References

Silva, N.D .; Taniwaki, M.H. ; Junqueira, V.C.A.;  Silveira, N.F.A. , Nasdcimento , M.D.D. and Gomes ,R.A.R .(2013) . Microbiological examination methods of food and water a laboratory Manual. Institute of Food Technology – ITAL, Campinas, SP, Brazil .