Spread plate technique

The main difference between surface plate and pour plate is that the sample and/or its dilutions are inoculated directly onto the surface of a solid medium, previously distributed over a certain number of Petri dishes. Surface inoculation is considered advantageous in some aspects, since it does not expose the microorganisms to the high temperature of the melted medium, allows visualization of the morphological and differential characteristics of the colonies, facilitates the transferring of colonies, allows to use media that may not be re-heated to melt the agar, and does not require that the culture media be transparent or translucent. Its main disadvantage is the volume to be inoculated, which is limited to the maximum liquid absorption capacity of the culture medium (0.5 ml per plate). The standard procedure is the inoculation of 0.1 ml/plate of each dilution, with a detection limit of 100 CFU/g for solid products or 10 CFU/ml for liquid products. This procedure can be adapted, if necessary, to a detection limit of 10 CFU/g for solid products or 1 CFU/ml for liquid products. Its main applications are total aerobic psychrotrophic counts, yeast and mold counts,  S. aureus counts and B. cereus counts.

1- Material required for the analyses

- Materials for preparing the sample and serial dilutions.

- Petri dishes containing the medium recommended for the test.

- Glass or plastic spreaders (Drigalski) immersed in ethanol 70%.

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

2- Procedure

Before starting the procedure, to ensure that all activities be carried out under aseptic conditions. Identify all the tubes and plates that will be inoculated with the sample code, the dilution and the standard abbreviation of the culture medium. Prepare the plates on beforehand and dry them in a laminar flow cabinet for 30–60 min with the lids partially open or in an incubator at 50°C/1.5–2 h with the lids partially open or in an incubator at 25–30°C/18–24 h with the lids closed.

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 sample to be inoculated. Using a pipette with a maximum holding capacity of 1 ml (and 0.1 ml graduation markings,(  inoculate 0.1 ml of each dilution onto the surface of previously prepared plates. Verify whether the identification of the plate actually corresponds to the sample and dilution that are being inoculated and whether the plate contains the correct culture medium. 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. Work in a laminar flow cabinet or in the proximity of the flame of a Bunsen burner. Spread the inoculum onto the entire surface of the medium as fast as possible, using glass or plastic spreader (Drigalski), and continue until all excess liquid is absorbed. Utilize a different spreader for each plate or, alternatively, flame-sterilize the spreader after each plate, starting with the greatest dilution plate and going to the smallest dilution plates.

 Note b.1)   When several tests are being performed simultaneously, the activities and teamwork should be organized and programmed so as to satisfy the following conditions the complete procedure, from the preparation of the first dilution until finishing the inoculation شof all the culture media, should not take longer than 20 minutes and the inoculum spreading should be started immediately after depositing the three dilutions onto the medium surface. recommends not exceed 45 minutes.

Note b.2)   As described for pour plating, select the dilutions as a function of the estimated contamination level of the sample, so as to obtain plates containing 25 to 250 colonies. However, it should be taken into account that the inoculated volume is ten times smaller. In the case of samples with a low level of contamination, a greater volume of the first dilution may be inoculated, distributing this volume over several plates. A distribution commonly used is inoculating three plates with 0.3 ml and one plate with 0.1 ml. The spreading of 0.3 ml onto the plates requires a longer time of absorption of the liquid, thus care and precautions must be taken to avoid that moisture films remain on the surface, with the consequent formation of spreading zones.

 Note b.3)  For the analysis of certain foods, the recommended initial dilution is greater than 1:10. If the expected counts in these products are 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 0.01 g of solid products or 0.1 ml of liquid products should be maintained, if possible.

 Note b.4)  To increase the accuracy of the counts, it is recommended not to utilize pipettes with a capacity greater than 1 ml to dispense volumes of 0.1 ml.

 Note b.5)   does not require the inoculation of three dilutions of the sample, establishing only two successive dilutions, without duplicate, or with a duplicate if only one dilution is to be used. How-ever, the way to calculate the results is somewhat different.

c) Incubation: Follow the same instructions and guidelines as those described for pour plating.

d)  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 .