Drop plate technique

The drop plate method is a surface inoculation technique that has the same advantages as the spread plating technique. The main difference is that the inoculum is not spread, but deposited onto the surface of the culture medium in the form of 0.01 ml-droplets. Since the droplets occupy a minimum amount of space, it is possible, on one and the same plate, to inoculate three dilutions in triplicate, three drops per dilution. This makes this technique extremely cost-friendly, with a detection limit 1,000 CFU/g for solid products or 100 CFU/ml for liquid products. Although the drop plate technique is not routinely used for the micro-biological examination of foods, it can be very useful in situations that require the inoculation of a large number of dilutions.

1-  Material required for the analyses

- Materials for preparing the sample and serial dilutions.

- Sterile 0.1 ml graduated pipettes or pipettes with disposable tips to dispense the droplets.

- Petri plates containing the medium recommended for the test, described in specific chapters.

- A laboratory incubator with the temperature set at the temperature specified by the test to be per-formed.

2  - Procedure

Before starting the procedure, to ensure that all activities be carried out under aseptic conditions. Identify by labeling 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 with the culture medium on beforehand and leave to dry in an incubator for 24 hours at 25–30°C with the lids closed.

a)  Preparation of the samples and serial dilutions: prepare the diluent supplemented with 0.1% agar, to make it easier to fix the droplets later on onto the surface of the culture medium.

b) Inoculation: Divide the plate into nine sectors, marking the bottom with a glass-marking pen (tracing three horizontal lines and three vertical lines). For each test that is being conducted, select three adequate dilutions of the sample to be inoculated .Before collecting the volume of each dilution to be inoculated onto the plates, vigorously agitate or shake the dilution tubes, by inverting them 25 times in an 30 cm-arc or with the aid of a vortex shaker. Using 0.1 ml (and 0.01 ml graduated) pipettes or pipettes with disposable tips, deposit three 0.01 ml drops of each dilution in three adjacent quadrates of the plate (triplicate). This procedure must be per-formed with care, to avoid any droplets from running out of their respective quadrate. Do not spread the drops. Keep the plates placed on a flat surface, wait until all the liquid is absorbed by the culture medium, which will require approximately 30 minutes.

Note b.1)   Select the dilutions as a function of the estimated contamination level of the sample, so as to obtain drops containing 30 colonies, at most. Take into account that drop plating cannot be used with samples with a contamination level lower than 103 CFU/g or 102 CFU/ml, except when the purpose of the test is not to quantify, but rather to show or prove that the count is below this limit.

c) Incubation: Wait until the liquid of the drops is completely absorbed by the culture medium and incubate under the same conditions recommended for pour plating.

d)  Counting the colonies and calculating the results: Follow the guidelines and instructions.

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 .

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Haemophilus influenzae

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Conventional Phenotypic Tests for Mycobacterial Infections

Genetic Sequencing and Nucleic Acid Amplification for Mycobacterial Infections

Acid-Fast Stains for Mycobacterial Infections

Vibrio Parahaemolyticus and Vibrio vulnificus

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