A variety of techniques has been developed for the isolation of microorganism, mainly the bacteria, from the specimen or from the sample cultures and pour-plate technique is among the simplest technique of isolating the bacteria.
The Culture techniques are commonly used in the laboratory for various purposes for which they are intended. The indications for the culture of the organism is mainly done –
- To demonstrate the cultural characteristics of the bacteria (e.g. color, texture, size, elevation etc.).
- To isolate the bacteria in discrete colonies from the specimen containing more than 1 bacterium.
- For determining the Sensitivity and/or Resistance of bacterium towards the particular Drug/Antibiotics or Test substances.
- To obtain the sufficient growth of the bacterium for various biochemical and other tests.
- To estimate the viable counts of the bacteria in the specimen.
- To maintain the stock cultures.
- To transport or short-term storage of the specimen (e.g. stab culture).
Three types of techniques are commonly employed for the isolations of microorganism from the specimen which are as follows:
- Pour Plate Technique
- Spread Plate Technique
- Streak Plate Technique
In this article, we’ll discuss the Pour Plate technique for the isolation of microorganism in the microbiology laboratory.
PRINCIPLE OF POUR PLATE TECHNIQUE
⇒ The modern pour plate culture method was initially developed in the laboratory of the famous bacteriologist and the father of bacteriology, Dr. Robert Koch.
⇒ In Pour Plate technique, successive dilutions of the inoculum (serially diluting the original specimen of old broth culture) is added to the sterile Petri plates containing the melted and cooled (40-45 °C) agar medium & thoroughly mixed by rotating the plates which are then allowed to solidify. After incubation, the plates are examined for the presence of individual colonies growing throughout the medium.
⇒ The pure colonies which are of different size, shape, and color may be isolated or transferred into test tubes containing liquid culture media (broth) or directly inoculated on the solid agar media by streak plate method for making pure cultures.
⇒ Pour Plate culture technique is also used as a means of determining the numbers of viable organisms in a liquid such as water, milk, Urine, or Broth cultures as well as to determine the hemolytic activity of deep colonies of some bacteria, such as the Streptococci, by using an agar medium containing blood.
REQUIREMENTS FOR THE POUR PLATE TECHNIQUE
- 24 hours old nutrient broth culture of two or more bacteria (Mixed Culture) or Sample/Specimen.
- Nutrient Agar Medium
- Six 9 ml Sterile Water Blanks
- Sterile Petri plates
- Graduated pipette (1ml)
PROCEDURE OF POUR PLATE TECHNIQUE
⇒ Melt the nutrient agar medium and keep it in the water bath set at 45 °C.
Serial Dilutions of the Specimen / Sample
⇒ Label the 6 Sterile Water blanks (9ml sterile water in each tube) as number 1 to 6 with the help of Marker. Also, label the Sterile Petri plates as number 1 to 6.
⇒ Place the labeled tubes in the test tube rack.
⇒ Mix well the 24 hours old broth culture to equally distribute the bacterial cells in the tube.
⇒ After mixing, Remove the Cotton plug and aseptically transfer the 1 ml of the bacterial suspension from the tube of culture to sterile water blank tube no. 1 using a graduated pipette.
⇒ Shake the tube no. 1 to mix well the content to uniformly distribute the bacterial cells. Transfer the 1 ml of this to the water blank tube no. 2 by using the graduated pipette.
Note: Use the separate sterile pipette each time to transfer the contents from one tube to another.
⇒ In this way, make serial dilutions till the six water blanks (no. 1 to no. 6).
Inoculating the Specimen / Plating of Specimen
⇒ Transfer 1 ml of the bacterial suspension each from the tube no. 1 to 6 to Petri Plates labeled as 1 to 6 by using separate sterile pipette each time.
⇒ Now, take out the Molten Nutrient Agar Medium (at 45 °C) from the water bath and pour the medium into the Plates no. 1-6 containing the specimen at different dilutions.
⇒ Rotate the plate gently to ensure the uniform distribution of cells in the media plates.
⇒ Allow the medium to solidify at room temperature.
⇒ Incubate the inoculated media plates for 24-48 hours at 37 °C in an inverted position.
Examine the plates for the appearance of individual colonies growing throughout the agar medium.
RESULTS OF THE POUR PLATE CULTURE TECHNIQUE
The colonies in the culture media plates inoculated by the serial dilutions of the specimen will show the lesser and lesser no. of the colonies as the dilution factor increased, which will be distributed more or less sparsely in the entire plate. These colonies may be transferred i.e. sub-cultured to the fresh media plates by streaking to obtain the pure culture of the bacterial cells for further study.
PRECAUTIONS TO BE TAKEN…..
⇒ The protocol should be followed under all aseptic conditions preferably in Laminar Air Flow (Safety cabinet) to avoid any contamination.
⇒ Accurately measure the quantity while preparing the serial dilutions of the specimen.
⇒ Use the sterile pipettes each time to avoid any contamination and errors in the result.
⇒ Accurately measure the quantity of Diluted specimen while inoculating onto the Solidified Media Plates.
⇒ Uniformly spread the specimen in the Media plate to get the discrete & well-developed colonies.