Microbial Technique Pour Plate Method :

Microbiology Lab Essentials Technique :
3)Pour Plate Technique :
Pour plate method is usually the method of choice for counting the number of colony-forming bacteria present in a liquid specimen.in this technique  fixed amount 1ml  of inoculum from a broth/sample is placed in the center of sterile Petri dish using a sterile pipette.

✓#REQUIREMENTS FOR THE POUR PLATE TECHNIQUE :
1)24 hours old nutrient broth culture of two or more bacteria (Mixed Culture) or Sample/Specimen.
2)Nutrient Agar Medium
3)Six 9 ml Sterile Water Blanks
4)Sterile Petri plates
5)Marker
6)Graduated pipette (1ml)

⇒ 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.

✓#PROCEDURE  :

⇒ 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.
Prepare the upto 6 Folds of Serial Dilutions

#Note :
Use Separate Steriled pippete each time.

✓#Inoculating the 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.

✓#RESULTS :
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.