Cultivation Techniques in Microbiology :

Microbiology Lab Essentials :
1)Spread Plate Technique :

✓Principle :

It is essentially a dilution technique that involves spreading a loopful of culture over the surface of an agar plate. ... On the initial region of the streak, many microorganisms are deposited resulting in confluent growth or the growth of culture over the entire surface of the streaked area.

#What #is #it ????
The spread plate method is a technique to plate a liquid sample containing bacteria so that the bacteria are easy to count and isolate. A successful spread plate will have a countable number of isolated bacterial colonies evenly distributed on the plate.

A perfect spread plate technique will results visible and isolated colonies of bacteria that are evenly distributed in the plate and are countable. The technique is most commonly applied for microbial testing of foods or any other samples or to isolate and identify variety of microbial flora present in the environmental samples e.g. soil , air or water

✓Requirements:

Glasswares: screw capped test tubes, sterile pipettes, glass spreader

Medium: Plate count agar or Nutrient agar

✓Serial Dilutions : 

1)Prepare a series of  at least 6 test tubes containing  9 ml of sterile distilled water.

2)Using a sterile pipette ,add 1ml of sample in the first tube of the set.Label it as 10-1

3(Mix the contents well by swirling the tube upside down few times.

4)From the first tube, take 1ml of the sample and transfer to second tube. Label it as 10 power in minus.

5)Repeat the procedure with all the remaining tubes labeling them until 10 folds.

✓Plating 

1)Pipette out 0.1 ml from the appropriate desired dilution series onto the center of the surface of an agar plate.

2)Dip the L-shaped glass spreader (hockey stick) into alcohol.

3)Flame the glass spreader over a bunsen burner.

4)Spread the sample evenly over the surface of agar using the sterile glass spreader, carefully rotating the Petri dish underneath at an angle of 45° at the same time.

5)Incubate the plate at 37°C for 24 hours.

Calculate the conlony forming units (CFU) value of the sample. 

6)Once you count the colonies, multiply by the appropriate dilution factor to determine the number of CFU/mL in the original sample.

✓Calculation of result : 

1)CFU/ml = (no. of colonies x dilution factor) / volume of culture plate

2)For example, suppose the plate of the 10^6 dilution yielded a count of 130 colonies.

3) Then, the number of bacteria in 1 ml of the original sample can be calculated as follows:

Bacteria/ml = (130) x (10^6) = 1.3 × 10^8 or 130,000,000.