SPREAD PLATE TECHNIQUE

SPREAD PLATE TECHNIQUE

AIM

           To isolate and enumerate the bacteria present in Water sample by Spread plate method.

PRINCIPLE   

          Spread plate technique is the method of isolation and enumeration of microorganisms in a mixed culture and distributing it evenly. The technique makes it easier to quantify bacteria in various samples. 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 and to isolate and identify variety of microbial flora present in the environmental samples e.g., water and soil.

MATERIALS REQUIRED

• Water sample
• Erlenmeyer flask
• Pipettes
• Test tubes
• Glass L – rod
• Distilled water
• Nutrient agar plates
• Incubator

PROCEDURE

I.  Serial Dilution Technique

a)     Prepare a series of 6 test tubes containing 9 ml of sterile Distilled water.

b)    Prepare the Master dilution by adding 1 ml of Water sample in the Erlenmeyer flask containing 99 ml of Sterile Distilled water.

c)     Mix the contents well by shaking the Master dilution flask for few times.

d)    From the First tube (10^-1 Dilution), take 1 ml of the sample and transfer to the Second tube (10^-2 Dilution).

e)     Repeat the procedure with all the remaining tubes labeling them until 10^-6 Dilution.

f)      Discard the final 1 ml from the 10^-6 Dilution.

II. Spread Plate Technique

a)   Pipette out 0.1 ml from the appropriate desired dilution series onto the center of the surface of the Nutrient agar plates.

b)   Dip the L-shaped glass spreader into alcohol and Flame the L - glass spreader over a Bunsen burner.

c)   Spread the sample evenly over the surface of Nutrient agar using the sterile glass spreader, carefully rotating the Petridish underneath at the same time.

d)     Incubate the Petriplates in an Incubator at 37 °C for 24 hours.

e)    Calculate the cfu value of the sample. Once you count the colonies, multiply by the appropriate dilution factor to determine the number of cfu/ml in the original sample.

 

Calculation

Colony forming unit (cfu/ml) = (Number of colonies × Dilution factor)/Volume of the sample

*Dilution factor – Reciprocal of the Dilution (For example, 10^-1 is the Dilution and 10¹ is the Dilution factor)

OBSERVATION AND RESULTS

               After incubation, count the bacterial population by using Quebec colony counter, Calculate using cfu/ml calculation and express the number of bacterial colonies as cfu/ml. 

Figure – 1: Serial dilution technique

Figure – 2: Spread plate method