Course: MCB 103 Lab

Section: 1
Name: Noor-E-Khadiza Shama
ID: 1921168
Date: 11/03/20

Experiment 9

SPREAD PLATE

PURPOSE
1. To demonstrate the cultural characteristics of the bacteria (e.g. color, texture, size,
elevation, etc.).

2. To isolate the bacteria in discrete colonies from the specimen containing more than 1
bacterium.
3. To estimate the viable counts of the bacteria in the specimen.

PRINCIPLE
During spread plating, the bacteria are introduced in the agar plates and each bacterium will form
individual isolated colony. After spreading, distance is sufficient to allow colonies to develop that are free
from each other. Each colony will represent one bacterium, so the number of colonies will be
proportional to the number of bacteria. Using this number, CFU calculation will be done which will give
an approximate idea about the amounts of bacteria in the sample

MATERIALS & EQUIPMENT

1. Micropipette
2. Tamarind water sample which has been diluted to 10 -3
 3. Agar plates of 4 different agar (Mac Conkey, TCBS, EMB and nutrient agar)
4. Glass spreader
5. Incubator

PROCEDURE
1. A sample of tamarind juice which has been diluted to 10 -3 is taken.
2. Using a micropipette,30 micro liter of sample is inoculated in each of the four agar plates.
3. Then a glass spreader is taken and dipped in ethanol to clean it.
4. Then the spreader is burned in Bunsen burner flame and held in the air to cool it down.
5. The spreader is then used to spread the inoculum over the surface of the agar plates evenly
6. While spreading, the plate should be rotated gently to evenly spread the sample.
7. After spreading, the spreader is cleaned by dipping it in ethanol.
8. The plates are covered with lids, then turned upside down and kept in an incubator at 37 o C for
24 hrs.
9. After 24 hours, all the visible colonies are calculated and represented as colony forming units

RESULT & OBSERVATION

The number of colonies observed in each plate is given below.

AGAR NUMBER OF
COLONIES
Mac Conkey 0
TCBS 0
EMB 0
Nutrient agar 17

Calculation of CFU using colonies found in Nutrient agar

Dilution factor – 10-3

Cells in 30ul sample - 17 cells

Cells in 1ul sample – (17 / 30)

1000ul = 1ml

Cells in 1ml = (17 X 1000 / 30)

Cells in the original sample = (17 X 1000 X 10-3 / 30) = 5.6 X 10-5 cfu/ml
DISCUSSION
As no colonies were found in Mac Conkey, TCBS and EMB, we can say that the sample had very
less contamination of bacteria. Colonies were only seen in Nutrient agar which suggest that
there must be some fastidious microorganisms in the sample or there was contamination during
transfer of sample.

LIMITATIONS
1. Strict aerobes are favored while microaerophilic tends to glow slower in spread plate
2. Volume no greater than 0.1 ml can be spread on the nutrient agar plate because it would
not soak well and may result in colonies to coalesce as they form.

PRECAUTION
1. The spreader should be gently rolled over the agar as the agar might break upon
exerting pressure.
2. While spreading, the plate should be rotated slowly using another hand to evenly
distribute the sample
3. Micropipette should not be pushed too hard while taking the sample
4. The tip of the micropipette should not touch anywhere
5. The alcohol should be used carefully to clean the spreader as introducing the hot
spreader in alcohol may catch fire.