Streak plate method
1. Sterilize the inoculating loop in the bunsen burner by putting the loop into the flame until it is
red hot. Allow it to cool.
2. Pick an isolated colony from the agar plate culture and spread it over the first quadrant
(approximately 1/4 of the plate) using close parallel streaks or Insert your loop into the
tube/culture bottle and remove some inoculum. You don’t need a huge chunk.
3. Immediately streak the inoculating loop very gently over a quarter of the plate using a back
and forth motion (see area 1 in the figure above).
4. Flame the loop again and allow it to cool. Going back to the edge of area 1 that you just
streaked, extend the streaks into the second quarter of the plate (area 2).
5. Flame the loop again and allow it to cool. Going back to the area that you just streaked (area
2), extend the streaks into the third quarter of the plate (area 3).
6. Flame the loop again and allow it to cool. Going back to the area that you just streaked (area
3), extend the streaks into the center fourth of the plate (area 4).
7. Flame your loop once more.
Results
Streaked plate are incubated at 37°C for 24 hours. Examine the colonies grown in the plate
carefully. All colonies should have the same general appearance. If there is more than one
type of colony, each type should be streaked again on a separate plate to obtain a pure
culture.
Spread Plate Technique
Procedure for Spread Plate Technique :
A: Serial Dilution
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 [Link] 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 10-2.
5. Repeat the procedure with all the remaining tubes labeling them until 10-6.
B. 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 45oat the same time.
5. Incubate the plate at 37°C for 24 hours.
6. Calculate the conlony forming units (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.
Pour plate Method
Procedure of Pour plate technique
1. Prepare the dilution of the test sample expected to contain between 30-300 CFU/mL. (Follow
serial dilution technique)
2. Inoculate labeled empty petri dish with specified mL (0.1 or 1.0 mL) of diluted specimen
3. Collect one bottle of sterile molten agar (containing 15 mL of melted Plate Count Agar
or any other standard culture media) from the water bath (45°C).
4. Hold the bottle in the right hand; remove the cap with the little finger of the left hand.
5. Flame the neck of the bottle.
6. Lift the lid of the Petri dish slightly with the left hand and pour the sterile molten
agar into the Petri dish and replace the the lid.
7. Flame the neck of the bottle and replace the cap.
8. Gently rotate the dish to mix the culture and the medium thoroughly and to ensure that the
medium covers the plate evenly. Do not slip the agar over the edge of the petri dish.
9. Allow the agar to completely gel without disturbing it, it will take approximately 10 minutes.
10. Seal and incubate the plate in an inverted position at 37°C for 24-48 hours.
Lawn Culture:
The lawn culture provides a uniform layer of bacterial growth on a solid medium.
It is carried out by flooding the surface of the solid media plate with a liquid culture
or suspension of bacteria, pipetting off the excess inoculum, and finally incubating
the plate overnight at 37°C.
The culture plate may be inoculated by a sterile swab soaked in liquid bacterial
culture or suspension and incubating overnight for the growth of the bacterial
colonies.