13 DE GUZMAN, Naomi Jianne B.

MT 6320 Laboratory

3DMT Standard Operating Procedure

Pure Culture - Spread Plate Method

1. From a sample, create a dilution series.

2. Pipette 0.1 ml from the appropriate required dilution series onto the agar plate's central
surface.
3. Dip the L-shaped glass spreader into alcohol.
4. Over a Bunsen burner, flame the glass spreader (hockey stick).
5. Using the sterile glass spreader, evenly distribute the sample across the agar surface while
gently rotating the Petri dish below.
6. Incubate the plate at 37°C for 24 hours.
7. Calculate the sample's CFU value. After you count the colonies, multiply by the
appropriate dilution factor to determine the number of CFU/mL in the original sample.

Mixed Culture – Clock method

1. With your non-dominant hand, lift the edge of the lid enough to enter the loop with your
other hand.
2. Using the loopful inoculum, swab a section of the agar plate (12 o'clock)
3. Close the lid and flame-sterilize the loop. Allow time for it to cool.
4. Move the loop from side to side until you've covered half of the surface area.
5. Close the lid and sterilize the loop with a flame. Quarter-rotate the plate in a clockwise
direction.
6. Open the lid once the loop has cooled and repeat the streak by touching the last two lines
of the previous streak and going towards the center to the side of the plate until more than
1/3 of the area is covered.
7. Close the lid and flame-sterilize the loop once again. Continue streaking by quarter-
rotating the plate clockwise.

References:

Aryal, S. (2019). Streak Plate Method- Principle, Methods, Significance, Limitations. Retrieved
from https://microbenotes.com/streak-plate-method-principle-methods-significance-limitations/