Method III

Plate streaking with inoculum from a liquid suspension:

This entire sequence must be repeatedly practiced with an empty Petri dish and plain water until it can be carried out smoothly without referring back to this write-up.

Gently shake the flask or tap the bottom of the culture tube with your finger to suspend microbial cells in the broth that are to be transferred. Watch out for the broth level and do not wet the cotton plug when shaking.

Light a Bunsen burner and flame an inoculation loop until the wire turns red hot. This will kill all the bugs on the loop surface. Slightly flame the handle as well to minimize the chance of contamination.

Cool the loop for about 5 seconds.

While the loop is cooling, with the thumb and index finger of the right hand holding the inoculation loop and with the left hand holding the flask or culture tube, take out and hold onto the plug with the baby finger and palm of the right hand. Quickly pass both the neck and rim of the flask through the flame.

Carefully dip the loop into the broth that contains the microorganism to be streaked. Note that only the small loop at the tip needs be dipped, not the entire extension of the wire leading to the loop and definitely not the handle. Since the handle is not really well sterilized, do not touch the side of the flask or culture tube with the handle.

Flame the neck and rim of the flask or tube as before. After lightly flaming the bottom and side of the plug, place the plug back into the mouth of the flask. Note that the side and bottom of the plug never comes in contact with anything but air; the plug is never placed on a table.

Put down the flask and pick up a Petri dish with the left hand. Flip open the cover plate with the middle finger and the thumb. Gently stroke 3-5 parallel lines at one corner of the plate with the loop to spread the cells. (Lines marked A in the following Figure.) Re-close the Petri dish; the best results are achieved when the dish is recovered immediately following the streaking. Do not gouge the agar with the loop; a light touch is all that is necessary to deposit the culture onto the plate.

Flame the loop as before and cool it for about 5 seconds. Gently touch the free surface of the agar plate to cool it further to avoid killing all the organisms upon the initial contact. Spread the cells further with 5-6 strokes (Lines B in the Figure); start each stroke by crossing the original set of lines.

Repeat the above step twice more to create Lines C and D, flaming the loop each time before streaking.

When done streaking, flame the loop for the last time before returning it to the bench. This is to prevent the spread of microorganisms on the bench. This last step is especially crucial when working with pathogenic strains.

Incubate the inoculated plate upside-down at 37ºC for 48 hours. Store the plate, also upside-down, in a refrigerator thereafter.

Yeast Culturing Page