Streak Plate Method and isolation techniques

In order to be able to properly identify a bacterial species, it is necessary to obtain well isolated colonies. The principle is to exhaust an initial deposit by making successive smears in different directions (hence the name of the technique: exhaustion by the quadrant method).

Several techniques are commonly applied: 3, 4 or 5 successive spreads

Ⅰ. Principle of isolation by streak plate method

Quadrant seeding (general purpose) is useful for most samples. The relative number of organisms can be estimated based on the extent of growth beyond the area of origin of the inoculum.

☰ Steps of streak plate method

1. Make sure that the content of the tube (broth or culture suspension) is mixed evenly.
2. Place a loop of culture broth on the surface of an agar dish, near the edge but without touching it. With the loop flat against the agar surface, lightly scratch the inoculum (see photo: ❶); do not dig up the agar.
3. Sterilize the loop and let it air cool.
4. Turn the open agar dish in your left hand so that you can draw a series of four lines back and forth, each passing through the inoculum and extending to one side of the agar dish (❷).
5. Sterilize the loop and let it air cool.
6. Rotate the agar dish and draw another set of four lines, each crossing the end of the last four lines and extending to the adjacent side of the agar dish (❸)..
7. Turn the plate over and repeat this parallel stripe once more.
8. Finally, make a few streaks in the intact center of the plate. Do not touch the original inoculum (❹).
9. Incubate the plate (inverted) at 35 ° C.

Ⅱ. Semi-quantitative seeding

Some specimens, such as urine, require a quantitative technique to determine the number of bacteria present.

The dishes are inoculated using a calibrated loop to deliver a specified volume. The urine is mixed well and the calibrated loop (1 or 10 µl) is inserted into the urine and transferred to the culture medium by making a single line in the center of the plate. Without flaming, the loop is streaked back and forth through the original inoculum.

The next day, count the number of colonies on the surface of each medium. Each colony growing on the agar plate represents one colony forming unit (cfu) / µL (depending on the size of the loop), which is equivalent to 1000 cfu / mL. Remember that nutrient agar is the primary medium used to count colonies.

Semi-quantitative streak plate method

« Example »

• 10 Colonies with a 1µl loop correspond to 10⁴ cfu/mL
• 10 Colonies with a 1µl loop correspond to 10³ cfu/mL

☰ bacteriological inoculation loop

öse (or loop ), the inoculation loop is a portable tool that ends in a small metal loop. The loop can be used to smear microorganisms on agar in a petri dish or to transfer them from one test tube to another. Before each use, the inoculation loop must be sterilized so that the cultures are not contaminated.