English: Isolation of pure microbiological cultures: Streak method and inverted droplet method

- Streak method

In order to isolate a pure culture from a mixed culture using the streak method, the following is to be performed:

1. The inoculation loop is to be sterilized before use using an open flame or other equipment.

2. The Petri dish is to be opened and the inoculation loop is cooled by touching the agar at a location where no growth has occurred.

3. Inoculum is to be selected from a colony.

4. The inoculum is initially to be transferred onto a sterile nutrient agar plate by performing zig-zag movements along the surface of the plate so as to cover up to half of the plate (streak A).

5. The inoculation loop is to be sterilized after streak A, cooled before use; followed by spreading the initial inoculum from streak A by moving the inoculation loop from the position where streak A ended, towards the remaining untouched surface of the plate, making sure to only touch streak A once (streak B).

6. The previous step is repeated in order to form streak C, making sure to only touch streak B once.

7. The final streak is to be performed as previously, making sure to touch streak C only once.

8. The plates are to be incubated at 37 degree centigrade for 24 hours.

RESULTS: Proper execution of the streaks should result in visibly distinct individual colonies from the latter streaks (D), due to the reduction in viable cell number as a result of spreading the initial inoculum.

- Inverted droplet method

1. Using a sterile inoculation loop or Pasteur pipette, a single drop of a suspension is to be applied onto a cover slide. The cover slide is immediately inverted (180 degrees), making sure the droplet remains hanging firmly from the underside of the cover slide.

2. Afterwards the counting chamber of choice is to be asserted underneath, hermetically sealed with Vaseline if possible.

3. Microscopic analysis of the slide at magnification of at least x450 should reveal if the suspension is of a pure culture or not.

RESULTS: The presence of a singular type of cell or spore is considered adequate confirmation of a pure culture.

Planned and performed by Sofija Kostandinovska, Institute of Biology, FNSM, Ss. Cyril and Methodius University, Skopje, Macedonia.