LAB: 4 Culture of microorganisms from environment

- Microorganisms are found throughout the environment - Microorganisms are found throughout the environment. For better study of these microorganisms pure culture is needed. Pure culture: a single kind of microorganism growing alone in a protected environment. There are two methods used widely for preparation of pure culture from mixed population. 1. streak plate method. 2. pour plate method.

Streak plate method ( Quadrant plate) This method is used for the isolation of pure culture of bacteria from mixed population e.g. sputum, urine, stool or from pus of infected wound or abscess.

Technique of Streak plate method : Sterilize the loop in a benzene flame, cool it and streak the specimen over an area-A. Re-sterilize the loop, cool it, then streak over an area-B from the distal part of area-A. Continue the streak in the same manner for areas C and D. Incubate the plates at 37°C for 24 hours.

A subculture is done after 24 hours incubation from one of the colonies needed for study on a sterile medium following the same technique mentioned previously. The culture produced is a pure one. coli                                     

Pour plate method -This method involved serial dilution of specimen and mixed with melted media. Then pour the content into sterile plate plates.

Cultural characteristics: Cultural characteristics involve both : 1. Growth requirement. 2. Colonial morphology. It is used to study the macroscopic characteristics of a pure culture on a solid medium.

Colonial morphology The following points have to be considered in describing a colony: 1. Size: measured by mm.

2. Shape: rhizoid ,circular, filamentous, irregular.

3. Elevation: flat, convex, raised, umbonate.

4. margin: entire lobulated ,filamentous

5. Consistency: dry, mucoid.

6. Surface texture: rough, smooth.

7. Color or pigmentation.

8. Optical density : opaque, translucent, glistening.

9. changes in the inoculated medium e.g heamolysis. 10. Odour : bad or sweat musty odour.

Stock culture :- Means preservation of a microorgansim in a culture medium for future study. It can be kept usually for 1 month at 4 °C.

Technique of stock culture: Use a universal bottle containing medium in slanted position ( to provide a wide surface for inoculation and good nutrition). Using a sterile loop inoculate the surface of the medium with one or few colonies of a pure culture. Incubate the inoculated medium at 37 °C for 24 hours, then store at 4 °C.