Explain Procedure for Isolation of Pure Culture?
Now carry out the exercise following the steps enumerated herewith.
1. Label the name of the organism on the bottom of the petri plate.
2. Sterilize the inoculating loop by holding it with right hand in flame till it red hot.
3. Hold the culture tube in left hand and remove the cotton plug with the help of little finger of right hand.
4. Sterilize the mouth of culture tube immediately in flame and take culture (loopful if the culture is liquid broth or small amount if the culture is solid medium) with the sterilized inoculating loop. Replace the cotton plug and keep the tube in test tube rack.
5. Transfer the culture to the labeled petri plate by streaking. For the same, hold the plate in the left hand at an angle of 60° C and open the lid (Petri plate cover) with the help of the thumb.
6. Place the culture at one end of the plate and spread it slightly in a rounded manner. It is the primary inoculum. (Look at area 1 in the margin Figure).
7. Flame the loop and cool it. Then streak the inoculums at an angle to the primary inoculum by making 5-6 parallel lines. 1-2 lines should pass through the primary inoculum (as shown in area 2 in margin Figure). The flaming of the loop results in desired dilution and fewer and fewer organisms are streaked in each successive area leading to final separation of the microbial cells.
8. Reflame and cool the loop. Turn the plate at right angle. Drag the inoculum in several parallel lines across the agar surface (as shown in area 3 of margin Figure) by touching the inoculum in area 2 only once or twice.
9. Again rotate the Petri plate at 90°C angle. After flaming and cooling the inoculating loop, touch the inoculum in area 3 once and streak the inoculum several times in parallel lines across the agar. Look at the margin Figure the area marked 4.
10. Without reflaming the loop again turn the Petri plate at an angle and drag the culture from previous streak series (area 4) across area 5 in a similar manner by making parallel lines.
11. Replace the lid on Petri plate and incubate the culture for 24 hours (overnight) at 37°C. Observe the plates for isolated colonies. Margin Figure shows the plate after incubation.
12. Pick and restreak the isolated colony on another nutrient agar plate in a similar manner to get the pure culture.