isolation of microorganisms

1. Isolation and Culturing of Different Microorganisms

2. Introduction: • Microorganisms are generally found in nature (air, soil and water) as mixed populations. • Many microbes are pathogenic. They cause a number of diseases with a variety of symptoms, depending on how they interact with the patient. • To study the specific role played by a specific microorganism in its environment, one must isolate in pure culture.

3. Pure vs Mixed Culture • A mixed culture is one that contains more than one type of organism growing in a medium. • A culture which contains just one species of microorganism is called a pure culture.

4. Culturing Techniques The different methods for obtaining pure cultures are: • Streak plate method • Spread plate methods • Pour plate method

5. Streak Plate Method • This method is routinely employed for the isolation of bacteria in pure culture. • In this method a sterilized inoculating loop is dipped into a suitable diluted suspension of microorganisms which is then streaked on the surface of an already solidified agar plate to make a series of parallel, non-overlapping streaks. • The process is known as streaking and the plate so prepared is called a streak plate. • The main objective of the streak plate method is to produce well separated colonies of bacteria from concentrated suspensions of cells.

6. Materials and Procedure Materials required: • Mixed culture • Inoculation loop • Bunsen burner • Agar plate (nutrient agar or any other agar medium) Procedure: • Sterilize the inoculating loop in the burner. • Put the loop into the flame until it is red hot. Allow it to cool. • Pick an isolated colony from the agar plate culture and spread it over the first quadrant using close parallel streaks. 

7. 4. Flame the loop.5. Turn the plate 90° and lightly sweep the loop 1-2 times through the inoculated area, then streak into the next quadrant.6. Flame the loop.7. Turn the plate 90°, overlap the previous area 1-2 times, and streakinto thenext quadrant.8. Flame the loop.9. Repeat #6, streaking the remainder of the plate. 10. Invert the plate and incubate at 37°C for 24 hr.

9. Spread Plate Method • The spread plate technique is used for the separation of a dilute, mixed population of the microorganisms so that individual colonies can be isolated. • In this technique, a small volume of dilute microbial mixture is transferred to the center of an agar plate and spread evenly over the surface with a sterile l-shaped bent glass rod. • Thepetri dish is spun, at some stage, and single cells will be deposited with the bent glass rod on the agar surface.

10. Procedure • Place the bent glass rod into the beaker and add a sufficient amount of 95% ethyl alcohol to cover the lower, bent portion. • With a sterile loop, place a loop full of the culture (e.G.Micrococcusluteus) in the center the nutrient agar plate. • Remove the glass rod from the beaker and pass it through the bunsen burner flame, with the bent portion of the rod pointing downward. • Allow the alcohol to burn off the rod completely.

11. 5. Cool the rod for 10 to 15 seconds. 6. Remove the petri dish cover and spin lightly touch the sterile bent rod to the surface of the agar and move it back and forth along with turnings the petri dish manually. This will spread the culture over the agar surface. 7. Immerse the rod in alcohol and flame it again. 8. Repeat the above process again to insure that the culture is completely spread.

13. Pour Plate Method • The pour-plate technique requires a serial dilution of the mixed culture by means of a loop or pipette. • The diluted inoculum is then added to a molten agar medium in a petri dish, mixed, and allowed to solidify. • This method also used to count the number of viable organisms in a liquid such as water, milk, urine or broth culture as well as to determine the hemolytic activity of deep colonies of some bacteria, such as the streptococci, by using an agar medium containing blood.

14. Procedure • Serial dilutions will be first performed in order to get a viable cell count. • Liquefy six agar deep tubes in an autoclave or by boiling. Cool them maintain in a water bath at 45°C. • Label the culture tubes (e.g. E. Coli) with the number 1 and the remaining water blanks (of 9ml) from 2 to 8. Label the petri dishes as well. • Mix the culture (tube 1) by rolling between the palms of your hands to ensure even dispersal of cells in the culture. • With a sterile pipette, aseptically transfer 1 ml from the bacterial suspension (tube 1), to water blank tube 2. The culture has been diluted 10 times to 10−1.

15. 6. Continue to transfer 1ml of suspension till test tube number 8 while returning back to the previous tube. 7. Transfer 1 ml of the bacterial suspension each from each tube each labelled petri plates numbers 8. Remove a nutrient agar tube from the water bath (at 45°C) and pour the medium into plate 1 and rotate the plate gently to ensure uniform distribution of cells in the medium. 9. Repeat the above step for the addition of medium to all the plates. 10. Allow the medium to solidify.  11. Incubate the inoculated plates for 24-48 hours at 37°C in an inverted position (lid on bottom).