Isolation and streak plate technique

During isolation, bacterial cells from a discrete colony that developed on the surface of an agar plate are transferred to an agar slant having the same composition. The culture developing on the surface of the agar slant after the isolation is called an isolate.

To aseptically transfer microorganisms from broth, slant or agar cultures to another medium, inoculating needles or loops are used. They are made up of a handle, a shaft, a turret and a straight or a loop-ended nickel-chromium needle (Fig. 20).

Fig. 20. Isolation.(a) Take the inoculating loop in one hand and hold it like a pencil. Flame the inoculating loop over a Bunsen burner until the wire becomes red-hot. (b) Make a gap on the Petri dish and choose a discrete colony to pick up a loopful of inoculum with the inoculating loop, then close the lid of the Petri dish. (c) After opening

and flaming the neck of the test tube, inoculate the surface of the agar slant in zigzag streaks using the infected inoculating loop. (d) Reflame the neck of the tube, close it and sterilise the loop with reflaming as well.

EXERCISE 27: ISOLATION OF CULTURES FROM THE AGAR SURFACE Object of study:

bacteria present in soil samples bacteria present in water samples Materials and equipment:

agar plates inoculated by spread plate technique agar slants

inoculating loop Bunsen burner incubator Practice:

1. Label slant to be inoculated with the date, your name and name/code/ number of isolate. Select adequate colonies from the plate culture by marking them on the bottom of the Petri dish.

2. Take the inoculating loop and hold it like a pencil. Flame the inoculating loop over a Bunsen burner until the wire becomes red-hot.

3. Open the lid of the Petri dish culture to a gap and cool the hot loop by inserting it into the agar without touching any colonies developed on the surface. Choose a discrete colony and pick a loopful of inoculum using the in-oculating loop, and then close the lid of the Petri dish.

4. Using the same hand that is holding the inoculating loop, remove the cap from a test tube, hold it between your fingers, and briefly flame the neck of the tube over a Bunsen burner by passing through the flame.

5. Inoculate the surface of the agar slant in zigzag streaks using the infected inoculating loop.

6. Flame the neck of the tube again and close it with the cap.

7. Sterilise the loop again by flaming over a Bunsen burner until the wire becomes red-hot. Take care to place the infected loop first into the core of the flame, and then slowly pull it upwards until it becomes red-hot.

8. Place the tube and the inoculating loop on the rack.

9. Incubate the slant at 28°C for one week.

10. Check the growth of the isolate after the incubation period.

(See also Supplementary Figure S15.)

An isolate is not necessarily a pure culture, i.e. containing cells of the same origin (derived from a single mother cell/a clone of cells). In mixed cultures, co-multiplication of two or more microbes occurs. This can happen acci-dentally, but not all microbes are able to grow independently from others; e.g. one of the microbes can produce a compound that enables the growth of another microbe in the culture medium (synergism), or a substance produced by one microbe inhibits the growth of another (antagonism), or one microbe can grow faster than the other, thus growth of the latter would be limited because of the use of essential nutrients. The microbiological examination of mixed cultures generally provides confusing and misleading results due to the different metabolic properties of various microbes. Therefore, it is necessary to create pure cultures. Pure (axenic) cultures are free from other mi-croorganisms, develop from single cells or colony forming units, and serve as the basis of species level identification of bacteria and other studies.

Pure cultures can be obtained by the streak-plate technique. This method is based on the creation of a dilution gradient on the surface of an agar plate. Due to an appropriate dilution of the inoculum (e.g. mixed culture), discrete

and visible colonies can develop at the end of the inoculation line. Reisolation of bacterial cells from these colonies (originating from a single cell) onto agar slants results in pure cultures (Fig. 21).

Fig. 21. Preparation of pure cultures by streak plate method.(a) Inoculate the mixed bacterial cultures on ap-proximately one-quarter of the surface of an agar plate with the inoculating loop (1). Sterilise the loop by reflaming, cross over the streaks of the first inoculation when streaking the second part of the agar surface (2). Repeat these steps on the third (3) and fourth quarter (4) of the agar surface. (b) Check the growth of discrete colonies with

different morphology, in this case colour, in the third and fourth quarter of the agar plate after the incubation period.

EXERCISE 28: PREPARATION OF PURE CULTURES BY THE STREAK PLATE METHOD Object of study, test organisms:

mixed suspension ofSerratia marcescensandMicrococcus luteusstrains Materials and equipment:

nutrient agar plates (see Appendix) inoculating loop

Bunsen burner incubator Practice:

1. Label a Petri plate to be inoculated with the date, your name, and the mark of the isolate to be purified.

2. Take the inoculating loop and hold it like a pencil. Flame the inoculating loop over a Bunsen burner until the wire becomes red-hot.

3. Holding the inoculating loop in one hand, take the test tube containing the suspension of mixed bacterial cultures in the other hand.

4. Using the same hand that is holding the inoculating loop, remove the cap from the test tube, hold it between your fingers, and briefly flame the neck of the tube over a Bunsen burner by passing through the flame.

5. Take a loopful of inoculum from the suspension.

6. Flame the neck of the tube again and close it with the cap. Place the tube in the rack.

7. Inoculate approximately one-third of the agar surface (at the edge) using the infected inoculating loop (without scratching the agar).

8. Sterilise the loop again by flaming until the wire becomes red-hot.

9. Cool the loop by thrusting it into the sterile agar.

10. Cross over the streaks of the first inoculation when streaking the second part of the agar surface.

11. Flame and cool the loop again before repeating the streaking process on the third part of the agar surface.

12. Sterilise the loop again by flaming and place it on the rack.

13. Incubate the culture at 28°C for one week.

14. Check the growth of discrete colonies with different morphology (see EXERCISE 35) after the incubation period. Perform re-isolation (see EXERCISE 27). (Fig. 21).

(See also Supplementary Figure S16.)