Pure Culture Techniques (Streak Plate, Pour Plate and Spread Plate Methods)
1. Streak Plate Method
Aim To isolate pure colonies of bacteria from a mixed culture by streaking on a solid agar medium so that individual cells are spatially separated and form isolated colonies.
Principle The inoculum is progressively diluted by streaking across successive sectors of the agar plate using a sterile loop. In the later streaks, individual bacterial cells are deposited far apart. Each viable cell multiplies during incubation to form a visible isolated colony that can be picked for pure culture.
Materials Required
· Mixed bacterial culture (broth or colony)
· Sterile nutrient agar (or appropriate selective/differential medium) plates
· Inoculating loop (nichrome or platinum)
· Bunsen burner / spirit lamp
· Incubator (37 °C)
· Marker pen
· Sterile forceps (optional)
Procedure
1. Label the bottom of a sterile agar plate with specimen details, date and your initials. Divide the plate mentally or lightly into 3–4 sectors.
2. Sterilise the inoculating loop by flaming until red-hot and allow it to cool.
3. Take a loopful of the mixed culture.
4. First streak (Sector 1): Open the plate lid just enough, streak the inoculum in parallel lines or zig-zag pattern covering about one-quarter of the plate.
5. Flame and cool the loop again.
6. Second streak (Sector 2): Drag the loop once or twice through the end of the first streak and continue streaking into the second sector (without reloading inoculum).
7. Flame and cool the loop.
8. Third streak (Sector 3): Drag through the end of the second streak and streak the third sector.
9. (Optional) Flame, cool and make a final streak into the remaining area for maximum isolation.
10. Close the lid, invert the plate and incubate at 37 °C for 18–24 hours (or appropriate temperature/time).
11. Observe isolated colonies in the later sectors. Pick a well-isolated colony with a sterile loop and subculture onto a fresh slant or plate to obtain pure culture.
Result / Observation Isolated discrete colonies appear in the later streak areas. Colonies of different morphology indicate different species; pick one type for pure culture.
2. Pour Plate Method
Aim To isolate pure colonies and also to enumerate viable bacteria (colony-forming units) in a liquid sample by embedding diluted cells in molten agar.
Principle Serial dilutions of the sample are mixed with molten agar (cooled to 45–50 °C) and poured into sterile Petri dishes. Individual cells become immobilised and grow into colonies both on the surface and within the agar (sub-surface colonies). Colonies can be counted and pure cultures obtained by picking isolated colonies.
Materials Required
· Mixed bacterial suspension / sample
· Sterile molten nutrient agar (kept at 45–50 °C in water bath)
· Sterile Petri dishes
· Sterile pipettes (1 ml, 10 ml) or micropipettes
· Sterile dilution blanks (9 ml saline or broth)
· Water bath (45–50 °C)
· Incubator
· Colony counter (optional)
Procedure
1. Prepare serial dilutions of the sample (e.g., 10⁻¹ to 10⁻⁶) in sterile diluent.
2. Label sterile Petri dishes with dilution numbers.
3. Aseptically transfer 1 ml of the appropriate dilution(s) into each empty sterile Petri dish.
4. Pour 15–20 ml of molten agar (cooled to ~45–50 °C) into each dish containing the inoculum.
5. Immediately mix by gentle rotation or swirling of the plate (in a figure-of-8 or circular motion) so that the inoculum is evenly distributed.
6. Allow the agar to solidify completely (10–15 minutes) on a level surface.
7. Invert the plates and incubate at 37 °C for 24–48 hours.
8. Observe colonies growing on the surface and within the agar. Count colonies (preferably plates with 30–300 colonies) for enumeration if required.
9. Pick well-isolated surface or sub-surface colonies for pure culture.
Result / Observation Colonies appear both on the surface and embedded in the agar. Isolated colonies can be subcultured. Colony count gives viable count (CFU/ml).
3. Spread Plate Method
Aim To isolate pure colonies and enumerate viable bacteria by spreading a diluted liquid sample evenly over the surface of a solid agar medium.
Principle A measured volume (usually 0.1 ml) of a diluted bacterial suspension is placed on the surface of a pre-poured, solidified agar plate and spread uniformly with a sterile spreader. Individual cells are separated on the surface and grow into discrete colonies after incubation. This method yields only surface colonies and is preferred when oxygen-requiring organisms or easy colony picking is needed.
Materials Required
· Mixed bacterial suspension / sample, Sterile pre-poured nutrient agar plates (dried surface), Sterile dilution blanks,Sterile pipettes or micropipettes
· Sterile glass or plastic L-shaped spreader (or sterile bent glass rod)
· Turntable, Alcohol (for flaming spreader)
· Bunsen burner, Incubator
Procedure
1. Prepare serial dilutions of the sample.
2. Label the bottom of dried agar plates with dilution numbers.
3. Aseptically pipette 0.1 ml of the chosen dilution onto the centre of the agar surface.
4. Sterilise the spreader by dipping in alcohol and flaming; allow to cool.
5. Place the plate on a turntable (or hold it) and spread the inoculum evenly over the entire surface by rotating the plate while moving the spreader back and forth.
6. Allow the plate to dry for a few minutes (lid slightly ajar if needed) so that the liquid is absorbed.
7. Invert the plate and incubate at 37 °C for 18–24 hours.
8. Observe discrete surface colonies. Count colonies on plates with 30–300 colonies for viable count if required.
9. Pick isolated colonies for pure culture.
Result / Observation Only surface colonies are formed. Well-isolated colonies can be easily picked for pure culture. Colony count × dilution factor × 10 gives CFU/ml (because 0.1 ml was plated).
