MICROBIOLOGY PLATING TECHNIQUES FOR COLONY
COUNTING AND PURE CULTURE ISOLATION
Channel: MicroChem's Experiments
Three Key Techniques Discussed:
1. Pour Plate Technique – for counting colony-forming bacteria.
2. Spread Plate Technique – for easy counting and isolation.
3. Streak Plate Technique – for obtaining a pure bacterial culture.
CULTURE MEDIA PREPARATION (COMMON STEPS)
Goal: Prepare agar culture media plates.
General Preparation Steps:
• Weigh agar media and mix with distilled water per label instructions.
• Sterilize according to manufacturer’s instructions.
Sterilization Methods:
• Boiling at 100°C to dissolve contents.
• Autoclaving at 121°C, 15 lbs for 15 min.
• Boiling in microwave oven for 2 minutes.
Post-Sterilization Handling:
• Spread/Streak Plates: pour, cool, solidify, and incubate to check contamination.
• Pour Plates: cool media to 45–48°C before use.
POUR PLATE TECHNIQUE FOR COLONY COUNTING
Purpose: Colony Counting
Key Materials: Biological safety cabinet, incubator, micro pipette, vortex mixer, tips, and petri dishes.
Inoculation Procedure:
1. Clean workstation in the biological safety cabinet.
2. Label two sterile petri dishes with test details.
3. Vortex the sample to mix uniformly.
4. Pipette 1 ml sample onto the petri dish.
5. Discard tips.
6. Add 15–17 ml of sterile media (45–48°C) and mix.
SPREAD PLATE TECHNIQUE FOR COLONY
COUNTING AND ISOLATION
Purpose: To count and isolate bacteria from a liquid sample.
Key Materials: Safety cabinet, culture media plate, Bunsen burner, glass rod spreader, colony counter.
Procedure:
1. Sterilize glass spreader with 70% ethanol, flame, and cool.
2. Vortex the sample.
3. Add 0.1 ml sample dropwise on agar surface.
4. Spread evenly using the spreader while rotating plate.
5. Allow sample to absorb/dry, then invert and incubate.
Result Interpretation:
• Colonies counted under colony counter.
• CFU example: 36 CFU from 0.1 ml = 360 CFU/ml.
STREAK PLATE TECHNIQUE FOR PURE CULTURE
ISOLATION
Purpose: To isolate pure bacterial cultures.
Key Materials: Safety cabinet, Bunsen burner, inoculating loop, TSA agar plate.
Four-Quadrant Streaking Method:
1. Flame loop until red hot; cool inside cabinet.
2. Pick one loop of bacterial sample.
3. Smear and streak Quarter 1.
4. Flame, cool, streak Quarter 2 (touch Quarter 1 once).
5. Streak Quarters 3 & 4 without flaming between them.
6. Invert and incubate (37°C for 24 hrs).
Observation: Colony density decreases from Q1 ? Q4; isolated colonies appear in Q3 & Q4.