1. Streak Plate Method
Objective: Isolate pure bacterial colonies from a mixed culture.
Materials Needed:
*Sterile nutrient agar plates.
*Inoculating loop.
*Bunsen burner.
*Bacterial culture.
Steps:
1. Sterilize the inoculating loop in a flame and let it cool.
2. Dip the loop into the bacterial culture.
3. Streak the loop gently across one quadrant of the agar plate.
4. Sterilize the loop, cool it, and streak the second quadrant by dragging from the first.
5. Repeat for the remaining quadrants.
6. Incubate the plate at 37°C for 24–48 hours.
Expected Result: Isolated colonies in the final quadrant.
Tips:
Avoid cutting the agar surface while streaking.
Ensure proper sterilization to prevent contamination.
2. Serial Dilution and Plating
Objective: Determine microbial load in a sample.
Materials Needed:
*Sterile test tubes with diluent (e.g., saline).
*Micropipette.
*Agar plates.
*Sample solution.
Steps:
1. Add 9 mL of diluent to each test tube.
2. Add 1 mL of the sample to the first tube and mix (10^-1 dilution).
3. Transfer 1 mL from the first tube to the second and mix (10^-2 dilution). Repeat for subsequent tubes.
4. Plate 0.1 mL from each dilution onto sterile agar plates.
5. Spread the sample using a sterile spreader.
6. Incubate at 37°C for 24–48 hours.
Expected Result: Countable colonies (30–300 CFUs) at a specific dilution.
Tips:
Use a vortex mixer for thorough mixing.
Always change pipette tips between dilutions.
3. Gram Staining
Objective: Differentiate bacteria into Gram-positive and Gram-negative groups.
Materials Needed:
*Gram stain reagents (crystal violet, iodine, alcohol, safranin).
*Microscope slides.
*Bacterial smear.
Steps:
1. Prepare a bacterial smear on a slide and heat-fix it.
2. Flood the slide with crystal violet for 1 minute.
3. Rinse with water and add iodine solution for 1 minute.
4. Decolorize with alcohol for 10–15 seconds and rinse.
5. Counterstain with safranin for 1 minute.
6. Rinse, blot dry, and observe under a microscope.
Expected Result:
Gram-positive: Purple.
Gram-negative: Pink.
Tips:
Do not over-decolorize.
Use fresh reagents for better results.
4. Pour Plate Method
Objective: Estimate microbial count by mixing samples with molten agar.
Materials Needed:
*Molten agar.
*Diluted microbial sample.
*Sterile petri dishes.
Steps:
1. Pipette 1 mL of the diluted sample into a sterile petri dish.
2. Pour molten agar (~45°C) into the dish.
3. Swirl gently to mix.
4. Let it solidify, then incubate at 37°C.
Expected Result:
Colonies grow both within and on the agar surface.
Tips:
Avoid overheating the agar; it can kill microorganisms.
Ensure uniform mixing to avoid uneven colony distribution.
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