Microbiology Lab Plan
Laboratory Goal
Grow and study safe bacteria while learning key microbiology skills aligned with BTEC Unit 13 requirements.
Safety First: Always
- Wear a lab coat and gloves at all times
- No eating, drinking, or applying cosmetics in the lab
- Tie back long hair and secure loose clothing
- Wash hands upon entering and before leaving the lab
- Disinfect work surfaces before and after use
- All used equipment must be sterilized properly
Laboratory Activities
Activity 1: Preparing Agar Plates
Objective: Create sterile nutrient agar plates for bacterial cultivation.
Steps:
- Mix: Weigh 2.8g of agar powder and add to 100ml distilled water in a flask
- Sterilize: Autoclave at 121°C for 15 minutes
- Pour: Carefully pour into sterile Petri dishes near a Bunsen burner
- Set: Allow to solidify for 30 minutes
- Store: Refrigerate plates upside down
Activity 2: Streak Plate Method
Objective: Isolate individual bacterial colonies using aseptic technique.
Steps:
- Sterilize inoculating loop in Bunsen burner flame
- Collect bacteria from broth culture
- Streak Zone 1: Smear loop back and forth in first section
- Sterilize loop and streak Zone 2: Drag through Zone 1 into new section
- Sterilize loop and streak Zone 3: Drag through Zone 2 into final section
- Incubate at 25°C for 24-48 hours
Activity 3: Gram Staining
Objective: Differentiate bacteria based on cell wall structure.
Steps:
- Prepare bacterial smear on slide and heat fix
- Apply Crystal Violet (60 seconds) → Rinse
- Apply Gram's Iodine (60 seconds) → Rinse
- Decolorize with Alcohol (5-10 seconds) → Rinse immediately
- Apply Safranin (60 seconds) → Rinse and blot dry
- Observe under microscope (1000x magnification)
Results: Gram-positive = Purple, Gram-negative = Pink/Red
Activity 4: Antibiotic Sensitivity Testing
Objective: Determine effectiveness of antibiotics against bacterial strains.
Steps:
- Create bacterial lawn on agar plate using sterile swab
- Place antibiotic discs evenly on agar surface
- Incubate at 25°C for 24-48 hours
- Measure zones of inhibition around each disc
- Compare to standardized chart for interpretation
Interactive Virtual Laboratory
Practice microbiology techniques safely in this virtual lab environment. Select an activity and follow the step-by-step instructions.
Activity 1: Preparing Agar Plates
Step 1 of 5
Click the button below to add nutrient agar powder to the flask.
Activity Complete!
You've successfully completed this activity. Well done!
Laboratory Safety Protocols
Essential Safety Rules
Following these rules is critical for preventing contamination and ensuring personal safety in the microbiology lab.
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Personal Protective Equipment
Always wear lab coats, gloves, and safety glasses. Tie back long hair and avoid loose clothing.
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No Food or Drink
Never consume food, drinks, or apply cosmetics in the laboratory to prevent ingestion of microorganisms.
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Hand Hygiene
Wash hands thoroughly with disinfectant soap upon entering and before leaving the laboratory.
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Aseptic Technique
Always work near a Bunsen burner flame to create an upward air current that prevents contamination.
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Surface Disinfection
Disinfect work surfaces with appropriate agents like 1% Virkon before and after all procedures.
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Proper Disposal
Place all used cultures and contaminated materials in biohazard bags for autoclaving before disposal.
Emergency Procedures
- Spills: Cover with disinfectant, leave for 15 minutes, then carefully wipe up
- Broken Glass: Use brush and dustpan, dispose in designated sharps container
- Fire: Turn off gas sources, use fire extinguifier for small fires, evacuate for large fires
- Exposure: Immediately wash affected area, report to supervisor, seek medical attention if needed
Safe Microorganism Use
This laboratory only uses Hazard Group 1 microorganisms that are unlikely to cause human disease. Examples include:
- Bacillus subtilis (Gram-positive rod)
- Escherichia coli K-12 (Non-pathogenic strain)
- Micrococcus luteus (Gram-positive coccus)
- Saccharomyces cerevisiae (Baker's yeast)
Laboratory Equipment
Familiarize yourself with the essential equipment used in microbiology laboratories.
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Petri Dishes
Shallow dishes for solid culture media
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Bunsen Burner
Creates sterile working environment
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Microscope
For observing microorganisms (1000x magnification)
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Autoclave
Sterilizes equipment and media (121°C, 15 psi)
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Incubator
Maintains optimal growth temperature (25-37°C)
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Inoculating Loop
For transferring microbial cultures
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Staining Reagents
Crystal violet, iodine, safranin for Gram staining
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Antibiotic Discs
For sensitivity testing
Culture Media
Nutrient Agar: General-purpose medium for growing a wide variety of bacteria
Composition: Peptone, beef extract, agar, water
Preparation: 28g per liter of distilled water, sterilize by autoclaving
Microscopy Techniques
Oil Immersion: Required for viewing bacteria at 1000x magnification
Gram Staining: Differential staining technique to classify bacteria
Wet Mounts: For observing live microorganisms
Knowledge Check
Test your understanding of microbiology concepts and laboratory procedures.
Discussion Questions
Select the best answer for each question from the dropdown menus. Click "Check Answer" for immediate feedback.
Questions Answered: 0/20
Current Score: 0/20
Activity 1: Preparing Agar Plates
1. Why is it critical to sterilize the nutrient agar in an autoclave before pouring it into Petri dishes?
2. The instructions say to pour the agar "near a Bunsen burner." What is the purpose of this specific instruction?
3. Why are prepared agar plates stored upside down in the refrigerator?
4. If fungal contamination appears on agar plates after storage, where was the most likely point of introduction?
5. What is the main advantage of using a selective medium over nutrient agar?
Activity 2: Streak Plate Method
6. What is the primary goal of the streak plate technique?
7. Why is the inoculating loop flamed between each streak zone?
8. If you observe dense growth in the first zone but no isolated colonies in the third zone, what is the most likely cause?
9. How does the streak plate method achieve bacterial dilution?
10. Why must the loop cool after flaming before touching bacterial culture?
Activity 3: Gram Staining
11. What does it mean that Gram stain is a "differential" stain?
12. What is the function of iodine in the Gram staining process?
13. If a known Gram-positive bacterium appears pink after staining, what error likely occurred?
14. Why is Gram staining clinically important?
15. What structural difference causes Gram-positive and Gram-negative bacteria to stain differently?
Activity 4: Antibiotic Sensitivity Testing
16. What is a "zone of inhibition"?
17. Why is a uniform bacterial lawn important for antibiotic testing?
18. Does a larger zone of inhibition always indicate a more effective antibiotic?
19. If there is no zone around an antibiotic disc, does this definitively prove bacterial resistance?
20. How do antibiotic sensitivity test results influence patient treatment?