Practical exam Flashcards
Why are aseptic techniques important in bacteriology?
They prevent contamination of pure cultures, keep growth media sterile and ensure reliable results by using sterile instruments and covering container openings
How do you maintain sterility during bacterial culture work?
Use only sterile instruments and fluids, keep culture containers covered, sterilise work surfaces before and after use
What is the purpose of streak plating?
To isolate single bacterial colonies from a mixed culture by spreading bacteria across an agar plate in a systematic pattern
How do you know if streak plating was successful?
You’ll see isolated, individual colonies in the final streaked sections of the plate
What does gram staining reveal?
It helps differentiate bacteria based on cell wall structure, revealing shape, size and arrangement of cells
What are the 4 key steps of gram staining
- Crystal violet (Primary stain): Binds to peptidoglycan.
- Iodine (Mordant): Fixes crystal violet in the cell wall.
- Alcohol (Decolorizer): Washes out stain from Gram-negative bacteria.
- Safranin (Counterstain): Stains Gram-negative bacteria pink/red.
What colour do gram-positive bacteria appear and why?
Purple - because they have a thick peptidoglycan layer without an outer membrane , retaining crystal violet
What colour do gram-negative bacteria appear and why?
Pink/red - because they have an outer membrane that blocks crystal violet, allowing safranin to stain them
Why is heat fixation important before gram staining?
It kills bacteria, helps dyes penetrate and makes cells stick tightly to the slide
Pseudomonas fluorescens - describe colony appearance
translucent , light brown colonies
Bacillus megaterium - describe colony appearance
Large, flat, brown colonies
Micrococcus luteus - describe colony appearance
Tiny, yellow colonies
Mystery ‘X’ bacteria
Pink colonies on dark malt agar
What is the purpose of gas vacuoles?
They provide buoyancy , allowing cells to float in water
What structures make up gas vacuoles?
Gas vesicles
Structure of gas vesicles?
Hollow, rigid and brittle structures made of protein ribs
How can gas vacuoles be distinguished from other inclusions?
They disappear under moderate pressure as the vesicles collapse
What happens under different intensities? (gas vesicles
Low intensity: cells form abundant vesicles and become buoyant
High intensity : vesicles collapse and cells sink
How do you prepare a 100-fold serial dilution?
Add 0.1 mL of culture to 9.9 mL of water (10⁻² dilution). Repeat:
10⁻² → 10⁻⁴ → 10⁻⁶ → 10⁻⁸, each by adding 0.1 mL of the previous dilution to 9.9 mL of water.
Plate 0.1 mL of chosen dilutions on agar plates.
How do you prepare a 1/2 dilution for turbidimetric counts?
Mix 5 mL of culture with 5 mL of nutrient broth (NB)
How do you measure bacterial turbidity?
Add 1 mL of the diluted culture to a spectrophotometer cuvette and measure attendance (OD600)
What is the volume of one haemocytometer square?
1 mm x 1mm x 0.1 mm = 0.1μL
How do you calculate cells per microlitre? (haemocytometer)
- Count cells in 1 small square.
- Multiply by 400 (small squares per medium square).
- Multiply by 10,000 (400 squares × 25 medium squares).
Cells/µL=n×400×25=n×10,000
What factors can affect haemocytometer counts?
Bacillus megatherium chains, pipetting errors and dead cells
