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
What type of bacteria thrive in a Winogradsky column?
Green sulfur photosynthetic anaerobic bacteria
What does CFU stand for?
Colony forming units
What is the purpose of CFU?
a measure of viable bacteria in a sample
How do you count CFUs?
- Count colonies on plates with 30–300 colonies.
- If overcrowded, count 1/4 of the plate and multiply by 4.
How do you calculate CFU/mL?
CFU/mL=(Colonies×10)×DilutionFactor
What is cytoplasmic streaming?
The rhythmic, back and forth flow of cytoplasm in Physarum , aiding nutrient transport
What materials are needed to calculate EPUs?
- Eyepiece Graticule: Scale inside the microscope eyepiece.
- Stage Micrometer: Slide with 100 µm divided into 100 divisions (1 division = 1 µm)
How do you calculate the size of 1 EPU?
- Align the eyepiece graticule and stage micrometer.
- Count how many EPUs match a known stage micrometer length.
- Apply the formula:
1EPU=StageMicrometerLength(µm)/EyepieceUnits(EPUs)
What is tropism in Phycomyces?
Growth in response to environmental stimuli, like light or gravity
What happens during Phycomyces mating?
Compatible mating types fuse to form a zygospore, a resistant structure for survival
Why do you need to recalibrate EPUs for each magnification?
The size of an EPU changes with magnification, so the rules slide (stage micrometer) must be lined up at each magnification
What does FLOs stand for?
Fungus-like Organisms/ Pseudofungi
What are FLOs?
Resemble fungi
Why are FLOs called pseudo fungi?
independantly evolved a mycelial growth form similar to fungi
How do FLOs differ from true fungi?
cell walls are made of cellulose, not chitin ; hyphae (aseptate - coenocytic) and spores (produce thick-walled sexual resting spores and asexual spores in sporangia)
What type of spores do FLOs produce? - development
Sporangiospores, which develop into motile zoospores
What is a zoospore?
A motile asexual spore that uses a flagellum for locomotion in aqueous or moist environments
What is unique about zoospores?
Have 2 flagella, formed at tips of coenocytic hyphae and they are released when the sporangia bursts (allows zoospores to swim erratically)
What are the types of flagella in zoospores?
Whiplash and Tinsel flagellum
What is the appearance of a Whiplash flagellum in zoospores?
Smooth for propulsion
What is the appearance of Tinsel flagellum in zoospores?
Hairy for steering
What defines Zygomycota? - earth
Terrestrial fungi producing non-motile sporangiospores
What are the hyphae of zygomycota like? - long
Coenocytic hyphae (aseptate), with sporangiophores ending in a single sporangium
How do Zygomycota reproduce sexually?
Through the formation of gametangia , where compatible strains fuse to form a zygosporangium
What is heterothallism? - sex
Presence of compatibility types, requiring opposite mating types for reproduction
What photoreceptor does P.blake use?
Flavin adenine dinucleotide (FAD) binds to the LOV domain of the MADA protein initiating gene transcription under blue light
What defines Ascomycota?
Production of an ascus, a sac-like structure containing usually 8 ascospores
How do ascospores form?
- Fusion of two compatible nuclei (+/-).
- Meiosis: First and second divisions → 4 nuclei.
- Mitosis: Each nucleus divides → 8 nuclei, forming 8 ascospores.
