Microbiology practicals and calculations guide
Cell biology • Cell structure
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Key concepts
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Aseptic technique - purpose and overview
Aseptic technique prevents unwanted microorganisms contaminating cultures and protects the handler from exposure. Sterile conditions reduce competition from background microbes, allowing target organisms to grow as intended. Clean benches, labelled plates, sterile inoculating loops and minimal opening of containers form the core controls that reduce contamination risk .
Preparing an uncontaminated culture - stepwise method
Label the underside of the agar plate before use to preserve readability after growth. Flame an inoculating loop in a blue Bunsen flame to sterilise it and allow it to cool before touching a culture; reheating the loop between transfers prevents transfer of unwanted microbes. Loosen but do not fully remove culture bottle lids, flame the bottle neck, transfer with the cooled loop and re-flame the neck before replacing the lid to reduce airborne entry of contaminants. Streak-plate technique produces isolated colonies by progressive dilution across the agar surface .
Sterilisation of Petri dishes and culture media
Sterilisation destroys any pre-existing microorganisms present on dishes or in media. Sterile Petri dishes and media ensure that colonies grown originate from the intended inoculum rather than from contaminants, which otherwise alter results and may pose safety risks. Autoclaving or factory-sterilised pre-poured agar removes viable microbes and spores before use .
Flaming inoculating loops - rationale
Passing the inoculating loop through a flame raises its temperature to levels that kill microorganisms present on the metal. Cooling the loop prevents killing of the inoculum and avoids heat damage to the agar. Repeated flaming between streaks prevents transfer of excess cells and helps to obtain single isolated colonies by stepwise dilution .
Sealing and inverting Petri dishes - reasons and limits
Securing the lid with light adhesive tape reduces accidental opening and limits airborne contamination while still allowing some gas exchange. Incubating plates upside down prevents condensation droplets from forming on the lid and falling onto the agar surface, which would spread colonies and distort colony morphology. Plates must not be fully sealed to avoid anaerobic conditions or pressure build-up; light tape placed at opposite sides is sufficient .
Incubation temperature in school laboratories
Incubation at 25°C restricts growth of many human-pathogenic microorganisms while allowing growth of environmental strains for investigation. Lower incubation temperatures reduce risk of culturing human pathogens and increase safety in educational settings. Higher temperatures increase growth rates but raise biosafety concerns and are avoided in school practicals .
Calculating cross-sectional area of colonies using πr²
The visible colony or inhibition clear zone approximates a circle whose area equals π × r², where r is the radius measured in the same units as the area requirement. Accurate measurement requires measuring diameter across the widest point, halving to obtain r, and then applying the formula. Area comparisons provide quantitative measures of colony size or antimicrobial effect and reduce reliance on subjective ranking .
Calculating bacterial population growth from mean division time
Bacterial growth through binary fission follows exponential increase. Population after time t equals initial population × 2^(t / d), where d is the mean division time in the same time units as t. Mean division time divides the total elapsed time into the number of generations; each generation doubles the population. Expressing answers in standard form simplifies very large numbers and reduces transcription errors .
Standard form for large bacterial counts (higher-tier)
Standard form represents numbers as a × 10^n, where 1 ≤ a < 10 and n is an integer. Very large bacterial populations are compactly written in standard form to show scale and facilitate comparisons. Converting between exact counts and standard form requires moving the decimal point and adjusting the exponent appropriately.
Investigating antiseptics or antibiotics - zones of inhibition
Agar diffusion tests compare the antibacterial effect of substances by placing samples or discs onto an inoculated agar lawn and incubating. Active substances diffuse into the agar and inhibit bacterial growth, producing a clear zone around the sample. Measuring the diameter (or radius) of the zone and calculating its area allows quantitative comparison of effectiveness. Controls without antiseptic/antibiotic provide baseline growth for comparison and ensure a fair test .
Key notes
Important points to keep in mind