Required practical 6: Use of aseptic techniques to investigate the effect…

The idea

Grow a bacterial lawn on sterile nutrient agar to test antimicrobial substances. Aseptic technique keeps unwanted microorganisms out. Sterilise a wire loop by flaming until red hot, or a glass spreader by dipping in alcohol and flaming, then cool. Work close to a lit Bunsen so the updraught carries spores away; lift the Petri dish lid at an angle; flame the neck of culture bottles. Spread the culture evenly, then place discs soaked in different antimicrobials, plus a sterile water control, well apart. Tape the lid at two points rather than sealing completely, invert the dish to prevent condensation dripping, and incubate at max 25 degrees Celsius. Measure the clear zone of inhibition around each disc and calculate its area. Repeating the plates and calculating a mean reduces the effect of random errors, improving repeatability.

What earns the marks

• Name an aseptic step with its reason, such as flaming the loop to kill microorganisms on it.
• Include a control disc soaked in sterile water or in the solvent alone.
• Quantify the result by measuring the diameter of the clear zone and calculating its area.
• Control a named variable such as disc size, volume of antimicrobial, incubation temperature and time, or the same culture and agar.
• Repeat the plates and calculate a mean to reduce the effect of random errors, improving repeatability.
• Explain why the lid is taped at two points rather than sealed completely, and why the dish is inverted during incubation.

Common mistakes and how to put them right

• Sealing the lid all the way round, which creates anaerobic conditions that favour the growth of pathogens.

  Put it right: Tape at two points only to allow some gas exchange and prevent anaerobic conditions.

• Incubating above 25 degrees Celsius in a school laboratory, which risks culturing human pathogens.

  Put it right: Incubate at or below 25 degrees Celsius to reduce the risk of growing pathogens.

• Comparing zone diameters when the question asks for area, or forgetting to halve the diameter before using pi r squared.

  Put it right: Measure the diameter, halve it to get the radius, then use area = pi r squared.

• Omitting the control disc, so a clear zone cannot be attributed to the antimicrobial rather than to the solvent.

  Put it right: Include a control disc soaked in sterile water or the solvent alone.

• Taking the lid fully off while placing the discs, allowing contamination from the air.

  Put it right: Lift the lid at an angle and only partially, to minimise entry of airborne contaminants.

Exam tips

• Every aseptic step needs its purpose attached; the reason is the mark, not the action.
• Treat the zone of inhibition as the measure of effectiveness, so a larger area means a more effective antimicrobial at that concentration.
• If asked why bacteria still grew on a plate containing an antibiotic, consider too low a concentration, contamination with resistant bacteria, uneven mixing, or mutation.