Growing microorganisms
Definition
Culture medium
A nutrient jelly (agar) or liquid broth that supplies microorganisms with the nutrients they need to grow.
- A culture medium supplies water and nutrients so microorganisms can grow. It may be a liquid broth or nutrient agar in a Petri dish.
- On solid agar, one microorganism can divide repeatedly to form a visible colony. A spread of bacteria across the agar is called a bacterial lawn.
Aseptic technique
Definition
Aseptic technique
A set of precautions used when culturing microorganisms to prevent contamination of the culture and to stop unwanted microbes spreading.
- Aseptic technique prevents unwanted microorganisms contaminating the culture and reduces the chance that cultured microorganisms escape into the surroundings.
- An autoclave uses pressurised steam at a high temperature to sterilise culture medium and Petri dishes before use. Sterilisation kills microorganisms and resistant spores.
- A sterile inoculating loop is used to transfer microorganisms. If a metal loop is used, it is heated in a Bunsen flame until red hot and allowed to cool before touching the culture.
- The Petri-dish lid is opened only slightly and briefly. After inoculation, it is secured at two or three points so it stays covered but is not sealed completely.
Exam technique
State the purpose as well as the step: sterilising equipment kills contaminating microorganisms, while keeping the lid covered reduces entry and escape of airborne microorganisms.
Antimicrobial cultures
Definition
Inhibition zone
The clear area around an antibiotic or antiseptic where bacteria have been killed or stopped from growing.
- A paper disc containing an antibiotic, antiseptic or plant extract is placed on a bacterial lawn. The substance diffuses through the agar.
- Where bacterial growth is killed or inhibited, a clear zone of inhibition forms. A larger zone usually indicates a more effective antimicrobial substance when all control variables are kept constant.
Practical
Investigating antimicrobial substances
- Aim: Compare the effectiveness of antiseptics, antibiotics or plant extracts by measuring the clear zones they produce in a bacterial culture.
- Apparatus: Sterile nutrient-agar plate, safe bacterial culture, sterile inoculating loop, sterile forceps, filter-paper or prepared antibiotic discs, test substances, sterile-water control, marker pen, ruler, sticky tape, incubator, Bunsen burner, ethanol and eye protection.
- Method:
- Disinfect the bench and work beside a lit Bunsen burner.
- Flame a metal inoculating loop until red hot, then let it cool without touching anything.
- Lift the Petri-dish lid only slightly and spread the bacteria evenly over the agar to form a lawn.
- Sterilise the forceps, allow them to cool, and place equal-sized discs containing equal volumes of the test substances onto the agar. Add a disc containing sterile water as a control.
- Label the base of the plate.
- Secure the lid with two or three pieces of tape, but do not seal it all the way around.
- Incubate the plate upside down at 25 ∘C25\,^{\circ}\mathrm{C}25∘C for 242424 to 484848 hours.
- Without opening the plate, measure each clear zone twice at right angles and calculate the mean diameter.
- Variables: Change the antimicrobial substance. Measure the area of the zone of inhibition. Keep the bacterium, agar depth, disc diameter, volume and concentration of substance, spacing of discs, incubation time and temperature constant.
- Results: A clear ring forms where bacteria have been killed or prevented from reproducing. The sterile-water control should have no clear zone, showing that any inhibition is caused by the test substance rather than the paper disc.
- Maths: For a circular zone, calculate the mean diameter, use r=d2r=\frac{d}{2}r=2d, then calculate A=πr2A=\pi r^2A=πr2. Measure the whole clear zone, including the disc, and give the area in mm2\mathrm{mm}^2mm2.
- Watch out: Unequal disc sizes, different volumes of substance, overlapping zones or an uneven bacterial lawn make the comparison invalid. Repeat each treatment and calculate a mean.
- Safety: Incubate at 25 ∘C25\,^{\circ}\mathrm{C}25∘C rather than body temperature, never reopen an incubated plate, keep ethanol away from the flame, and autoclave cultures after use.
Interpreting the investigation
- The plate is inverted during incubation so condensation collects on the lid rather than dripping onto the agar and spreading colonies.
- School cultures are incubated at 25 ∘C25\,^{\circ}\mathrm{C}25∘C because temperatures closer to 37 ∘C37\,^{\circ}\mathrm{C}37∘C would favour microorganisms adapted to the human body, including potential pathogens.
Self review
- Why must culture medium and Petri dishes be sterilised before use?
- Why is a sterile-water disc included?
- Why is the plate incubated upside down at 25 ∘C25\,^{\circ}\mathrm{C}25∘C?
- A clear zone has a mean diameter of 18 mm18\,\mathrm{mm}18mm. Calculate its area.
