Aseptic Techniques & Culturing

Preparing the Uncontaminated Culture

• Understanding how heat changes airflow explains why we use fire to keep a workspace clean.
• Preparing an uncontaminated culture requires aseptic technique, a strict set of step-by-step procedures to prevent contamination.
• All work must be performed directly near a lit Bunsen burner. The heat creates a convection current of warm air that constantly draws airborne microorganisms upwards and away from the open workspace.
• The metal inoculating loop used to transfer bacteria is sterilised by passing it through a roaring blue Bunsen burner flame until it glows red hot.
• The loop must be allowed to cool in the air for a few seconds; if used while too hot, it will instantly kill the desired bacteria you are trying to transfer.
• The neck of the culture bottle should also be passed through the flame immediately after opening and before closing to prevent airborne microbes from entering.

Inoculation and Sealing the Petri Dish

• Why do we tape a Petri dish shut, but specifically avoid sealing it completely?
• During inoculation, the Petri dish lid should be opened as little as possible (held at an angle) and for the shortest time possible to reduce exposure to airborne microbes.
• Once the bacteria are transferred, the lid must be secured to the base using two pieces of adhesive tape.
• The lid must specifically NOT be sealed all the way around, meaning it must not be airtight.
• A complete seal would create an oxygen-free environment, which promotes the growth of dangerous anaerobic pathogens.
• Finally, dishes should be labelled on the bottom (base) rather than the lid so that identification is not lost if lids get swapped.

Safe Incubation and Storage

• Industrial labs routinely grow bacteria at human body temperature, but doing this in a school lab is strictly banned.
• In school laboratories, cultures must be incubated at a maximum temperature of 25°C.
• This temperature limit is a strict safety precaution to prevent the growth of harmful human pathogens, which are highly adapted to thrive at human body temperature (37°C).
• Petri dishes must always be placed into the incubator using inverted storage (upside down).
• Storing plates upside down prevents condensation drops from forming on the lid and dripping down onto the agar surface.
• Dripping water would cause distinct bacterial colonies to merge and spread together, ruining the results of the culture.

Calculating Bacterial Growth and Inhibition Zones

• Under optimal conditions with sufficient nutrients and an ideal temperature, a single bacterial cell can multiply into thousands in just a few hours.
• Bacteria reproduce asexually through a process called binary fission, with some species dividing as often as once every 20 minutes.
• The effectiveness of an antibiotic or antiseptic is evaluated by measuring the inhibition zone—the clear area around a chemical disc where bacteria have been killed or prevented from growing.

Worked Example: Bacterial Population Calculation

To find the final number of bacteria after a set time, use the formula:

$$\text{Total number of bacteria} = \text{Initial number of bacteria} \times 2^n$$

(Where $n$ is the number of divisions)

If 1 bacterium divides every 20 minutes, how many will there be after 2 hours?

• Step 1: Convert total time to minutes. 2 hours = 120 minutes.

• Step 2: Calculate the number of divisions ($n$). $120 / 20 = 6$ divisions.

• Step 3: Substitute into the formula. $1 \times 2^6 = 64$ bacteria.

Worked Example: Area of an Inhibition Zone

To find the area of the clear zone, use the formula for the area of a circle:

$$Area = \pi r^2$$

Calculate the area of an inhibition zone with a diameter of 20 mm.

• Step 1: Find the radius ($r$). $20 / 2 = 10$ mm.

• Step 2: Substitute into the formula. $Area = \pi \times 10^2$

• Step 3: Calculate the final answer. $3.14 \times 100 = 314$ mm$^2$.