Aseptic Techniques & Culturing

Growing microorganisms in a culture

To study microorganisms, scientists often need to grow them in large numbers. This is called culturing. Bacteria may be grown in a liquid nutrient broth or as visible colonies on solid agar gel in a Petri dish. The culture medium supplies nutrients needed for growth.

For an investigation to be valid, the culture should contain only the microorganism that was deliberately introduced. An uncontaminated culture is therefore essential when investigating substances such as disinfectants or antibiotics: if other microorganisms grow as well, it becomes unclear which organism produced the results. Contamination may also introduce potentially harmful microorganisms.

An uncontaminated culture is not the same as a sterile culture. A sterile dish contains no living microorganisms; once the intended bacteria have been added, the culture is no longer sterile, but it should remain uncontaminated.

What aseptic technique achieves

Aseptic technique is the collection of precautions used to prevent unwanted microorganisms entering a culture and to prevent cultured microorganisms escaping. Microorganisms are common on surfaces, skin and equipment and in the air, so contamination could occur at several stages.

Sterilisation is one part of aseptic technique. It kills microorganisms that are already present. Petri dishes and culture media must be sterilised before use; otherwise, existing microorganisms could multiply alongside the organism being studied. Glass dishes and media can be sterilised using heat, while plastic Petri dishes are often supplied ready-sterilised.

Preparing an uncontaminated agar culture

Begin with a sterile Petri dish containing sterile nutrient agar. The agar provides a moist, solid surface containing nutrients on which colonies can grow.

1. Pass the inoculating loop through a Bunsen-burner flame until it is red hot. This kills microorganisms on the loop.
2. Allow the loop to cool without putting it down or blowing on it. A hot loop could kill the bacteria that are meant to be transferred.
3. Dip the sterile loop into a suspension of the chosen microorganism.
4. Lift the Petri-dish lid only slightly and for as short a time as possible. Use the loop to make streaks across the agar surface, then replace the lid promptly. Limiting how far and how long the dish is opened reduces contamination from airborne microorganisms.
5. Secure the lid with adhesive tape. This reduces the chance of microorganisms entering or escaping. The source method does not seal the entire edge because oxygen should still enter, reducing the chance of harmful anaerobic bacteria growing.
6. Label the dish and store it upside down.

The loop is sterilised immediately before transfer because it could have picked up microorganisms since its previous use. Sterilising only the Petri dish and agar would therefore not be enough.

Why the dish is stored upside down

Water can evaporate from the warm agar and condense on the cooler lid. If the dish were stored upright, droplets could fall onto the agar, spreading microorganisms across its surface and mixing colonies. Storing it upside down keeps the agar above the lid, so condensation does not drip onto the culture.

Safe incubation in school

The secured, inverted dish is incubated so that the microorganisms can grow. In school and college laboratories, cultures should be incubated at a maximum temperature of 25°C.

Many microorganisms that cause human disease grow well near body temperature, about 37°C. Incubating at the lower school temperature reduces the likelihood that dangerous human pathogens will grow. The intended microorganisms can still form colonies, although growth may be slower than at their optimum temperature.

The dish should remain secured during and after incubation. Opening a grown culture could release large numbers of microorganisms and expose it to further contamination.

Purpose

• Bacteria can be grown in nutrient broth or as colonies on an agar plate.
• Uncontaminated cultures are needed so that results can be attributed to the intended microorganism.
• Aseptic technique prevents unwanted microorganisms entering and cultured microorganisms escaping.

Method recall

1. Use a sterilised Petri dish and sterile nutrient agar.
2. Flame the inoculating loop until red hot; allow it to cool without putting it down or blowing on it.
3. Transfer the chosen microorganisms to the agar and streak the surface.
4. Open the lid as little and as briefly as possible.
5. Replace and secure the lid with adhesive tape.
6. Label and store the dish upside down.
7. Incubate school cultures at no more than 25°C.

Reasons to remember

• Sterilise dish and medium: kill microorganisms already present.
• Flame the loop: prevent it transferring unwanted microorganisms.
• Secure the lid: reduce entry and escape of microorganisms.
• Store upside down: stop condensation falling onto the agar.
• Use a maximum of 25°C: reduce the likelihood of culturing harmful human pathogens that grow well near 37°C.