Aseptic techniques are precise laboratory procedures used to prevent the accidental introduction of unwanted microorganisms, known as contamination. They also stop the cultured microbes from escaping into the environment.
The ultimate goal is to grow a pure culture, which contains only one single species of microorganism. Maintaining a pure culture ensures that any results you observe—such as the size of a clear inhibition zone around an antibiotic disc—are valid and caused entirely by the specific microbe being tested.
It also prevents other species from competing for nutrients and space, while avoiding the accidental growth of a dangerous pathogen. To further prevent contamination, scientists work close to a lit Bunsen burner on a roaring blue flame.
This creates convection currents (updrafts) that draw dust and airborne microbes away from the open workspace. This simple step is vital for keeping the growth medium sterile during the inoculation process.
To achieve complete sterilisation—meaning the equipment is entirely free from all living microorganisms and their spores—laboratories use an autoclave. An autoclave is a machine that uses moist steam under high pressure (usually psi).
This pressure raises the boiling point of water, allowing the machine to reach . At this extreme temperature, the proteins and enzymes inside microbes denature, completely killing them alongside highly heat-resistant bacterial spores.
Schools often use a pressure cooker to achieve the exact same result for sterilising nutrient agar and Petri dishes. When performing inoculation (transferring microbes to a sterile medium), a wire inoculating loop is used.
The loop must be held in the roaring blue flame of a Bunsen burner until the metal glows red-hot, which incinerates any existing microbes. Crucially, the loop must then be allowed to cool for to seconds.
If it is too hot, it will instantly kill the bacterial sample you are trying to transfer. The neck of any culture bottle being used should also be passed through the flame to kill microbes near the opening.
During inoculation, the lid of the Petri dish should only ever be lifted slightly and at an angle. This acts as a physical shield against airborne contamination falling into the agar.
Once the microbes are transferred, the lid is secured with just two pieces of adhesive tape on opposite sides (e.g., North and South). The dish must never be completely sealed all the way around.
Leaving gaps allows oxygen to enter so the bacteria can respire aerobically. This actively prevents the growth of highly dangerous anaerobic pathogens that thrive in oxygen-free environments.
After sealing, the Petri dishes are always incubated upside down (inverted). This prevents condensation from forming on the lid and dripping down onto the agar surface, which would cause the bacterial colonies to merge together.
In school laboratories, incubation is strictly limited to a maximum temperature of . This is significantly lower than human body temperature (), safely reducing the risk of accidentally cultivating human pathogens.
Bacteria multiply by a type of asexual reproduction called binary fission, where one cell divides into two. Under ideal conditions (adequate nutrients and temperature), some bacteria can multiply as often as once every minutes.
The population increases exponentially, and you can calculate the total number of bacteria using the mean division time.
First, calculate the number of divisions () that occur in the given time:
Then, calculate the total population size:
Worked Example: A bacterium has a mean division time of minutes. How many bacteria will be produced from one cell after hours?
Students often state that the Petri dish lid is sealed completely to 'keep air out', but examiners want you to explain that it is taped only partially to allow oxygen in and prevent the growth of dangerous anaerobic pathogens.
When answering 6-mark questions on preparing uncontaminated cultures, examiners always look for you to explicitly state that the inoculating loop is heated until 'red-hot' and must be allowed to 'cool' before touching the bacterial sample.
Be prepared to explain the difference in incubation temperatures; schools use a maximum of to prevent the growth of human pathogens, whereas industry uses higher temperatures (like ) for faster microbial growth.
In sterilisation questions, mentioning that an autoclave destroys highly resistant bacterial 'spores' will often secure you an extra mark that boiling water alone would not achieve.
When calculating bacterial populations, always ensure your units for 'total time' and 'mean division time' match (e.g., both in minutes) before dividing.
Aseptic techniques
A set of laboratory procedures designed to prevent the introduction of unwanted microorganisms into a culture or the environment.
Contamination
The accidental introduction of unwanted microbes or pathogens into a culture, which can invalidate experimental results.
Pure culture
A population of microorganisms that contains only one specific species or strain.
Inhibition zone
The clear area on an agar plate where bacteria have been killed or their growth prevented by a substance like an antibiotic.
Pathogen
A microorganism that is capable of causing disease.
Sterilisation
The process of making equipment or media completely free from all living microorganisms and their highly resistant spores.
Autoclave
A piece of laboratory equipment that uses steam under high pressure and temperature (typically ) to sterilise equipment and kill spores.
Inoculation
The process of purposefully introducing microorganisms into a sterile growth medium using a sterilised tool.
Inoculating loop
A wire tool used to transfer microorganisms, which is sterilised by heating it in a Bunsen burner flame until red-hot.
Airborne contamination
The introduction of unwanted microorganisms into a culture via dust, air currents, or gravity.
Anaerobic pathogens
Microorganisms capable of causing disease that specifically grow and thrive in environments lacking oxygen.
Binary fission
A form of asexual reproduction in which a single-celled organism divides into two approximately equal parts.
Mean division time
The average time it takes for a population of bacteria to double in number.
Put your knowledge into practice — try past paper questions for Biology
Aseptic techniques
A set of laboratory procedures designed to prevent the introduction of unwanted microorganisms into a culture or the environment.
Contamination
The accidental introduction of unwanted microbes or pathogens into a culture, which can invalidate experimental results.
Pure culture
A population of microorganisms that contains only one specific species or strain.
Inhibition zone
The clear area on an agar plate where bacteria have been killed or their growth prevented by a substance like an antibiotic.
Pathogen
A microorganism that is capable of causing disease.
Sterilisation
The process of making equipment or media completely free from all living microorganisms and their highly resistant spores.
Autoclave
A piece of laboratory equipment that uses steam under high pressure and temperature (typically ) to sterilise equipment and kill spores.
Inoculation
The process of purposefully introducing microorganisms into a sterile growth medium using a sterilised tool.
Inoculating loop
A wire tool used to transfer microorganisms, which is sterilised by heating it in a Bunsen burner flame until red-hot.
Airborne contamination
The introduction of unwanted microorganisms into a culture via dust, air currents, or gravity.
Anaerobic pathogens
Microorganisms capable of causing disease that specifically grow and thrive in environments lacking oxygen.
Binary fission
A form of asexual reproduction in which a single-celled organism divides into two approximately equal parts.
Mean division time
The average time it takes for a population of bacteria to double in number.