Aseptic Techniques

Aseptic techniques describe the sterile methods used in a laboratory to prevent the cultivation of unwanted microorganisms. We will explore why this is important and the different techniques used.

What is the importance of aseptic techniques?

Aseptic techniques ensure that we do not contaminate our samples with microorganisms that we are not investigating. Our goal is to keep our microorganism samples pure. These techniques also discourage disease-causing microorganisms (pathogens), which are harmful to humans, from growing in a laboratory.

What are the aseptic techniques in microbiology?

Many aseptic techniques are used in a microbiology laboratory. Take a look at the list below.

In the practical we will be explaining in the following section, we will be investigating how antimicrobial substances affect microbial growth. First, let's explore the stages of microbial growth by looking at their growth curve.

Bacteria are prokaryotes; unlike eukaryotes (e.g., humans), they undergo asexual reproduction, also known as binary fission. In other words, bacterial populations grow because the bacteria divide by themselves.

In the growth curve above, you will see different microbial growth phases. It is important to mention that this curve occurs when bacteria are cultivated in a closed system, meaning no nutrients are added or removed. Although the practical we will perform later in this article will not use a closed system, it is useful to appreciate how bacteria grow.

The different phases of bacterial growth are (Figure 1):

• Lag phase
• Exponential phase
• Stationary phase
• Death phase

Fig. 1 - The phases of bacterial growth

The lag phase involves a small number of bacteria growing in size and synthesizing proteins. This process is seen at the beginning of the curve, where not much growth is occurring yet, and the bacteria are adjusting to the new environment.

The exponential phase (often called the log phase in some textbooks) involves the bacteria rapidly dividing by binary fission. The conditions for bacterial growth are optimum, and this causes the population number to increase exponentially.

The stationary phase occurs when bacterial growth is inhibited by the accumulation of waste products and the depletion of nutrients. Remember, this occurs in a closed system, and therefore waste cannot be removed, and nutrients cannot be added. As a result, the rate of bacterial growth is equal to the rate of bacterial death.

The death phase is the final phase of the growth phase and, as its name suggests, signals the death of the bacterial population numbers. The accumulation of waste products and starvation of nutrients has become toxic and has caused bacterial death to exceed the rate of bacterial growth.

Investigating the effect of antimicrobial substances on microbial growth

Antimicrobial substances describe chemical agents that kill bacteria. In this practical, we will investigate the use of these substances in culture and implement the aseptic techniques we learned earlier.

Equipment :

Method :

• Implement aseptic techniques as described in the earlier section of this article.
• Flame the wire loop using the bunsen burner to transfer bacteria from the bacterial sample broth into the nutrient agar plate.
• Use the glass spreader to distribute the bacteria across the agar plate.
• Use the forceps to lay the antimicrobial disc on the agar plate.
• Secure the lid on the agar plate with tape.
• Incubate the agar plates at 25 ° C for 24 hours upside down.
• Implement aseptic techniques to tidy up the workspace.

Throughout the 24 hour incubation period, the different antibiotics on the antimicrobial disc will act on the bacteria that have been transferred on the agar plate. If the bacteria are susceptible to the antibiotic, you will observe a clear area around the disc. This is called the zone of inhibition, and this indicates that there are no bacteria present as the antibiotic has inhibited their growth and killed the cells.

The areas that appear cloudy and white indicate the presence of bacterial colonies that have survived.

Results of the investigation

After the incubation period, measure the area of the zone of inhibition around each antibiotic. Do this by measuring from the base of the agar plate, as you must not remove the lid!

To visualize your data, plot a bar chart of area (Y-axis) against the different antibiotics (X-axis). This graph shows which antibiotic killed the most bacteria and which antibiotic killed the least bacteria.

The antibiotics which have killed the most bacteria and therefore have the largest zones of inhibition are the most effective antibiotics. The antibiotics with a small zone of inhibition indicate that the bacteria may have resistance to these agents.