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Aseptic Techniques

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Biology

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

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 :

  • Antibacterial disinfectant
  • Bunsen burner and heatproof mat
  • Bacterial sample in broth
  • Nutrient agar plates
  • Wire loop
  • Glass spreader
  • Forceps
  • Antimicrobial discs
  • Tape
  • Ruler

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.

To calculate the area, find the diameter of the zone of inhibition first. Then use πd / 4 to find the area.

Aseptic Techniques - Key takeaways

  • Aseptic techniques are methods that prevent the growth of unwanted microorganisms. This is important in practice as it prevents contamination and the growth of pathogenic microorganisms.

  • Aseptic techniques include disinfecting work surfaces, using a bunsen burner and flaming bottleneck of bacterial samples.

  • The stages of microbial growth involve the lag phase, exponential (log) phase, stationary phase and death phase.

  • To investigate the effect of antimicrobial substances on bacteria, you can experiment using antibiotic discs and bacterial culture on agar plates.

Aseptic Techniques

Aseptic techniques describe the methods by which a sterile environment is maintained to prevent the cultivation of unwanted microorganisms. The techniques include using an antibacterial disinfectant to wipe down laboratory surfaces, using a bunsen burner and using a flamed wire loop when transferring bacteria.

Aseptic techniques are important in a laboratory as these methods prevent your culture from being contaminated with other microorganisms. Additionally, it prevents the growth of pathogenic microorganisms, which can be harmful to humans.

Aseptic techniques are used in a laboratory when handling samples of bacterial samples. 

The 4 stages of microbial growth include the lag phase, exponential (log) phase, stationary phase and death phase. 


The lag phase involves bacteria adjusting to their environment and synthesizing proteins. The exponential phase occurs when the bacteria actively divide by binary fission and the environment is optimal for bacterial growth. The stationary phase involves the plateau of growth, meaning the rate of bacterial growth is equivalent to the rate of bacterial death. The death phase occurs when the environment becomes too toxic, the rate of bacterial death exceeds their growth. 

To test for microbial growth, use aseptic techniques to transfer bacteria from a sample to a nutrient agar plate. Incubate this at 25ºC for 24 hours. The presence of microbes is indicated by cloudy-white zones on the agar plate. These are bacterial colonies. 

Final Aseptic Techniques Quiz

Question

What is the importance of aseptic techniques?

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Answer

Aseptic techniques ensure that bacterial samples are not contaminated. They also ensure that pathogenic microorganisms do not grow. 

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Question

Identify 4 examples of aseptic techniques. 

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Answer

Wash hands before handling agar plates. Disinfect workspaces with an antibacterial cleanser. Use a bunsen burner to flame wire hoops. Flame bottle necks of bacterial broth. 

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Question

Why are bunsen burners used in aseptic techniques?


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Answer

Bunsen burners prevent the entry of microorganisms into your culture. 

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Question

What piece of equipment is used to transfer bacteria into agar plates?


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Answer

Flamed wire hoops. 

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Question

Identify the 4 stages of microbial growth. Under what conditions does the microbial growth curve occur?


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Answer

The lag phase, exponential (log) phase, stationary phase and death phase. This occurs in a closed system. 

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Question

Describe the lag phase of microbial growth. 


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Answer

The lag phase involves a small number of bacteria adjusting to their environment. There is very little growth happening at this stage. 

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Question

Describe the exponential phase of microbial growth. 


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Answer

The exponential phase involves the rapid increase of the bacterial population due to binary fission. The bacteria grow in optimal conditions. 

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Question

Describe the exponential phase of microbial growth. 


Show answer

Answer

The exponential phase involves the rapid increase of the bacterial population due to binary fission. The bacteria grow in optimal conditions. 

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Question

Describe the stationary phase of microbial growth. 


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Answer

The stationary phase involves the plateau of bacterial growth. At this stage, the rate of bacterial growth is equivalent to the rate of bacterial death. This is because of the accumulation of waste products and the depletion of nutrients. 

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Question

Describe the death phase of microbial growth. 


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Answer

The death phase involves the toxic accumulation of waste products and the starvation of nutrients. The rate of bacterial death exceeds the rate of bacterial growth. 

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Question

What is a zone of inhibition?


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Answer

A zone of inhibition describes the clear area around an antibiotic to indicate the killing of bacterial colonies. 

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Question

How do you calculate the area of a zone of inhibition?


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Answer

Find the diameter first. Then use πd/4 to find the area. 

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Question

What are antimicrobial substances?


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Answer

Antimicrobial substances inhibit the growth of bacteria and kill bacteria. 

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Question

What do you observe on an agar plate that indicates the presence of bacteria?


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Answer

The presence of bacteria is indicated by cloudy-white areas on an agar plate.

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Question

What does a small zone of inhibition indicate?


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Answer

A small zone of inhibition indicates that bacteria may be resistant to the antibiotic. 

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Question

What does a large zone of inhibition indicate?


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Answer

A large zone of inhibitions indicates that bacteria are highly susceptible to the antibiotic.

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Question

What does a large zone of inhibition indicate?


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Answer

A large zone of inhibitions indicates that bacteria are highly susceptible to the antibiotic.

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