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In this exercise you will complete a virtual laboratory simulation

Biology

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In this exercise you will complete a virtual laboratory simulation.  Begin by reading the Lab Handout titled 3 Aseptic Technique Labster.  You will need this information to answer questions throughout the simulation.

Learning Objective

Understand the principles of aseptic technique for the prevention of infection and contamination

• • Create and maintain a sterile work area

• • Use sterile equipment and consumables correctly

• • State potential sources of microbial contamination

• • Assess whether a sample was contaminated

 

Once you are ready, you can start the simulation by pressing the simulation launch button/link below.

NOTE:  Chrome or Firefox are the recommended browsers.  For technical issues, contact Labster support (link in Start Here module)

Virtual Lab Manual

Aseptic Technique: Culture your sample without contamination

Synopsis

This simulation, along with “Fermentation: Optimize bio-ethanol production,” was adapted from learning objectives in the original “Fermentation” simulation. For more information on this topic, see Labster’s Microbiology simulations.

A patient sample has arrived in the microbiology lab. Will you be able to culture it using good aseptic technique? Learn about aseptic technique and what you should pay attention to when preparing a sterile work area, sterilizing equipment and reagents, and decontaminating the work area after you finish your experiment.

Preparing a sterile field

You will start o by preparing the sterile work area. Use your own microbiology knowledge, the tooltips, and theory pages to ensure you are using good aseptic technique. Don’t worry if you make a mistake and your work area is not quite sterile. In the virtual lab, Dr. One can guide you to repeat some steps without losing several hours like you would in the physical lab.

Culturing and cleaning up

Next, you will culture your sample and the appropriate control using sterile equipment and reagents. Once you have placed your samples in the incubator: it’s clean up time! Dr. One will again guide you if you accidentally contaminated one of the samples or accidentally set the lab on fire.

Analyzing your results

After the incubation, check your samples to see if you have used correct aseptic technique throughout the experiment. Will you be able to create a pure culture that the microbiologist can use to identify an unknown microbe?

Techniques in Lab

• Aseptic technique
• Culturing

Learning Objectives

At the end of this simulation, you will be able to…

• Understand the principles of aseptic technique for the prevention of infection and contamination
• Create and maintain a sterile work area
• Use sterile equipment and consumables correctly
• State potential sources of microbial contamination
• Assess whether a sample was contaminated

Theory

Microbiological culture

Culturing is the practice of growing microorganisms, such as yeast or bacteria, in the lab. The nutrients the microbes need are provided by the culture medium. Microorganisms can be grown in liquid culture or on media made solid by the addition of agar, which gives culture media a gel-like consistency. When culturing microorganisms, good aseptic technique is required to keep the culture free from external contaminations. Correct aseptic technique also keeps those working in the lab safe, which is particularly important when working with pathogenic organisms.

Setting up liquid and solid cultures

To create a liquid culture, a colony from an agar plate is added to liquid medium using a culture loop, or a small volume of liquid culture is added using a pipette. To create a solid culture, a colony is streaked across a petri dish containing solid medium (known as an agar plate) using a culture loop or a small volume of liquid culture is added to the plate and spread across the surface using a spreader. All equipment must be sterilized before and after each use.

Controls

To ensure good aseptic technique is used, the best negative control is uncultured medium. Since the medium and equipment should be sterile, microbial growth in this control indicates contamination. Everything else should be treated the same across the sample and control.

Aseptic technique

Sterile technique is used to ensure a "clean" lab environment. It is essential to ensure the reliability of experimental results.

Sterile practices are especially important when working with microorganisms. A single spore or tiny bacterium can overgrow your whole medium and destroy your experiment.

The following steps are used to keep laboratory work sterile: Keep calm and assess the situation:

• Laboratory doors and windows are kept closed to prevent air currents, preventing surface microorganisms becoming airborne.
• The wire loop and glass spreader are sterilized before and after use with a Bunsen burner to prevent the introduction of unwanted microorganisms.
• Lids from bottles and tubes are held when removed, and not placed on the bench during material transfer from one bottle or tube to another.
• The neck of a bottle or tube must be immediately heated using the Bunsen burner so that any air movement is outward.
• The bottle or tube are opened for the minimum time possible, and while open, all work is performed close to the Bunsen burner flame.
• Media    and        equipment          are         sterilized              to prevent the         growth  of           unwanted microorganisms.

Culturing with a Bunsen burner

A sterile work area for microbiological culturing can be created around a Bunsen burner or in a biosafety cabinet. The techniques used to work aseptically near a Bunsen burner and in a biosafety cabinet are similar.

The Bunsen burner creates a radial sterile field around it. Items can also be sterilized by passing them through the flame. This allows for the use of reusable equipment such as wire loops and glass spreaders. The key steps to correct aseptic technique using a Bunsen burner are listed below.

Preparing the sterile work area

• Decontaminating the work surface with ethanol
• Light the Bunsen burner and adjust to produce a roaring blue flame
• Place only necessary materials in the sterile work area and arrange them so the updraft that is creating the sterile field is not disturbed

Culturing technique

• Minimize movements that can disturb the sterile field around the Bunsen burner
• Hold any lids and closures in the hand in the sterile field rather than placing them on the work surface
• Sterilize any equipment, including the necks of bottles, by holding it in the flame
• Use either a new disposable item (such as pipette tips) for each action or sterilize reusable equipment (such as glass spreaders) before and after each action

Decontamination and waste disposal

• Sterilize all reusable equipment, close all containers and dispose of all disposable items in the biological waste bin
• Turn o the Bunsen burner and decontaminate the work surface with ethanol ? Disinfect your hands

Bunsen Burner safety measures

• Tie back long hair and avoid loose items of clothing near the flame
• Gloves should never be worn, instead, wash hands thoroughly before and after working with microbiological samples or disinfect hands with hand sanitizer
• Ethanol should only be used while the Bunsen burner is o

Culturing in a biosafety cabinet

A sterile work area for microbiological culturing can be created around a Bunsen burner or in a biosafety cabinet. The techniques used to work aseptically near a Bunsen burner and in a biosafety cabinet are similar. Open flames can not be used in a biosafety cabinet. All equipment must, therefore, be pre-sterilized. It is also important to understand the airflow in the biosafety cabinet to avoid blocking the airflow and allowing air to mix.

The key steps to using the biosafety cabinet correctly are outlined below.

Bunsen Burner safety measures

• Tie back long hair and avoid loose items of clothing near the flame
• Gloves should never be worn, instead, wash hands thoroughly before and after working with microbiological samples or disinfect hands with hand sanitizer
• Ethanol should only be used while the Bunsen burner is o

Preparing the biosafety cabinet

• Establish laminar airflow and decontaminate surfaces before starting work
• Ensure all items in the biosafety cabinet have been pre-sterilized

Culturing technique

?	Place only necessary items in the biosafety cabinet and maintain the minimum distance from the sash to avoid disturbing the laminar airflow
?	Organize materials in such a way that movements are minimized
?	Keep contaminated items away from sterile items and dispose of contaminated

materials correctly as soon as possible

Decontamination and waste disposal

• After completing work, dispose of all contaminated materials, and decontaminate the surface
• Change gloves

Sterile equipment and reagents

Aseptic technique requires that all surfaces, equipment, and reagents that may come into contact with the sample are sterilized or disinfected to avoid contamination. Di erent sterilization and disinfection techniques exist depending on the application. The most common techniques when culturing a sample in the lab are listed below.