For example, microbes on or in tools, glassware, and liquid media can be heat-killed in an autoclave, which is a chamber that sterilizes contents via exposure to high-temperature pressurized steam. In addition, some tools can be heat-sterilized using a flame source, such as a Bunsen burner. The use of a flame source is also one of the most common ways to establish an aseptic working environment. Subscription Required.
Please recommend JoVE to your librarian. Aseptic technique is a fundamental skill in microbiology, and has crucial applications in environmental research.
If microbiological cultures are contaminated, the time, labor, and financial costs that would be required of a lab to "clean up" or replace the cultures, particularly rare isolates from unique environments, could be very high and prohibitive, and some samples may be irreplaceable. Proper use of aseptic techniques reduces the likelihood of bacterial and fungal contamination of reagents, culture media, and environmental samples, and also avoids cross-contamination between samples. It is also a safety measure that diminishes the potential transmission of microbes to the experimenter, which is especially important when working with pathogens.
This video will introduce the principles of asepsis; several important techniques to maintain sterile reagents and cultures; and finally, some of their uses in environmental science. Common sources of contamination include airborne microorganisms, microbes present on the laboratory bench or equipment, and those from the hair, body, and clothing of the researcher. Two types of agents are central to removing or preventing contamination in the laboratory: disinfectant chemicals and heat.
At the same time, microbes on or in tools, glassware, and liquid media can be heat-killed in an autoclave, which is a chamber that sterilizes contents via exposure to high-temperature pressurized steam. In addition, tools such as glass rods used for spread plating and metal inoculation loops can be heat-sterilized using a flame source, such as a Bunsen burner.
The heat from the flame causes air convection, generating an updraft that lifts any airborne contaminants away from the vicinity of the burner, and creating a "sterile field" in which to conduct aseptic experimental work. Now that you understand the principles behind aseptic techniques and why they are important, let's go through a protocol for creating an aseptic working environment, making up sterile growth media, and aseptically transferring bacteria between different culturing conditions.
Before beginning aseptic work, it is important for the experimenter to don proper personal protective equipment, or PPE. The purpose of this is both to prevent the experimenter from contaminating the samples and lab cultures, and also to prevent the transfer of potentially pathogenic microbes to the researcher. PPE items include a lab coat, gloves, and safety goggles. The next step is to properly sterilize and store the growth media to be used for culturing the microbial samples.
First, weigh out the proper amount of solid medium components, and add them to the proper volume of liquid solvent specified by the manufacturer, such as deionized water, in an autoclavable container Add a magnetic stir bar, place the container on a hot plate stirrer, and dissolve the solid medium components with low heat and stirring. Close the medium containers.
If using a glass vessel with screw-on cap, be sure to not tighten the cap completely, as the air inside the vessels will expand due to heating during autoclaving and needs to escape.
Without escape, the gas could cause the vessel to rupture. After autoclaving, verify that the stripes on the autoclave tapes turned black, indicating the proper temperature was reached.
For liquid growth media, let them cool to room temperature, and store them at room temperature or with refrigeration as appropriate. Allow the agar to cool and solidify before storing at the appropriate temperature.
For media that cannot be autoclaved due to the presence of heat-sensitive components, filter sterilize using a 0. A core technique in microbiological work is to aseptically transfer bacterial cultures between different growth media, both solid and liquid. Prior to beginning, clean the lab bench surface with a disinfectant. This lowers the risk of contaminating cultures or sterile media.
Transferring bacterial cultures commonly makes use of a tool called an inoculating loop, which needs to be sterilized prior to use by heating in a flame. Turn on a flame source. Slowly pass the inoculating loop through the tip of the flame.
The loop will turn red hot. To transfer a bacterial colony from a solid agar plate, open the Petri plate slightly, and gently tap the hot inoculating loop onto an empty part of the agar surface to avoid heat-killing the bacteria.
Scrape a single colony with the inoculating loop, and close the plate. To transfer bacteria from a liquid growth medium, remove the cap from the culture container.
To help prevent contamination, avoiding setting the cap down onto the bench. Pass the mouth of the container times through the hottest portion of the flame. Then, carefully touch the hot, sterilized inoculation loop onto the inside of the container and let it cool before inserting it into the broth culture.
Remove one loopful of the culture, and immediately close the cap. For transferring the obtained bacteria to a sterile growth medium, remove the cap from a container with the sterile broth and pass the container's opening through the flame times.
Then, carefully lower the inoculation loop into the medium, and agitate gently to release the bacteria. Immediately close the cap. Sterilize the inoculation loop after use.
If transferring bacteria onto a sterile agar plate, open the lid of a fresh Petri plate with uninoculated agar. Streak the inoculation loop with the bacterial culture back-and-forth across one sector of the agar. Sterilize the loop and cool it by touching an empty part of the agar, then make another streak on the agar at an obtuse angle to the first streak, making sure to cross the first streak on the first strokes but avoid touching the first streak on subsequent strokes.
Repeat the sterilization and streaking 2 more times. Close the Petri plate, and sterilize the inoculation loop. Once inoculated, the broth or agar plate should then be incubated at the ideal growth temperature for the given microorganism to obtain viable culture. On solid medium, a lawn or continuous strand of bacteria would be visible on agar covered by the first two streaks, but individual colonies should be obtained on the final streak.
Poor aseptic techniques would result in the growth of mold and other contaminants on the plate. Aseptic techniques are important in many experiments involving microbial samples from the environment. In this study, researchers isolated bacteriophages, which are bacteria-infecting viruses, from the common soil bacterium Arthrobacter.
Arthrobacter cultures were first grown under aseptic conditions. Soil samples were then washed and filtered in phage buffer, and the phage solution was mixed with the bacterial culture and plated onto agar plates.
A bacterial lawn would form on the plate, but there would be clearings, or "plaques", at spots where the virus had infected and killed the bacteria. Phage could then be purified from these plaques for further study.
Other than using Bunsen burners, aseptic working environments can also be maintained in specialized workstations known as laminar flow hoods, which use directed airflow and filters to maintain sterility. Aseptic transfers are often conducted in a laminar flow cabinet that has surfaces that can be sterilised before use with alcohol swabbing and ultraviolet light, and sterile filtered air is supplied to maintain postive air pressure in the working area of the cabinet to prevent settling of any potential contaminants.
Every aseptic procedure in the microbiological laboratory will be done using similar techniques, with modifications as necessary. Remember Me. Lost your password? Sustainable Winegrowing Australia: click here to log in. The procedure for aseptically transferring microorganisms is as follows: Sterilise the inoculating loop.
The inoculating loop is sterilised by passing it at an angle through the flame of a gas burner until the entire length of the wire becomes orange from the heat. In this way all contaminants on the wire are incinerated. Never lay the loop down once it is sterilised, or it may again become contaminated.
Allow the loop to cool a few seconds before contacting the inoculum to avoid killing the microorganisms. The pressure will go up over 15 pounds per square inch psi : at this point the timer begins to count down usually for 15 minutes, depending on the type of media. Fifteen minutes is the thermal death time for most organisms except some really hardy sporeformers. The prepared media is distributed in different ways, depending on the form one is making.
Broths and agar deeps are dispensed into tubes and then sterilized. Agar slant tubes are sterilized and then the rack is tilted to allow the agar to solidify in a slanted fashion. Agar medium to be be poured into plates is sterilized in a flask, and then poured afterward. Not all media or solutions can be sterilized via an autoclave. Certain high-protein solutions such as urea, vaccines, and serum will denature in the extreme heat, and so they may have to be filter-sterilized without heat.
You will be making slant and broth media, but not plate media in this lab. If the agar solidifies in the tip of the pipet, dispose of the pipet in the pipet jar and get another one. To prevent this from happening, either pipet out all the tubes at the same time, or leave the pipet in the flask of melted agar.
Learning Objectives Understand how to make media, how to sterilize it, and how to distribute it in different formats. Understand the basics of an autoclave and how it sterilizes, including parameters. MATERIALS NEEDED per table 2 plastic weigh boats 1 test tube rack liter Erlenmeyer flask 1 pipet pump 1 graduated cylinder several nonsterile glass10 ml pipets 1 spatula 28 medium, nonsterile test tubes 1 jar agar powder 15 green caps 1 jar nutrient trypticase soy broth powder 15 yellow caps 1 magnetic stir bar 1 pipet disposal jar.
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