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.
Here, scientists worked in a flow hood to isolate potential pathogenic bacteria and viruses from water samples. These isolates were then cultured together with amoebae. Because amoebae normally eat or "phagocytose" bacteria, any bacteria that were able to resist amoebal digestion and remain in these organisms can also potentially remain viable in human cells and cause diseases.
Finally, sterile conditions permit detailed study of ecological mechanisms such as the formation of root nodules in legume plants - bacteria-filled organs that "fix" atmospheric nitrogen into ammonia, which is used by the plant for growth. Researchers here created "microcosms" for studying the nodulation process using notched Petri plates with plant growth medium, placed seedlings into them and inoculated the seedlings with nodule-forming rhizobial bacteria.
The aseptic environment of the flow hood prevents contamination of the cultures with other bacteria or fungi. You've just watched JoVE's video on aseptic techniques in environmental science.
You should now understand why aseptic working conditions are important; how to aseptically perform microbiological experiments; and some applications of aseptic techniques to environmental research.
As always, thanks for watching! The outcome of the procedure demonstrates proper aseptic technique and poor aseptic technique. Figure 7 illustrates the contamination that can arise from poor aseptic technique when pouring agarose plates top plate: sterile medium; bottom plates: contaminated media.
Figure 7: Contamination that can arise from poor aseptic technique when pouring agarose plates. Top plate: sterile medium; bottom plates: contaminated media. Proper use of aseptic technique is vital for environmental microbiologists when sampling in the field and in the laboratory when working with media, reagents, and cultured isolates.
Poor aseptic technique in the field can result in the transfer of microorganisms from the technician to critical environmental samples, as well as the cross-contamination of microbes from one sample to another. Such events are of importance, for example, in microbial ecology studies seeking to identify and compare bacterial and fungal populations that may be present in a given biome.
Contamination of such samples can result in a loss of data integrity. Aseptic technique is also critical for the maintenance of laboratory culture isolates originating from field sampling or from well-established microbial and cell culture repositories.
Environmental Microbiology. Aseptic Technique in Environmental Science. To learn more about our GDPR policies click here. If you want more info regarding data storage, please contact gdpr jove. Your access has now expired.
Provide feedback to your librarian. If you have any questions, please do not hesitate to reach out to our customer success team. Login processing This is a sample clip. Sign in or start your free trial. Previous Video Next Video. Overview Source: Laboratories of Dr. Charles Gerba - The University of Arizona Demonstrating Author: Luisa Ikner Aseptic technique is a fundamental skill widely practiced in the field of environmental microbiology that requires a balance of mindfulness and practice in the laboratory.
Log in or Start trial to access full content. Preparation for Aseptic Work Obtain and apply the following PPE items: lab coat, latex or nitrile gloves free from tears or holes , and safety goggles Figure 1.
For safety in the event of using an open flame, tie back long hair. Figure 1: PPE: A lab coat, latex gloves, and safety goggles. Prepare liquid broth medium e. For the broth medium, dissolve the powder on a hot plate with low heat applied, and dispense the liquid either in mL volumes into glass screw-top flasks, or in mL volumes into glass screw-top test tubes.
Using a magnetic stir bar, stir the agarose medium on the hot plate stirrer until the powder is fully dissolved. Note that the color of stripes on the autoclave tape should change from white pre-autoclave to black post-autoclave. Although the color change generally indicates that sterilization was successful, sterility checks using spore strip kits can be conducted to verify the autoclaving process. Figure 2: Autoclave tape being applied to material. Figure 3: Note the color change of stripes on autoclave tape from white pre-autoclave to black post-autoclave.
Once cooled, the media can be poured into sterile Petri dishes. Allow the medium to cool and solidify, then consolidate for storage under temperatures specified by the manufacturer. There are several varieties of culture media that cannot be autoclaved as the high temperatures degrade critical ingredients. Sterilizing these require filter-sterilization using a vacuum filtration system employing a 0. Prior to performing work on the benchtop, disinfect the surface using an appropriate solution e.
This lowers the risk of transferring contaminants from the working surface to cultures and sterile media. To establish a sterile field, turn on a Bunsen burner. When transferring a culture from a plate, cool the loop by touching on the very edge of agar. When transferring from a broth, the red-hot loop will make a sizzling noise as soon as you insert it into the culture.
The loop will automatically cool once it makes contact with the broth culture, but wait a one or two seconds before removing the loopful of inoculum from the tube. The hot loop may create aerosols when it touches the media containing microorganisms. It will cause some of the broth and bacteria to boil briefly, creating a bacteria-containing aerosol.
This airborne bacteria have the chances of entering into the respiratory tract or into the body parts. If you hear a hissing sound when you place the heat sterilized loop into the broth culture indicates that the loop is not cooled sufficiently. Flaming the Mouth of the Test Tube: Passing the mouth of a tube through the flame of a Bunsen burner creates a convection current which forces air out of the tube.
This prevents airborne contaminants from entering the tube. The heat of the Bunsen burner also causes the air around your work area to rise, reducing the chance of airborne microorganisms contaminating your cultures.
Agar Slants: Cultures are often transferred to agar slants, in addition to broth tubes and agar plates.
You will spend a lot of time in lab transferring organisms from one tube to another, or to slides or to plates. It is imperative that you do this quickly and safely. After you have practiced these procedures several times your instructor or IA will assess your proficiency. It is essential that you grasp these skills before you proceed to working with actual microorganisms. Special precautions must be taken to prevent the formation of aerosols when working with BSL2 organisms.
An incinerator sterilizes inoculating utensils much the same way as a Bunsen burner does except the risk of aerosol production is reduced. If you are using screw cap tubes, be sure to loosen them before you start this procedure. Once your instructor or IA has observed your aseptic technique using the practice materials, you can begin to work with real organisms.
The next step is learning proper aseptic technique for handling BSL 2 organisms. Have your two plates on your lab bench. You will be given a plate with streaked organisms on it. These are the real thing. Look closely at it and select an area that has individual colonies. They will look like small dots on your plate.
Each dot represents one or a few cells that multiplied to form a colony — also called a colony forming unit CFU. Use the same plate of bacteria you did for your plate-to-plate transfer. Find another well-isolated colony. Microbiology Resource Center. Introduction In the microbiology lab we use aseptic technique to: Prevent contamination of the specific microorganism we are working with.
Prevent contamination of the room and personnel with the microorganism we are working with.
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