1.4: Aseptic Technique
- Define aseptic technique, sterilization, and contamination.
- Discuss the importance of growth media and keeping it sterile.
- Recognize potential sources of contamination.
- Complete the Aseptic Technique Procedure.
Aseptic Technique and Sterile Environments
The goal of microbiology is to generate and study pure cultures : growth media containing a single microbial species. Pure cultures provide objective, unbiased data that can be used in clinical analysis and research. If unwanted growth of other microbial species is present in a culture, the sample is contaminated. To prevent sample contamination, maintaining a sterile environment through aseptic technique is vital. Sterile environments are free from all unwanted microbes including exogenous fungi, yeasts, endospores, vegetative cells, and viruses. (OpenStax, 2024) Sterilization can be accomplished through chemicals, like 10% bleach solutions, and physical means, like exposur e to high heat and pressure in an autoclave. All steps and procedures that help prevent contamination are a part of aseptic technique, "aseptic" meaning "without contamination". Routine disinfection, media sterilization, equipment sterilization, and proper transfer of samples/microbes are all key aseptic techniques that you will complete in this lab course to maintain a sterile working environment.
Routine Disinfection
Disinfection uses antimicrobial chemicals to kill microbes on surfaces. These chemicals should be fast acting, easy to prepare and use, and shelf stable. (OpenStax, 2024) Natural disinf ectants include vinegar whose high acidity is capable of inactivating most microbes. In this microbiology lab, we will use the chemical disinfectant chlorine bleach. 10% bleach soluti ons will be used to clean your work area (lab bench) daily, before and after experiments. Undiluted Bleach will be used to clean up any spills as described in "1.1 Laboratory Safety". We will also use mild antimicrobial soap to wash hands during the lab. While disinfection does not completely sterilize a work area or hands, some microbes will always remain, it is still a key component of aseptic technique.
Growth Media
Microbes are grown on or in substances composed of nutrients needed for cellular metabolism. These substances are called media or growth media. Growth media can be liquid, solid, or semi-solid and is ma de with different carbon sources, sugar sources, and any other nutrient needed to support microbial growth. Liquid media is also called "broth". Broth cultures can be used to observe metabolic pathways in bacteria, such as urease production, and are a good tool for growing large amounts of a microbe quickly. Both semi-solid and solid media are made using a polysaccharide, called agar, in different amounts to create a solid substance. Agar is naturally solid at room temperature, contains no nutrients, and is not digestible by microbes (specifically bacteria) making it an ideal solidification agent. (Hartline, 2023) Semi-solid agar can be used to test for microbial motility. The semi-solid state of the media allows the microbes to penetrate the media and move out from the area of inoculation, serving as a quick method to determine if motility structures are present. Finally, solid media has the greatest variety of experimental usage. As seen in Figure 1, solid agar can be used to make slants, agar deeps, and petri plates. Typically, slants are used for making stock cultures, agar deeps for metabolism experiments, and plates for observing colony morphologies. Media in test tubes should always be covered with caps to prevent contamination and kept in test tube racks. Petri dishes are kept inverted, see Figure 2. Petri dishes are kept in incubators with the lid-side down and agar-side up to prevent any dirt, dust, condensation, or foreign microbes from falling onto the agar during incubation.
Figure 1. Media can be liquid (broth), semi-solid, or solid depending on the needs of an experiment.
Figure 2. Petri dishes should be inverted in incubators to decrease the chance of contamination.
Microbial Transfer Procedures and Media Inoculation
Growth media needs to be sterile prior to the experiment and remain uncontaminated during lab use. Growth media is sterilized using physical means, specifcally, an autoclave. Autoclaves use high heat (steam) and pressure to inactivate or terminate microbes including vegetative cells and spores. The autoclave functions with a pressure of 15-20lbs/ in 2 and reaches temperatures between 121 o C and 132 o C. Media and other BSL1 liquids, used petri dishes, and used perishable items are autoclaved for 15 to 20 minutes depending on the volume of material present. (OpenStax, 2024 and Hartline, 2023) To keep growth media uncontaminated during an experiment, specific transfer procedures are used to minimize microbial transmission. A key step in microbial transfer is equipment sterilization. Sterile swabs, inoculation loops, and inoculation needles can be used to move microbes between different medias. Inoculation loops and needles must be sterilized before and after direct contact with a microbe. Either a Bunsen burner or table-top "mini" incinerator should be used to sterilize loops and needles. The wire must be kept in the Bunsen burner flame or incinerator body for at least 10 seconds until the wire has turned red/orange. Never leave the wire unattended in either heat source, the wire will become hot and may cause burns or melt. The inoculation loop or needle should be allowed to cool for 10 to 15 seconds before picking up any microbe. If the wire is still hot, it can burn, damage, or kill the microbe being worked with. Once a sterile swab, loop, or needle is ready to use, microbes should be moved/transferred in the following order:
- Stock Broth to Sample Broth Tube
- Sample Broth Tube to Stock Plate (Petri Dish)
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Stock Plate to Sample Plate
- In this lab, you will be provided with pre-made stock plates to use for sample plate preparation.
The process of transferring microbes to d ifferent medias and spreading them across a media's surface is called inoculation. Before transferring and inoculating microbes, test tubes and petri dishes must be properly labeled. Test tubes should be labeled using labeling tape and include: student initials, date, course section, and sample type. Petri dishes can be written on directly using a sharpie. To label a petri dish, write in small letters around the edge of the bottom plate and include: student initials, date, course section, and sample type. Do not write on the lid, the lid will not be visible in the incubator.
Figure 3. Example for how to label the bottom of a petri-dish. Write in small letters around the edge of the bottom dish. Never write on the lid of a petri dish. The lid my be damaged, lost, or not visible in an incubator.
Once everything is properly labeled, to collect microbes from a stock plate:
- Place the plate lid-side up and agar-side down on the work bench.
- Using a non-dominant hand, lift the lid slightly. Never take the lid fully off, always keep it partially covering the plate to shield the agar.
- Touch a sterile swab, loop, or needle to an area with no microbial growth to test that the metal has cooled down.
- Gently, touch part of a microbial colony. A complete colony is not needed, never swipe a large mass of cells. There are thousands of cells in a single colony. A little bacteria goes a long way!
- Be careful to not press too hard on the agar, it can break.
- After microbes have been collected, replace the lid on the stock plate and prepare to transfer the microbes to a secondary media: broths, slants, deeps, or sample plates.
Figure 4. This image shows the proper technique for opening petri dishes. Always keep the lid over the exposed media to prevent contamination.
To inoculate a sample plate:
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In this lab we will complete quad streak inoculations. A more in-depth lesson on completing a quad streak can be found in "2.1: Introduction to Streak Plate Techniques". This chapter will present a brief description of proper microbial streaking.
- We will also discuss slant, semi-solid, and deep agar inoculations in "Biochemical Characterization Part I and II".
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With a non-dominant hand, lift the lid slightly on the sample plate.
- Do Not sterilize the wire at this point, this will kill the microbes and there will be no bacteria to actually transfer.
- With the swab, loop, or needle containing microbes swipe the bacteria in a zig-zag pattern across one region/quadrant of the plate.
- Now, sterilize the wire and let it cool. When the wire or swab is ready, drag a small portion of bacteria from the first quadrant and swipe in a new zig-zag pattern in a new secondary quadrant.
- Repeat this process until four quadrants of bacteria have been created on one plate.
Inoculated petri dishes should be wrapped in parafilm. Parafilm keeps samples uncontaminated while also protecting the user from direct transfer contamination. Potential sources of contamination include (CDC, 2016):
- People: students, instructors, and anyone else who may enter the lab space.
- Surfaces: tables, counter tops, personal items like bags or books, phones and other electronics, sinks and faucets.
- Equipment: incinerators, inoculation loops or needles, and microscopes.
- Foreign microbes, dust, dirt, and other biological materials.
Sample plates should always be stored in an incubator, a temperature controlled growth environment, to promote growth of the microbe. In this lab, our incubators are set to 37°C as we are working with mesophiles, microbes that prefer to grow in or on human hosts. After 24 to 48 hours, microbial growth should be visible on or in all media types and ready to use for follow-up experiments.
Medical Asepsis
Aseptic technique in a healthcare setting, between patient and healthcare worker, uses a strict set of hygiene practices known as medical asepsis. Healthcare workers closely monitor patient hygiene and mobility to determine what intervention is necessary to treat infections, prevent new infections, and promote patient health. Patient health and hygiene in medical asepsis pays close attention to the skin, hair, nails, oral cavity, and perineum. (Openstax, 2024). These cells and tissues can easily become infected or transmit diseases if aseptic techniques are not used correctly. Here, we will focus on the skin.
A patient’s skin is their largest organ and first defense against external pathogens. Normal, healthy skin will help a patient regulate their body temperature, produce sebum to stop pathogens, and prevent water lose. The skin can become weakened and damaged in healthcare settings. Long-term immobility or even medications like anticoagulants can affect skin health. Changes to skin health put the patient at risk for developing infections like Scabies.
Scabies is a contagious skin infection caused by the Sarcoptes scabiei mite. Scabies outbreaks are common in nursing facilities, childcare facilities, and detention facilities. (CDC, 2023). The mite targets a patient’s weakened skin barrier and burrows into the upper skin layer. It causes intense itching and pain and appears as a red pimple-like rash. Scabies will be found most often in skin folds near the armpits or knees and other locations where patients may have difficulty cleaning themselves. The mite is spread by direct contact and can spread from patient to healthcare worker if proper aseptic technique is not followed. Washing clothing and bedding, wearing proper PPE, and limited physical contact will decrease the risk of transmission. Scabies can be prevented with regular cleaning, exfoliation, and moisturizing of the patient’s skin.
Attributions and Text Citations
"Microbiology Laboratory Manual: Labs, 1.7 Aseptic Technique" by Dr. Rosanna Hartline , LibreTexts: Biology , West Hills College Lemoore is licensed under CC BY-NC 4.0
"Microbiology Textbook: Chapter 9, Microbial Growth" by OpenStax , Digital ISBN 13: 978-1-947172-23-4 is licensed under CC BY 4.0
"Microbiology Textbook: Chapter 13, Control of Microbial Growth" by OpenStax , Digital ISBN 13: 978-1-947172-23-4 is licensed under CC BY 4.0
"Microbiology Labs 1: 2 , Aseptic Transfers " by Jackie Reynolds, LibreText Biology , Dallas College, Richland Campus is licensed under CC BY-NC 4.0
"Microbiology for Allied Health Students: Lab Manual, 3.5 Motility Agar" by Molly Smith and Sara Shelby, LibreText Biology , South Georgia State College via GALILEO Open Learning Materials is licensed under CC BY 4.0
"How Infections Spread" by Centers for Diseases Control and Prevention , Use of CDC Materials Statement is in the Public Domain
"Clinical Nursing Skills Textbook: Chapter 7, Hygiene" by Openstax , Digital ISBN: 13: 978-1-961584-40-2 is licensed under CC BY 4.0
"Venous Thromboembolism (Blood Clots): Testing and Diagnosis" by Centers for Disease Control and Prevention , Use of CDC Materials Statement is in the Public Domain
"Scabies: About Scabies" by Centers for Disease Control and Prevention , Use of CDC Materials Statement is in the Public Domain
"Scabies: Public Health Strategies for Scabies Outbreaks in Institutional Settings" by Centers for Disease Control and Prevention , Use of CDC Materials Statement is in the Public Domain
Image Citations
Figure 1, "Microbiology Laboratory Manual: Labs, 1.7 Aseptic Technique" by Dr. Rosanna Hartline , LibreTexts: Biology , West Hills College Lemoore is licensed under CC BY-NC 4.0
Figure 2, Modified From:
- "Petri Dish or Culture Plate containing agar medium used for stool culture.jpg" by Frankincense Diala, Wikimedia Commons is in the Public Domain, CC0
Figure 3, Modified From:
- "Bio 225 OER Images" by Madison Rost, Associate Professor , HGTC and are in the Public Domain
- "File:Streptomyces mirabilis NRRL B-2400 (Type Strain).jpg" by Agricultural Research Service (NRRL)Culture Collection, Wikimedia Commons is in the Public Domain
Figure 4, "Microbiology Laboratory Manual: Labs, 1.7 Aseptic Technique" by Dr. Rosanna Hartline , LibreTexts: Biology , West Hills College Lemoore is licensed under CC BY-NC 4.0
Video 1, "HOW TO: Streak Bacteria " by jeanhuanglab , YouTube, Creative Commons Attribution license (reuse allowed) is licensed under CC BY 4.0