Lab 2: Aseptic Technique
Some of the organisms we use in this lab are considered to be Bio Safety Level Two (BS2). BS2 microbes are potentially pathogenic (cause disease) if a person is inoculated with a large dose or if the person is immuno-compromised. They require autoclaving (steam heat sterilization) and have to be handled by aseptic technique (we will discuss that later today). BS3 microbes have to be handled with safety equipment under the hood. BS4 microbes require a space suit type of gear. This room has negative pressure, so when you open a door, air moves into the room, to prevent airborne pathogens from escaping. All the air in this room is circulated about every eight minutes. We need to eliminate contamination within the lab as well. We need to grow and study these organisms without contaminating anything.
GROWTH MEDIUMS
Nutrients are added to agar (a seaweed product) and heated to a liquid, and sterilized. The sterile liquid agar can be poured into a Petri dish and cooled. The Petri dish is then referred to as a “plate.” Sometimes liquid agar is placed in a tube which is tilted 45° while cooling. This is called a slant. Sometimes the nutrient medium is a broth which does not solidify. Tubes with this medium are called nutrient broth tubes. All of these types of mediums are made for the purpose of being inoculated, which means having a tiny amount of a bacterial culture streaked across the surface. After inoculation, the plate or tube is usually incubated for at least 24 hours to encourage growth of the sample.
Broths and Slants
Broths are the same nutrients as NA (nutrient agar), but without the agar. They do not have to be boiled, just stirred. They are placed in tubes instead of plates and autoclaved as usual.
Slants are made exactly as NA, except they are poured into tubes instead of plates. After they are removed from the autoclave, they are placed on slant racks to cool so the agar in the tube stays at a slant.
Why use a slant?
We can inoculate just the top of a slant to get growth of aerobic bacteria, or we can stab a needle of bacteria into the tube to see if there are any microbes that can grow without air (anaerobic). Also, Petri dishes will only keep fresh for 3 weeks and then they dry out. Tubes will last for a long time in the refrigerator, and are useful for making stock cultures (pure cultures).
CULTURE MEDIA CLASSIFICATION
1. By Consistency
2. By Contents
- All-purpose
- Selective
- Enriched
Consistency refers to a liquid, solid, or semi-solid. The use of a semi-solid allows us to do two things: see if the microbe is motile (can move), and to see if the microbe is aerobic or anaerobic.
All Purpose media
Nutrient agar (NA) does not support fastidious organisms (those that are hard to grow because they need special nutrients), so NA is a good all-purpose media. It will support the growth of a wide variety of organisms. It is also inexpensive.
Selective media
This type of media selects for a particular type of organism to grow.
An example is Sabouraud’s Dextrose Agar (SDA), which has a high sugar content and an acidic pH. SDA isolates molds and yeasts, which do well in high sugar content, whereas bacteria are inhibited. Therefore, sugar acts as a bacterial preservative; that’s why jams, jellies, and preserves don’t get bacteria growth. However, molds are aerobic and they like sugar. They can get into your jelly jar when their microscopic air-borne spores drift in whenever the jelly jar is opened. If you see mold in your jelly, toss out the whole jar because some molds produce cancer-causing toxins! Molds and yeast also grow well in an acidic environment.
SDA was originally designed to isolate molds of skin, nails, and hair, called dermatophytes, which are opportunistic pathogens (organisms that only cause disease if the skin in broken, or the person is diabetic or otherwise ill). Dermatophytes produce ringworm, especially athlete’s foot. Yeasts are known for causing infections in women, diabetic and cancer patients. Since people with diabetes have a high sugar content, they are susceptible to such infections. Scrape off a little skin and place in SDA to isolate the microbe. SDA has 40 grams of glucose and a pH of 5.6.
Enriched Media
This type of media has added nutrients such as vitamins and amino acids. It is used to grow bacteria which are hard to grow, known as FASTIDIOUS organisms. Many pathogens are fastidious. An example of enriched media is Blood Agar, which has Trypic Soy Agar (made from soy) and 5% sheep’s blood (defibrinated to keep it from clotting).
BACTERIAL CULTURES
We may get the bacterial inoculate from a stock culture of a pure organism. We might use an unknown culture of organisms that you need to grow and run tests on for identification. In either case, you need to learn to transfer the bacteria from the culture to the sterile medium. The bacterial inoculate can be transferred using an inoculation loop (a wire with a small loop at one end and a handle at the other) or an inoculation needle (wire on a handle, but without a loop on the end). Since these tools are made of metal, they can be used and then re-sterilized in a flame repeatedly.
CONTAMINANTS
The media on which you want to grow your new culture may also grow undesirable contaminants, especially molds and other types of fungus, and bacteria from your skin and hair. It is therefore essential that you protect your cultures from contamination from airborne spores and living microorganisms, surface contaminants that may be on your instruments, and from skin contact. Bacteria and other contaminants cannot fly. Nearly all forms of contamination are carried on microscopic dust particles that make their way onto sterile surfaces when they are carelessly handled. Therefore, we must maintain the rules of aseptic technique, which decrease the chance of contamination. Aseptic technique is not as pure as sterile technique. It means that we are using caution to prevent infection or contamination, but it is not as strict as sterile technique. When a nurse or doctor cleans an infected wound, the site is considered contaminated already (since it is infected), so aseptic technique is used. Clinically, using aseptic technique means you don’t have to scrub the patient’s skin with Betadine (antimicrobial scrub) or use sterile gauze. You also don’t have to wear a gown or face mask. However, if the patient is having a sterile surgery, sterile technique is used.
In lab, we are not dealing with wounds, just the organisms that infect them. Using the phrase “aseptic technique” now refers to being able to safely transfer organisms from one location to another, without spreading the organism to other locations, and without contaminating a pure culture that we are trying to grow.
ASEPTIC TECHNIQUE FOR TRANSFERRING BACTERIA
General preparations
· Never leave a culture dish open, even for a short time when viewing colonies of organisms. During your procedure, keep the lid close to the dish, open it only as far and as long as is necessary to accomplish the procedure, and keep the lid between your face (and your germs!) and the agar surface.
· For most bacterial cultures you will use a sterile loop or needle to inoculate or to obtain an inoculum. Never reach for a loop or needle to grasp the metal…it might be scalding hot from a previous student. Always grab the instrument by the handle.
· Prepare the equipment: Place the Bunsen burner in front of you and assemble all necessary equipment with in arms reach. Position everything so that you will not burn yourself while trying to inoculate your tubes.
· Label the tube or plate to be inoculated with the date, the test type, your lab period, and your name. If it is a tube, place it in a rack in front of you.
· Prepare the Bunsen burner by adjusting the flame so the yellow flame disappears and just the blue flame is visible. Then decrease the blue flame until a blue cone of flame is seen near the tip of the Bunsen burner. Be careful…this flame is hard to see, so never reach over a Bunsen burner, since you can never be sure whether it is on or not.
· Flame a loop or needle by holding it angled downward into the blue part of the flame. Start the flame at the loop. When the lower 1/3 of the instrument glows red, push the instrument further into the flame so the middle 1/3 is in the flame. When that glows red, push it in further so the upper 1/3 of the instrument can be flamed. It is now sterile. Don’t let any part of the metal touch any surface until you are ready to obtain your scoop of bacteria (called the inoculum). Also, don’t flame a loop that is wet, or it will spatter, scattering bacteria with it!
· Let the instrument cool by holding it for 30 seconds. You can make sure it is cool by touching it to the agar or liquid surface in your culture before you obtain your culture. If the instrument is too hot, it will kill the culture on contact. While waiting for the loop to cool, do not wave it around to hasten cooling, and certainly don’t blow on it. Either action could introduce bacterial contamination.
· When the loop or needle is cool (30 seconds), pick up the tube that contains the culture that you want to transfer, and hold it by the base in your NON-dominant hand. Your sterile loop is in your dominant hand. Take off the top of the culture tube with your dominant hand, between your pinky and ring finger….this takes a while to develop the skill. Never place a lid on another surface. You must hold the lid in your hand during the transfer. Make sure the open side of the lid is facing downward, so no contaminants can float down into it. The lids and the sterile loops should be in your dominant hand, and the culture tube is in your non-dominant hand.
· Flame the tube: Pass the neck of the glass culture tube through a flame (two quick, back-and-forth strokes), with the container at about a 45° angle toward the flame. Use the blue part of the flame. This tube should be in your non-dom. hand.
· The sterile, cooled loop or needle is in your dominant hand. Stick it into the pure culture tube, obtain your culture inoculum, then withdraw the loop or needle.
· Flame the tube again, then place the lid back on. In the meantime, the pure culture inoculum is on the loop, being held by your right hand…be aware of it at all times so you don’t accidentally touch it to your shirt or any other surface, or you will have to flame it and start over.
Obtaining the inoculum
· If you are obtaining the inoculum from a tube, the tube may contain solid agar or nutrient broth. In either case, you usually use a loop instead of a needle. Hold the instrument like a pencil in your dominant hand. If it is a broth, shake the tube a little, then just dip the loop into the broth and the liquid will spread out across the loop. If it is solid agar, gently scrape the top of the loop across the surface of the culture in the tube. Be careful not to dig into the agar. If the agar in the tube was solidified in a slanted position, keep the loop parallel to the agar surface to prevent digging into the medium.
o To obtain a large inoculum from a broth culture, you need to use a sterile disposable pipettes, so read your procedure carefully to see when you need to use this technique.
· If you are obtaining inoculum from a Petri dish with a loop, scrape the top of the loop across the surface of the culture, being careful to keep the loop parallel to the agar surface to prevent digging into the medium. Remember, don’t take the lid all the way off the Petri dish, just lift it a little to obtain the sample.
· If you are obtaining inoculum from a Petri dish with a needle, just touch the tip of the needle to a single colony, being careful not to dig into the medium. Don’t pick up more than one colony. This technique is used when the Petri dish contains multiple types of bacteria and you want to subculture one pure colony to run tests on it for identification.
Performing the inoculation
· If you are inoculating a Petri dish, pick up the lid with your non-dominant hand, and only raise it a little, keeping the lid face down and directly above the Petri dish. The inoculum on the loop should still be in your dominant hand. Streak the plate with the loop (or needle), being careful not to touch the outer surface of the Petri dish. Since both hands are occupied, the Petri dish base must be left on the table.
· Replace the Petri dish lid, then re-flame the loop or needle after performing the procedure, putting down safely without burning the bench, you, or another student.
o To inoculate a Petri dish, there are several techniques (streak, streak for isolation, etc) which will be described later.
· If you are inoculating a tube instead of a Petri dish, you must keep the tube in the rack (since your dominant hand is occupied), take the lid off with the two little fingers of your non-dominant hand, then pick up the tube also in your non-dominant hand, flame the glass neck, then inoculate, flame the neck again, set the tube down in the rack, then replace the lid. Then you can re-flame the loop or needle.
· There are so many things to keep track of while doing this! Pay the most attention to the culture until the sterile Petri dish (or sterile tube) is exposed, then pay the most attention to keeping the inoculated medium free from contamination until the lid is closed, then pay attention to sterilizing the loop carefully.