Biology 481

Sterile method and sterile technique are very important concepts when working with microbes. We want to be confident that what we see growing on our plates and in our tubes and flasks are the organisms we placed there. Contamination costs time and money and may sometimes result in the devastating loss of irreplaceable material. Cutting too many corners can compound errors and increase the likelihood for contamination. Implementing sterile technique requires conscious thought about the process of contamination and the potential points at which contamination could possibly be introduced. Always Be Aware.

There are several steps that we take to ensure the media and media components that we prepare or handle remain contaminant free.

1. Wash your hands with a high level of frequency, wear gloves and always make sure your gloves have been recently rinsed with 70% EtOH before handling media components, pipets, glassware etc.
   1. When you have gloves on you should never touch anything you would touch without gloves on (with the exception of pens), whether you just put the gloves on or not. The gloves are not to protect you; they are to protect your experiment from becoming contaminated.
      1. Gloves on for: Glassware or plastic ware, pipets, pipet tip boxes, any media container, plates, etc.
      2. Gloves off for: your clothes, your hair, your bag, the computer, door handles, freezer door, ipad, phone, face or skin
   2. If you use a pen that has not been wiped down with 70% EtOH, your gloves are no longer sterile. If you touch a faucet handle, a drawer pull, etc., your gloves are no longer sterile.
2. Autoclave media and media components, and only use components you are sure were properly sterilized.
   1. We always autoclave media, but you should try to use your best sterile technique while preparing the media as well. Clean the bench with dH2O and then 70% EtOH before and after you make media.
   2. Examine the lid of the container and confirm that it has stayed in place during the autoclave process. If the lid is made of foil, is it intact? Does it cover enough of the neck of the container to prevent contaminants from entering? If a lid has popped off then it is no longer considered sterile.
   3. Once a container of medium has come out of the autoclave, the introduction of contaminants into the container becomes an issue. Avoid touching the autoclaved container near the neck or the lid with non-sterile gloves.
   4. Make sure autoclaved containers remain properly covered with aluminum foil or a lid at all times.
3. Do not speak when you have open plates or media containers in front of you (for instance while making SA tubes or pouring plates).

Good sterile technique is the first and most important step in ensuring consistent results when working with microbes. Sterile technique refers to procedures by which cultures may be manipulated without infecting the worker or contaminating the cultures or the laboratory environment.

Because contaminating microbes are ubiquitous and are found on fingertips, bench tops, etc., it is important to minimize contact with these contaminating surfaces. When students are working with the inoculation loops and agar plates, you should stress that the round circle at the end of the loop, the tip of the pipetter, and the surface of the agar plate should not be touched or placed onto contaminating surfaces.

The flaming of lips of tubes and flasks must ALWAYS be done whenever culture liquid is to be poured from a container (e.g., pouring plates). Flaming should be routinely done when caps are removed from tubes during transfer of cultures. The purpose of flaming is not to sterilize, but to warm the tube and create warm air convection currents up and away from the opening. This "umbrella" of warm, rising air will help to prevent the entrance of dust particles upon which contaminating bacteria reside.

Petri dish lids prevent dust from falling directly onto plates but allow diffusion of air around the edges. There are no direct air currents into the plate, and to enter, dust particles would have to rise vertically more than a centimeter. This does not often occur because of the density of the particles. Whenever the lid is removed, it should be held over the plate as a shield. Do not place the lid on the bench top. Do not leave plates uncovered. Do not walk around the room with an open plate.

When working with cultures in test tubes, work rapidly while maintaining sterile technique. Keep the tubes open a minimum amount of time. While the tubes are open, hold them at a 45 degree angle so that dust cannot fall into the open tube. Hold the tubes away from your face while transferring.

Test tubes are handled in the following manner:

• The test tube is held in the left hand (for a right-handed person).
• The instrument (loop, pipet, or needle) is held in the right hand.
• The test tube cap is grasped by the little finger of the right hand, and removed.
• While continuing to hold the cap with the little finger, the tube is lightly flamed and the instrument is manipulated appropriately, and withdrawn.
• The cap is replaced on the test tube and the test tube is put back into the rack.

Always clean all work areas (your bench, balance area, sink area, gel area, etc.) thoroughly before leaving the laboratory! The last step before leaving the lab is to wash your hands thoroughly.

These are guidelines. You may find a set of techniques that best suite your working style. This is fine as long as you adhere to the basic concepts of good sterile technique.

Adapted from Chazen Lab Sterile Technique at Vanderbbilt University