Throughout the semester, we will be working with potential
human pathogens. Although the actual risk of becoming infected with any of the
bacteria we will use in this course is very remote, we must be vigilant about
safety. Luckily, paying attention to a few common-sense rules will reduce the
chance of infection to almost zero and will reduce any fears other occupants
of this building or your roommates may have. So . . .
Absolutely no eating or drinking is allowed in the lab at any time.
Avoid sticking stuff (pens, fingers, etc.) in your mouth.
Wear gloves whenever you may come in contact with any type of microbe.
If your gloves become contaminated, remove them and put on a new pair (you would be surprised how quickly you can contaminate many things if your gloves are contaminated!).
Do not remove materials from the lab.
Report any spills to me immediately
All non-glass disposables (gloves, Petri plates, plastics, etc.) that may have come in contact with any microbe should be placed in an orange autoclave bag. This material will be autoclaved after each lab session.
Any glass disposables that may have come in contact with any microbe should be placed in a separate autoclave bag.
Any non-disposable items that have come in contact with any microbe should be placed in the appropriately marked container.
Wash your hands with soap after each lab session.
Think about what you are doing. Most accidents in the lab occur because people are rushing through a procedure, are preoccupied, or haven't spent time planning their experiment. A few minutes of planning before beginning a procedure usually saves you time in the end and makes accidents much less likely.
Background Reading: Chapter 3 (Salyers and Whitt)
Sterile (Aseptic) Technique
As you probably know, bacteria are everywhere - on your hands, in your mouth, in the air. And these bacteria would be very happy to wind up in our culture plates and tubes. Unfortunately, their presence would negatively impact our attempts to study the microbes we are interested in studying. How can we prevent these "environmental" microbes from entering our experimental system? And how can we limit the "escape" of our experimental microbes into the environment? The answer is sterile technique.
Sterile technique involves following a few simple rules:
Disinfect the work area before beginning
Sterilize all materials before beginning
Keep cultures closed whenever possible
Never lay caps or lids on the bench top
When removing a cap or lid, keep it over the open tube or plate
Avoid creating aerosols (which often result from bursting bubbles)
Keep tubes and plates away from your mouth.
Streaking for Isolated Colonies
Purposes: Become familiar with aseptic technique and obtain pure cultures of bacterial species
Tryptic soy agar
Sterile inoculation loops
A common microbiological technique for the isolation of pure bacterial cultures is the streak-plate technique. This technique allows one to easily separate individual bacterial cells on a solid medium. As we will see, this technique is essential for isolating clonal populations of bacteria and, through the observation of the resulting bacterial colonies, is an important component of bacterial identification. Basically, this technique involves streaking a loopful of bacteria across a solid medium, such that individual bacterial cells fall off the loop and are deposited on the medium. Each individual cell, then, will replicate in that location, giving rise to a colony of genetically identical cells - a clonal population. As you probably can imagine, this technique can be used for many purposes, from separating different species growing in a mixture to determining how many bacterial cells are present in a solution.
Generally, bacteria are obtained by sticking a sterile loop into the bacterial culture (either a liquid culture, a culture growing on a solid medium, or an 'environmental' culture) . This loop then is streaked repeatedly across about 1/3rd of the plate. The plate then is rotated and a new, sterile loop or needle is used to spread some bacteria from the primary region across another 1/3rd of the plate. Finally, this step is repeated for the final 1/3rd of the plate. The result is that many bacteria are deposited in the primary region, fewer bacteria are deposited in the secondary region, and even fewer bacteria are deposited in the final region. These plates then are incubated, allowing the individual cells to replicate and give rise to colonies. Ideally, the plate will contain many colonies in the primary region (perhaps even a confluent lawn of bacteria) and well-isolated, individual colonies in the final region (see Figure 3.3 in Salyers and Whitt).
In this exercise, we will gain experience in streaking for isolated colonies by streaking bacteria on tryptic soy agar plates. Each group will be given a tube of tryptic soy broth, containing an overnight growth of a mixture of bacterial species. Each person will plate the mixture on an agar plates. Make sure you label your plates. After the bacteria have been plated, place the plates upside down (why?) in the 37oC incubator. After a 24 hour incubation period, transfer the plates from the incubator to the refrigerator (why?). We will examine them next week.