Introduction to Microbiology, Sterile Technique and Micropipetting Background: Bacteria represent the most widely distributed and metabolically diverse group of organisms. Because bacteria are relatively simple organisms and are amenable to experimental manipulation, they have provided elegant systems for basic research. Similarly, yeast, which are unicellular eukaryotic organisms, are a good model system for the study of basic cellular processes of eukaryotic cells. Additionally, because a small culture of bacteria or yeast has millions of individual cells, the researcher can quickly screen for rare genetic mutations. Some of the ways that bacteria or yeast are used in research laboratories include the following: 1. simple biochemical models 2. hosts for recombinant DNA 3. as the actual object of study, for example bacterial pathogenesis 4. for biotechnology purposes, such as insulin production or control of oil spills Bacteria and yeast are typically grown on solid media (agar plates) or in liquid culture. Cells are deposited on a plate by streaking from a liquid culture, or by moving dividing cells from one plate to a new plate. The transfer of bacteria or yeast to a sterile substrate in this fashion is called inoculation. Single cells inoculated on a plate grow and divide, forming a mass of cells called a colony. All the bacteria or yeast in a colony are genetically identical, with the exception of rare spontaneous mutants. There are wide variations in growth rates for different strains and species of bacteria. Many strains can double the number of cells in a liquid culture in less than 3 minutes under optimal conditions. On solid media, a single bacterial cell will form a visible colony 1-24 hours after inoculation. Saccharomyces cerevisiae (Baker s yeast) is commonly used in research laboratories around the world, and it has a generation time of 2 hours when grown at 3 º C. STERILE TECHNIQUE: In many laboratory exercises, sterile technique (which will be demonstrated in lab) is absolutely necessary. Sterile technique is a method of careful lab manipulations that prevents foreign cells (from the air or from you) from getting into your plates and cultures and prevents the bacteria in your exercises from escaping into the environment. This requires common sense and a bit of practice. In general, the following manipulations should be used when working with bacterial or yeast cultures (or for preparing samples for PCR): 1. The key to successful sterile technique is to work quickly and efficiently. Before beginning, clear off and disinfect the lab bench with 7% ethanol and arrange the tubes, pipet tips, and culture media within easy reach. Locate the Bunsen burner in a central position on the lab bench to avoid reaching over the flame. The barrel of your micropipettors should be cleaned using 7% ethanol and a Kimwipe. 2. Many of the materials you will use, such as solutions, pipette tips and microcentrifuge tubes, are already sterilized. Be sure these items remain sterile by covering or closing them immediately after use (i.e., close pipette tip boxes, put lids back on jars of solutions, etc.). 1
3. Never touch anything sterile with your fingers or clothes, and avoid directly breathing onto sterile items. Be aware especially of pipette tips once they are on your micropipettor - if they contact you or the bench as you perform manipulations, they are no longer sterile! 4. Wire loops used for transfer of bacteria must be flamed until they are red hot before and after each manipulation. Let the loop cool before it actually contacts the cultures you are working with to insure that you do not kill the cells that you wish to transfer. Alternatively, sterile plastic loops may be used once (without flaming!) and then disposed of. 5. When working with agar plates, minimize the time that plates are left uncovered. Work with the plate close to the base of a lit Bunsen burner when removing the lid (see diagram on right). The rising heat creates a convection current, which prevents microorganisms in the air from dropping onto the open plate. 5. Any pipette tips or materials that are contaminated with bacteria should be placed in Bio-Hazard bags or in the disinfectant jars on the bench tops. HEAT 6. Wipe down the bench with disinfectant when finished. Re-clean the barrel of your micropipettors with 7% ethanol and a Kimwipe. Wash your hands. 2
Exercise 3: Use of Micropipettors (adapted from DNA Science pp. 325-327) Micropipettors are instruments used to accurately transfer small volumes (1 µl to 1 ml) of solution. Because of their accuracy, ease of use, and convenience in sterile techniques they are a practically universal lab tool. In this week s lab you will learn how to properly use this instrument. In the rack at your lab bench, you will find three micropipettors; each one is appropriate for a specific volume range. If you look at the dot on the plunger of each micropipettor you will see a number that represents the maximum volume, in microliters (µl), that can be transferred by that micropipettor. The minimum volume appropriate for each micropipettor is typically ten percent of the maximum. The dot on the plunger is also color-coded and generally matches the color of the disposable tips used with that micropipettor. The table below shows the volume range, expected accuracy, and the appropriate tips for the micropipettors that you will be using. Micropipettor Volume Range (µl) Accuracy Disposable Tip P1 1-1 ± 1. µl Blue P2 2-2 ± 1. µl Yellow P2 1-2 ±.5 µl Yellow 3
In most experiments, accuracy is important when transferring small volumes of liquid with a micropipettor. A researcher needs to be sure that he or she is transferring the volume desired with a reasonable degree of accuracy. The researcher can be confident of this provided two conditions are satisfied. First, the micropipettor has been calibrated and tested for accuracy (this is usually done on a yearly or semi-yearly basis). Second, the researcher must be using the micropipettor properly. Proper use of the Micropipettor Use the text below and the illustration on the previous page to become familiar with the parts of a micropipettor: Barrel - the working end of the micropipettor; a disposable tip is seated on the lower end of the barrel before each use Plunger - the plunger is pressed and released to withdraw and expel liquid Tip ejector button - is used to remove a tip from the barrel without direct handling of the disposable tip Digital volume setting - displays the volume the micropipettor is currently set to deliver Volume adjustment knob - is rotated to change the digital volume setting Part 1: Familiarization with the micropipettor Materials: set of micropipettors Pick up a P2 and set the digital volume setting for 5 µl (reading down, the setting should be 5 ). Push the plunger down and notice that at some point the plunger becomes more resistant and requires more effort to push further. This point is called the first stop. Notice that the plunger can be pushed well beyond the first stop until it reaches the second stop. Be sure you can feel the first stop point and notice how far the plunger travels before reaching this point. Reset the micropipettor to 2 µl ( 2 ). Again push the plunger to the first stop. You should notice now that the plunger travels further to reach the first stop than it did when the micropipettor was set for 5 µl. Examine the P2 and P1 setting windows and plunger tension at various volumes as well. The table below shows how the digital volume display looks for each micropipettor when it is set at its maximum volume. Micropipettor P1 P2 P2 Maximum Setting 1 2 2 4
To transfer solution using a micropipettor: 1. Set the digital volume setting to the desired volume by rotating the volume adjustment knob. Note: the digital volume setting should never be adjusted above the maximum volume specified for a particular micropipettor. Remember that the maximum volume is the largest volume shown on the plunger. 2. Seat a disposable tip on the micropipettor by firmly placing the end of the barrel into a tip. 3. Depress the plunger to the first stop and immerse the end of the tip into the solution to be transferred. SLOWLY release the pressure of your thumb on the plunger to SMOOTHLY draw the solution up into the tip. 4. To expel the solution, put the tip into the next tube (or just above a sheet of paper). Then depress the plunger ALL THE WAY TO THE SECOND STOP. 5. If expelling into a liquid solution, REMOVE the end of the tip from the solution BEFORE releasing the pressure on the plunger with your thumb. 6. You should generally use a new tip for each transfer (or whenever the tip becomes clogged, if you are repeat pipetting from the same, non-sterile solution). When you wish to eject the tip you are using, place the tip over the appropriate waste container (small plastic beaker) and press the tip ejector button. If the tip is difficult to eject it is likely that you are jamming the tips onto the micropipettor harder than necessary. Part 2: Transferring small volumes with a micropipettor Materials: Piece of blotting paper 2X Loading dye P2 and rack of yellow tips Obtain a 4 x 5 inch piece of blotting paper and a microcentrifuge tube containing 2X loading dye. Following the protocol above, use a P2 to spot the following volumes of dye directly onto the paper in a linear order: 1 µl, 3 µl, 5 µl, 7 µl, 1 µl, 15 µl, and 2 µl. Spot each volume twice to check your consistency. Compare the spots you made with those of the instructor. If the spots do not look like those of the instructor, try to determine the cause. 5
Part 3: Transferring large volumes with a micropipettor (adapted from DNA Science page 328) Materials: 2 x 1.5 ml microcentrifuge tubes Solutions I-IV P1 and rack of blue tips This exercise simulates parts of a bacterial transformation or plasmid preparation for which a 1-1 µl micropipettor is used. It is far easier to mismeasure when using a large-volume micropipettor. If the plunger is not released slowly, an air bubble may form or solution may be drawn into the piston. 1. Use a permanent marker to label two 1.5 ml reaction tubes E and F. 2. Use matrix below as a checklist while adding solutions to each reaction tube. Tube Solution 1 Solution II Solution III Solution IV E 1 µl 2 µl 15 µl 55 µl F 15 µl 25 µl 35 µl 25 µl 3. Set micropipettor to add appropriate volumes of Solutions I-IV to tubes E and F. Follow the same procedure as for small-volume pipettor. 4. A total of 1 µl of reactants was added to each tube. To check that your measurements were accurate, set the micropipettor to 1 µl and carefully withdraw solution from each tube. a. Is the tip just filled? or b. Is a small volume of fluid left in the tube? or c. After extracting all fluid, is an air space left in tip's end? (The air can be displaced and actual volume determined simply by rotating the volume adjustment to push the fluid to the very end of the tip. Then, read the volume directly.) 5. If the measurements were inaccurate, repeat the exercise to obtain a nearly perfect result. 6