Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles
Sanders, E. R. Aseptic Laboratory Techniques: Volume Transfers with Serological Pipettes and Micropipettors. J. Vis. Exp. (63), e2754, doi:10.3791/2754 (2012).
Microorganisms are everywhere - in the air, soil, and human body as well as on inanimate surfaces like laboratory benches and computer keyboards. The ubiquity of microbes creates a copious supply of potential contaminants in a laboratory. To ensure experimental success, the number of contaminants on equipment and work surfaces must be minimized. Common among many experiments in microbiology are techniques involving the measurement and transfer of cultures containing bacterial cells or viral particles. To do so without contacting non-sterile surfaces or contaminating sterile media requires (1) preparing a sterile workspace, (2) precisely setting and accurately reading instruments for aseptic transfer of liquids, and (3) properly manipulating instruments, cultures flasks, bottles and tubes within a sterile field. Learning these procedures calls for training and practice. At first, actions should be slow, deliberate, and controlled with the goal being for aseptic technique to become second nature when working at the bench. Here we present the steps for measuring volumes using serological pipettes and micropipettors within a sterile field created by a Bunsen burner. Volumes range from microliters (μl) to milliliters (ml) depending on the instrument used. Liquids commonly transferred include sterile broth or chemical solutions as well as bacterial cultures and phage stocks. By following these procedures, students should be able to:
1. Prepare a Sterile Workspace
2. Transferring Liquids Using Serological Pipettes
3. Transferring Liquids Using Micropipettors
The volumeter shows three numbers. Depending on the micropipettor, the numbers are interpreted differently. Note that each micropipettor is only as accurate as the smallest graduation mark.
P2: For volumes between 0.2-2.0 μl. The top number denotes volume in microliters. The second number indicates tenths of a microliter (0.1 μl), and the third number represents hundredths of a microliter (0.01 μl). Each graduation mark equals an increment of two one-thousandths (0.002 μl) of a microliter.
P10: For volumes between 1.0-10.0 μl. The top number is for tens of microliters; this usually is set at "0" and should only be set at "1" with the other two numbers set at "0" when dispensing 10.0 μl. The middle number denotes volume in microliters. The third number indicates tenths of a microliter (0.1 μl). Each graduation mark equals an increment of two one-hundredths (0.02 μl) of a microliter.
P20: For volumes between 2.0-20.0 μl. The top number in black is for tens of microliters; this should only be set at "2" with the other two numbers set at "0" when dispensing 20.0 μl. The second number in black denotes the volume in microliters. The third number in red indicates tenths of a microliter (0.1 μl). Each graduation mark equals an increment of two one-hundredths (0.02 μl) of a microliter.
P200: For volumes between 20.0-200 μl. The top number is for hundreds of microliters; this should only be set at "2" with the other two numbers set at "0" when dispensing 200 μl. The middle number indicates the dispensed volume in tens of microliters, and the third number denotes volume in microliters. Each graduation mark equals an increment of two one-tenths (0.2 μl) of a microliter.
P1000: For volumes between 200-1000 μl. The top number is for thousands of microliters; this usually is set at "0" and should only be set at "1" with the other two numbers set at "0" when dispensing 1000 μl. The middle number is for hundreds of microliters. The bottom number is for tens of microliters. Each graduation mark equals an increment of two (2 μl) microliters.
4. Cleaning Up The Work Space
5. Representative Results
A sample application for using serological pipettes to transfer liquids is shown in Figure 7. These pipettes often are used in the microbiology laboratory to prepare media for inoculation with bacterial cultures. For example, sterile flasks first are filled with a specified volume of culture broth, in this case Luria Broth (LB), then a small number of cells (such as E. coli) are added to the media. Using a serological pipette, first the broth must be aseptically transferred from the media bottle to the flask. In this case, 25 ml of LB was added to a 125 ml sterile flask using a 25 ml serological pipette. Next, the broth must be inoculated with E. coli cells. Here, 10 μl of cells were transferred aseptically using a P20 micropipettor from a previously growing culture flask to the 25 ml of fresh LB. The flask is incubated in a growth chamber for a particular amount of time, allowing the cells to replicate (for this example, the E. coli cells were incubated overnight at 37 °C on a shaking platform). The result is a turbid bacterial cell culture that can be used for subsequent experiments.
Serological pipettes also may be used to transfer media originally supplied in a bottle to test tubes, or between test tubes, as is done when making dilutions of a bacterial culture. If aseptic technique is not maintained throughout these types of media manipulations, then cultures will become contaminated, and subsequent experiments using those cultures will be delayed because fresh, uncontaminated cultures will need to be prepared. Errors occur because a sterile field is not maintained throughout the procedure. For instance, you may forget to disinfect the laboratory bench or flame the rim of a culture bottle or tube. You may touch the tip of the pipette or set the cap of a bottle or test tube on the bench instead of holding it in your hand. Proper procedure is critical for keeping contamination of media and cultures to a minimum. Figure 8A provides an example of a pure versus contaminated culture of E. coli in a tube containing 5 ml of LB. The left panel shows a culture displaying uniform fine turbidity typical of a pure E. coli culture. In contrast, the right panel shows a contaminated culture in which the growth characteristics deviate from those expected for this bacterial strain.
Technical errors may occur when manipulating serological pipettes resulting in transfer of incorrect volumes of media between test tubes. For instance, you may read the volume on the pipette incorrectly (i.e., top versus bottom of the meniscus) or you may expel the media completely from a TD pipette, which was designed to leave a tiny bit in the tip not to be delivered. When performing a point-to-point delivery of media, you may use the wrong calibration marks and dispense the incorrect volume. Figure 8B shows an example of test tubes with correct versus incorrect volumes of media. The tube on the left contains 3.5 ml of LB measured with a 5 ml serological pipette. The student conducted a point-to-point delivery of the media in which LB was drawn up to the 5.0 ml graduation mark and dispensed to the 1.5 ml mark. The tube on the right contains 2.5 ml of LB measured with a pipette of the same size because the student who performed the point-to-point delivery of media incorrectly dispensed it from the 5.0 ml mark to the 2.5 ml mark. This mistake will result in a bacterial culture that will be at a higher concentration than planned, causing subsequent dilutions to be incorrect. This propagation of errors can result in a failed experiment that would need to be repeated with the correct cell concentrations.
A sample application for using micropipettors to transfer liquids is shown in Figure 9. These pipettors are used for a variety of experiments in molecular biology and microbiology including preparing samples for PCR and gel electrophoresis or inoculating sterile media or buffer with small volumes (less than 1.0 ml) of bacterial cells or phage particles. In the example provided, the student transferred 12.5 μl of TE buffer into a 1.8 ml microcentrifuge tube (left tube in panel A; note that dye has been added to the buffer to facilitate visualization of the liquid inside the clear microcentrifuge tubes). This procedure required the student first to select the correct micropipettor, in this case a P20, and next to set the volumeter to the correct volume (panel B). A tip was used that contains a cotton wool plug at the end to prevent possible contamination that could be expelled from the barrel of the micropipettor from reaching the buffer sample in the tip. This precaution is not necessary if care is taken when aspirating liquids into the tips, depressing the plunger slowly so the liquid does not splash into the pipettor barrel. Technical errors may occur that result in transfer of incorrect volumes. For example, you may select the wrong micropipettor for the job or set the volumeter on the correct micropipettor to an incorrect volume. Before immersing the tip into the buffer, you may push the plunger past the first stop, causing an excess of buffer to be drawn into the tip when releasing the plunger. Alternatively, you may not immerse the tip far enough into the buffer, so air is drawn into the tip instead of buffer. You may forget to push the plunger to the second stop when dispensing buffer into the microcentrifuge tube causing less than the desired volume to be released from the tip. The right tube in panel A of Figure 9 shows a microcentrifuge tube containing the incorrect volume of buffer relative to the tube on the left. Instead of dispensing 12.5 μl of buffer, the student dispensed 125 μl. In this case, although the numbers are set identically on the volumeter, the wrong micropipettor was selected for the job (the student used a P200 instead of a P20; panel B) resulting in the delivery of a substantially larger volume of buffer. If this solution was being used to prepare a mixture of reagents for an application such as PCR, then this mistake will change the final concentration of all reagents subsequently added to the same tube. Consequently, it is unlikely that the experiment will be successful, since molecular biology procedures such as PCR require all components to be at specific concentrations for the reaction to work properly.
Because it is not always feasible to ensure micropipettors (especially the inside of the barrel) are sterile, stock solutions can become contaminated causing even troubleshooting efforts to fail when performing experiments. If using micropipettors to transfer sterile solutions, it is strongly recommended that aliquots of stock solutions (media, buffer, water) be made using aseptic technique with serological pipettes. It is common to maintain working stock solutions in 15 ml or 50 ml sterile conical tubes. These are often easier to manipulate while operating a micropipettor and can be replaced with a fresh aliquot of the stock solution if contaminated during volume transfers.
Figure 1. Sterile field created by updraft of Bunsen burner flame. To minimize contamination of sterile solutions and cultures, it is critical that all manipulations be conducted within the sterile field. The rims of glass culture tubes and flasks should be passed through the tip of the blue cone, the hottest part of the flame. Plastic tubes and tips cannot be flamed - these should be pre-sterilized by alternative methods prior to use.
Figure 2. Serological pipettes used for aseptic transfer of liquids. (A) Shown from left to right are drawings of 25 ml, 10 ml, and 5 ml pipettes. (B) Serological pipettes may be plastic or glass. Plastic pipettes are disposable (one-time use) and typically are individually wrapped in paper and plastic sleeves in which all inside surfaces are sterile (left side). Glass pipettes can be used multiple times provided they are cleaned and sterilized between uses; these typically are stored in metal canisters (right side).
Figure 3. Serological pipettes are of two types: TC ("to contain") or TD ("to deliver"). Shown is the explanatory label of a TD 5 ml pipette.
Figure 4. Aseptic technique. When aspirating liquids from a bottle, flask, or tube with caps, never place the cap on the bench. Instead, hold the cap in the same hand as pipette aid while manipulating the vessel containing the liquid with the opposite hand as shown.
Figure 5. Meniscus formed when drawing liquid into serological pipette. The volume corresponds to the graduation mark on the pipette where the bottom of the meniscus aligns. In this example, the meniscus aligns with the 2.5 ml graduation mark.
Figure 6. Single channel micropipettor. (A) Shown is a sample micropipettor with a plastic tip attached to the bottom of the barrel tip holder. Indicated are the locations of the volumeter, the thumb wheel for changing the volumeter setting, the barrel tip holder, the tip ejector button, and the pushbutton for the plunger. (B) Two-stop plunger system on a micropipettor.
Figure 7. Using serological pipettes to transfer media into sterile 125 ml flasks. The left flask has 25 ml of media only (LB), while the right flask is a culture of E. coli resulting from inoculating LB with cells then incubating overnight at 37 °C. Note how the media in the flask on the right is turbid due to cell growth.
Figure 8. Using serological pipettes to transfer media into sterile test tubes. (A) The left tube contains 5 ml of a pure E. coli culture, while the right tube contains 5 ml of a contaminated bacterial cell culture. Note the differences in growth characteristics between the two cultures. Although both are turbid, the culture on the right has been contaminated with a fungus or other airborne microorganisms giving the culture a different color and consistency from that expected for E. coli cells. (B) The left culture tube contains 3.5 ml LB while the right tube contains only 2.5 ml LB. This volume difference resulted from a mistake made while conducting a point-to-point delivery of media to the tubes.
Figure 9. Using micropipettors to transfer buffer into sterile microcentrifuge tubes. (A) The left microcentrifuge tube contains only 12.5 μl of TE buffer, while the right tube contains 125 μl. Note that a dye has been added to the buffer to facilitate visualization of the liquid inside the clear microcentrifuge tubes. (B) The left volumeter is from a P20 micropipettor, while the right volumeter is from a P200 micropipettor. A common mistake is selecting the wrong micropipettor. Although the numbers are set identically on the P20 and P200 volumeter, selection of the wrong micropipettor results in transfer of incorrect volumes.
Figure 10. Laminar flow hood used to prevent contamination of solutions and cultures. Shown is a biosafety cabinet approved for work with BSL-2 organisms.
Aseptic technique refers to a set of routine procedures done to prevent sterile solutions and cultures from becoming contaminated by unwanted microorganisms in the laboratory. Such techniques are essential for experiments that require growing cells. Although a work setting that is completely sterile cannot be achieved, procedures such as disinfecting laboratory surfaces, creating a sterile field using a Bunsen burner, limiting exposure of uncapped cultures and media to the air, sterilizing materials such as bottles, tubes and glass pipettes, and avoiding contact of sterile instruments with non-sterile surfaces reduces the possibility of contaminating solutions and cultures in an experiment. The goal is for these precautionary procedures to become second nature; this comes with training and practice while working in a laboratory.
Volume transfers with sterile solutions and cultures using instruments such as serological pipettes and micropipettors are one of many types of routine techniques done in a laboratory. Different experimental applications call for instruments capable of transferring distinct, yet precise and accurate, volumes. Serological pipettes are used in microbiology laboratories to prepare cell cultures requiring media preparations involving milliliter volumes, while micropipettors are essential to molecular biology experiments that need only microliter amounts of solutions. When aseptic technique is practiced with these instruments, contamination is minimized during volume transfers regardless of amount of liquid or type of experiment.
Although not discussed in this protocol, one other means commonly used to prevent contamination is to work within a laminar flow hood (Figure 10). This equipment is critical for tissue culture and for experiments performed with microorganisms classified as BLS-2 or higher. A laminar flow hood contains a HEPA (high-efficiency particulate air) filter that removes airborne contaminants from the air flowing into the hood while preventing unfiltered air from the room from permeating the workspace. Of note, a Bunsen burner cannot be used inside a laminar flow hood because the heat from the flame disrupts air flow essential to the functionality of the hood.
It is often helpful to check the quality of your aseptic technique when performing experiments. To confirm solutions and culture media do not get contaminated during experimental manipulations, always prepare a negative control. For example, if preparing tubes of broth for growth of bacterial cultures, do not inoculate one tube leaving only sterile media. Incubate the medium alongside the inoculated tubes then inspect uninoculated control tube for signs of contamination such as turbidity from growth of unwanted cells unintentionally introduced into the tube. If the control tube is contaminated, the experimental tubes likely are contaminated as well, and the experiment will have to be repeated. These precautionary measures should be done with every experiment.
I have nothing to disclose.
Special thanks to Cori Sanders at Iroc Designs for preparing illustrations and to Kris Reddi at UCLA for setting up sample cultures for figures. Funding for this project was provided by HHMI (HHMI Grant No. 52006944).
|LB Broth||Difco Laboratories||244620||Recipe also available in reference 6|
|EDTA disodium salt dihydrate||Sigma-Aldrich||E5134|
|Ethanol||Fisher Scientific||A406||For use as disinfectant, prepare 70%(v/v) with distilled water|