Use an analytical scale to measure water, making sure the minimum and maximum settings correspond to the intended volume. Since water has a density of 1, then 1 ml of water is equivalent to 1 gram g.
Also, make sure the tip does not leak and can maintain the desired volume until dispensed using plunger system. Micropipettors must be used with plastic disposable tips at all times. Fit a tip tightly onto the end of the barrel of the micropipettor. Press down and twist slightly to ensure an airtight seal. Tips are usually packed into plastic boxes that can be sterilized by autoclaving. Open the tip box to retrieve a tip, then close the tip box to minimize contact with contaminants in the air.
Some tips have filters similar to the cotton wool plugs on serological pipettes. These tips are often more expensive than regular tips and thus are used for specialized applications. For instance, when measuring volatile chemicals such as chloroform or radioactive liquids such as 32 P-labeled DNA, using filter tips helps prevent the barrel of the micropipettor from getting contaminated.
Keeping the micropipettor upright will prevent liquids from running inside and contaminating the barrel of the micropipettor. The micropipettor has three positions: 1 Rest position, 2 First stop, and 3 Second stop Figure 6 , panel B. The instrument has a two-stop plunger system. The first stop has two functions. The first is to draw in the desired volume of liquid into the tip when releasing the plunger from the first stop to the rest position.
The second function is to dispense the majority of liquid from the tip when depressing the plunger from the rest position to the first stop. Further depressing the plunger to the second stop dispenses whatever liquid remains in the tip. Depress the pushbutton on the plunger from the rest position to the first stop. Air equal to the volume of the setting will be displaced.
Do not touch the micropipettor itself to the sides of bottles, tubes and flasks; otherwise the inside surfaces of these vessels will become contaminated. Only the tips are sterile. Release the pushbutton slowly to aspirate the liquid into the tip. Stop once the pushbutton is back to the rest position.
Wait a moment so liquid can be drawn into the tip. The volume of liquid in the tip will equal the volume of the setting of the micropipettor. Remove the tip from the liquid, and visually inspect the tip to confirm that the liquid drawn up has reached the expected level in the tip and there are no air bubbles in the tip. If necessary, expel the liquid and manually tighten the tips onto the micropipettor. Draw up the liquid and check again. To expel the liquid, slowly depress the pushbutton on the plunger to the first stop.
Wait a moment then press the pushbutton to the second stop to expel any residual liquid in the tip. Depressing the plunger too quickly may cause the liquid being expelled to splatter or will produce undesirable bubbles in the tube. Discard tips into designated sharps waste container by pressing the ejection button on the micropipettor. When finished with an experiment requiring use of aseptic technique, turn off the Bunsen burner, then put away all supplies and reagents.
Wipe down the outside surfaces of labware bottles, micropipettors, pipette tip boxes with a pre-moistened disinfectant wipe to ensure contaminants are not transferred to the storage location. Place contaminated glassware and hazardous waste materials into the proper disposal receptacle. Laboratory waste includes labware such as gloves, pipettes, tips, and tubes.
Non-infectious hazardous waste is generated when performing experiments with non-pathogenic organisms BSL-1 while infectious hazardous waste is generated when using pathogenic organisms BSL-2 or above.
Infectious waste must be autoclaved or disinfected before it is discarded. Wipe down the entire work area on the laboratory bench with a pre-moistened disinfectant wipe from the canister, once again allowing the disinfectant to evaporate. Wash hands thoroughly with antiseptic soap and warm water before leaving the laboratory. 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. 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 ml sterile flask using a 25 ml serological pipette. Next, the broth must be inoculated with E. The flask is incubated in a growth chamber for a particular amount of time, allowing the cells to replicate for this example, the E.
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. The left panel shows a culture displaying uniform fine turbidity typical of a pure E. 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.
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. The student conducted a point-to-point delivery of the media in which LB was drawn up to the 5. The tube on the right contains 2. 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.
In the example provided, the student transferred 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 In this case, although the numbers are set identically on the volumeter, the wrong micropipettor was selected for the job the student used a P 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.
Four types of flames can be distinguished:. It is particularly used in biology to sterilise instruments by passing them into the flame and the air within a radius of 20 centimetres around the flame essential for microbiology experiments.
The Bunsen burner was invented in by Peter Desdega. Our services Dictionary. Accueil Dictionary. Do not discard glass slides in the waste cans. All materials used for handling or culturing microorganisms are to be disposed of as follows: test tubes placed in racks in a bin for autoclaving; petri dishes in the other bin for autoclaving and disposal. Any living culture material that is spilled, either on tables or on the floor, is to be treated immediately with disinfectant and cleaned up with paper towels.
Notify the instructor of any spills. The paper towels that you use to clean up the spill should be placed in the bin with the petri dishes for autoclaving. Prepared slides that are used during the semester must be returned clean to the trays from which they were taken. Any papers on the floor at the end of the laboratory period are to be picked up and discarded in the wastebasket. The same is true for your laboratory bench area. DO NOT throw plates, tubes, swabs, slides, pipets, pipet tips, broken glass, etc.
These items need to be disposed of properly. Throwing potentially contaminated items into the regular garbage is a safety issue for students, instructors, lab techs and the cleaning staff. If you are unsure about where an item should go, always ask your instructor. Instructions for Good Laboratory Practice and Care of Laboratory Equipment Correct use and care of the laboratory equipment is considered a fundamental part of good laboratory technique. Please read over and follow the instructions listed below: Microscopes The most critical and most expensive piece of equipment in the microbiology laboratory is the microscope.
Inoculating loops and inoculating needles Inoculating loops and needles are used to transfer bacteria into and from culture media. Bunsen burners A Bunsen burner is a source of open flame that is used to sterilize loops and needles, as well as flaming the lips of test tubes during inoculations.
Microscope slides Any disposable glass slides should be discarded in the sharps container. Have your two plates on your lab bench. You will be given a plate with streaked organisms on it. These are the real thing. Look closely at it and select an area that has individual colonies.
They will look like small dots on your plate. Each dot represents one or a few cells that multiplied to form a colony — also called a colony forming unit CFU. Use the same plate of bacteria you did for your plate-to-plate transfer. Find another well-isolated colony. Microbiology Resource Center. Introduction In the microbiology lab we use aseptic technique to: Prevent contamination of the specific microorganism we are working with.
Prevent contamination of the room and personnel with the microorganism we are working with. Wear your lab coat and gloves. Tie back long hair. Leave all food and drink in your backpack. Do not chew gum in lab.
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