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Lab Safety, How to Keep a Lab Notebook, How to use Lab Equipment
Lab Safety, How to Keep a Lab Notebook, How to use Lab Equipment
Safety First!
Safety First!
Introduction
Laboratory work is exciting and the skills we’ll learn will be important for success! One of the most important things we can learn is how to be safe in the lab. Safety is our number one goal when working in a laboratory. Properly operating laboratory equipment and following safety protocols and regulations keeps everyone safe and ready to work on extended research projects.
It’s not just us in the lab either, there are three important things to consider when it comes to safety:
1. Keeping individuals safe.
a. If we all follow the safety procedures closely, the rest of the safety considerations become almost second nature. As the airplane emergency safety analogy goes, we must put our own oxygen mask on, in case of an emergency, before helping others, the same sentiment goes for individual safety in the lab.
2. Safety of those around us in lab.
a. Science is a collaborative process, one that invites close interactions with those around us. With that comes a certain amount of risk, which we need to take into consideration. Not only must we keep ourselves safe, but we must also keep those around us safe too.
3. The community beyond the lab.
a. Some of the reagents in the lab can cause significant damage to the environment around us if not handled properly. We must be good stewards of science and handle and dispose of these reagents in the appropriate way to keep everyone safe and the environment healthy for the future.
We’ll showcase how to be safe in our laboratory (PSC 718) but also how to be safe in other laboratories too. It should be noted that depending on the lab that we work in, there are different levels of risk and various safety considerations. For example, if we work in a lab that has live virus (like HIV-1), we’d have different considerations than in a lab that studies the behavior of microcrustaceans. We’d also have different safety procedures and training so that we are prepared for the particular hazards.
It’s important to talk with your supervisor when first starting in a new lab to know which specific safety trainings are required. At UofSC, an entire program called Environmental Health and Safety oversees the training of individuals in basic lab safety and other specialized trainings that may be needed (depending on the lab’s research focus). From chemical safety to biohazard training, they provide an essential resource for UofSC and beyond. More information on Environmental Health and Safety and the training they provide can be found at the following link: EH&S.
As a part of our workshop, we’d love for you to complete the “Chemical and Lab Safety” training offered through EH&S. They offer both in person and virtual options that will teach you the basics when it comes to lab safety. Please sign up here: Training Safety Schedule. Not only will this help you in our workshop, but it’ll be also a great addition when it comes to applying to labs within the department. You can show that you’ve already taken the initiative by being training in basic chemical and lab safety.
Figure 1. Safety shower. Located in the far right corner of the room in PSC 718, this eye wash/safety shower combination safety equipment allows us to quickly remove chemicals that may have spilled on ourselves. Note the shower and eye wash stations are checked periodically to ensure their proper operation continues.
Figure 2. Eye wash station. Located near the exit in PSC 718, this is one area that you can wash chemicals from your eyes if an accident happens. Typically, there’s an eye wash station and a safety shower combo in laboratories.
Figure 3. Broken glass container. These are located on each bench in PSC 718. Only broken glass belongs in these containers, all other items should be disposed of in the proper location.
Figure 4. Biohazard symbol.
Figure 5. Fire Extinguisher.
Figure 6. Lab Coats.
Workshop Safety Procedures
Workshop Safety Procedures
No food or drink are allowed in the lab.
If you have a meal or beverage, please leave it in the hallway. This is for your protection as there are various chemicals that could cause serious harm to your health. This will be a strict rule for PSC 718 and for other labs.
Closed-toe, solid construction shoes must be worn at all times.
If you wear shoes with open toes, or mesh top, we will ask you to please find another form of footwear. This is to protect yourself from potential spills as various chemicals could cause permanent damage.
You are required to notify the instructor immediately if are injured in lab.
The first aid kit is in the front of the classroom near the exit.
It’s important to identify and know how to use the safety equipment in the laboratory. The first aid kit contains various medical relevant items that can be used in case of an injury. In PSC 718, the first aid kid is by the exit, typically on the counter but may also be in the cabinet under the sink. In other laboratories, one of the first things to do is find where the first aid kit is located (along with other safety equipment).
Any spills of chemicals on your skin, need to be washed immediately. If chemicals get in your eye, we’d use the eye wash station. If chemicals get on your body, we'd use the safety shower.
In PSC 718, the eye wash station is in the front of the room toward the right (near the refrigerator) (figure 1). There’s an additional eyewash station located at the sink in the front left of the room near the door (figure 2). The safety shower is in the front right of the room (near the refrigerator, figure 1).
These pieces of equipment must be located when entering another laboratory quickly to know where to go if any chemical spills occur.
Waste materials should be properly disposed of in the appropriate waste receptacles.
Broken glass should be placed in the broken glassware container. In PSC 718 we have a broken glass container at each set of benches. Look for the cardboard box with a round hole and plastic bag labeled “glass disposal” (figure 3). In other labs, broken glass containers could range from cardboard boxes to hard plastic containers such as used five gallon buckets.
Biological materials should go in the biohazard waste disposal. Typically this will be red in color and contain the biohazard symbol (figure 4). In PSC 718 we have a reserve biohazard waste bag if needed near the instructor station (we typically don’t have biohazard waste in our lab). What goes in biohazardous waste? A wide variety of substances and tissues (EH&S offers another training for those who need it).
If you break a piece of glassware, please don’t try to clean it up yourself. Call the instructor for assistance.
In PSC 718, please always contact the instructor. For other labs, check with the supervisor for the appropriate procedure.
Please use extreme caution when handling sharp objects (razor blades, scalpels, etc) and dispose of used sharps in the red sharps container.
A sharps container is located near the fume hood at the back left of the lab in PSC 718. We shouldn’t need to use sharps in our workshop.
In other labs, sharps containers are typically located close to where sharps will be used. A word of caution, sharps go into the waste container but never come back out by the user (and one never attempts to go into the sharps container for any reason).
Use caution when working around hot plates. Open flames like Bunsen burners require special attention and caution.
If there is ever a fire in PSC 718, please alert the instructor immediately. There’s a fire extinguisher mounted on the wall near the door that the instructor (or another trained professional) can use to potentially extinguish the fire.
Wash your hands before leaving the laboratory classroom.
Gloves will be used for your protection during lab exercises that have potentially harmful chemicals or biological materials.
This is a general rule that applies to all labs (and is a great hygiene tip anyway).
Lab coats and goggles are provided for your use. Wear lab coats during lab procedures.
Lab coats are located in the middle right of the room near the safety shower (figure 6). These lab coats help protect your clothing from various chemicals and stains. Depending on the lab this may be a hard and fast rule or not. Check with the supervisor to be sure.
Tie back long hair during lab procedures.’
Long hair may inadvertently fall into various chemicals, open flames, or could potentially cause other issues.
By tying it back, we provide another layer of safety for ourselves and those around us.
Conclusion
Want even more detailed information on biological lab safety here at UofSC? Check out EH&S’s manual at this link: Biological Safety Manual. Together, by following the safety regulations above, we can create a safe learning environment for everyone in our workshop and in the research lab. Safety should always be on our mind when doing anything in the lab.
Laboratory Notebooks
Laboratory Notebooks
I. What is a lab notebook?
A. A lab notebook is a complete record of the procedures (what you did), the reagents (what you used), your observations (what you saw), and the results of the experiments you perform in the lab.
1. It should describe why an experiment was performed, how it was carried out, and the results of the experiment.
2. It should be clearly written and understandable to another scientist, most importantly your PI (principal investigator-the head of the lab).
3. Your laboratory notebook should help you figure out what might have gone wrong when your experiment does not work.
4. Your lab notebook may need to be used by others in the lab after you have left.
B. Your lab notebook is a legal document.
1. It will be closely examined if any of your work contributes to the filing of a patent application.
2. It can be used to validate your results in the case of fraud allegations.
II. Types of lab notebooks
A. Bound notebooks
1. Advantages: there are no lost pages; it is legally strong in cases of alleged fraud
2. Disadvantage: it is difficult to copy; it is organized only by the order in which you do things
B. Loose leaf/Three ring binder notebook
1. Advantages: you can organize it in a logical manner; it is easy to record information as you are performing an experiment by making notes using a clipboard
2. Disadvantage: sheets can be lost or removed; it is harder to authenticate data
C. Electronic notebooks
1. Advantages: it can be searched; it has a large capacity for data storage
2. Disadvantage: it requires frequent backups; there can be compatibility issues with new versions of the software; it is hard to prove authenticity
III. Content of notebook
A. Put your name and project name on the cover of the notebook
B. Include a Table of Contents with the date, title of the experiment, and page numbers
C. Experimental Entries
1. Date of the experiment
2. Title of the experiment
3. Hypothesis or Goal: What is the purpose of the experiment?
4. Protocols, reagents, equipment
a. written protocol or reference to a previously used protocol
b. calculations performed
c. list of reagents (source; expiration date; location in lab) and equipment used (location)
d. solutions (how they were made)
e. other details (gel percentages; cells used, antibody titer etc.); anything that will help you duplicate the experiment
5. Observations
a. everything that happened (any deviations from planned protocol; errors; note any oddities)
b. raw experimental data (readings from equipment; qualitative observations; tape in pictures of data such as gel images)
c. any data analysis performed; graphs; names of corresponding data files on computers
6. Summary of results: Include a one sentence summary of the results of the experiment; if the experiment did not work include any thoughts you have on why it failed
IV. Ethics
A. Your lab notebook is not yours; it belongs to the lab. It should not leave the lab for any reason. You may make photocopies of your work and take the photocopies with you.
B. All data should go in the notebook
1. Do not eliminate "bad" data or failed experiments
C. Do not remove pages from your notebook
D. Cross out mistakes with a single line
E. Use a pen (not pencil)
V. A good lab notebook
• Promotes accurate collaboration.
• Promotes reproducibility not just for other researchers but for yourself as well.
• Maintains the reasoning behind your experimental flow.
• Serves as the building blocks of your research paper's methods, results and conclusions.
• Serves as a log of all observations and anomalies.
• Becomes a helpful troubleshooting tool.
• Allows you to answer specific experimental questions.
• Can help defend your intellectual property, particularly when it comes to patents.
from https://www.goldbio.com/articles/article/15-Laboratory-Notebook-Tips-to-Help-with-your-Research-Manuscript)
VI. Useful References
https://www.training.nih.gov/assets/Lab_Notebook_508_(new).pdf
Barker, K. (2005) At the Bench: A Laboratory Navigator
How to use: Microscopes, Pipettors, Balances, and Micro-centrifuges.
How to use: Microscopes, Pipettors, Balances, and Micro-centrifuges.
Figure 7. Compound Light Microscope. Numbers correspond to the various parts of the microscope. They are listed in the text of the document above. Note these are general parts of a microscope, the model we use may differ slightly (and may change depending on which lab/specific use is needed for that model of microscope).
Parts of a Compound Light Microscope
Parts of a Compound Light Microscope
Figure 7
Arm: The curved part of the microscope that contains the lenses in the body tube.
Ocular lens (Eyepiece): Lens through which the specimen is viewed; typically, a magnification of 10x.
Rotating Head (Body tube): Piece on which the ocular lenses are mounted. Rotates for ease of viewing and storage.
Mechanical Stage Control: Used to position the slide by moving the stage.
Nosepiece: Rotating piece that holds the objective lenses.
Coarse Adjustment: Used to roughly bring the specimen into focus.
Objective Lens: Lenses that are closest to the specimen; magnification ranges from 4x-100x.
Fine Adjustment: Used to sharpen an image after it has been coarsely focused.
Stage: The flat surface where slides are placed for viewing.
Illuminator Switch: turns the light and the microscope on.
Condenser: Device on the underside of the stage that condenses and focuses incoming light before it is passes through the specimen. Iris Diaphragm: Located under the stage with the condenser, this device regulates the amount of light that passes through the specimen.
Dimmer (Illuminator): The light source at the base of the microscope.
Base: The bottom of the microscope; contains the light source and supports the rest of the scope.
Microscopes
Microscopes
Introduction
In cellular and molecular biology, we need to see the cells that we’re using for experimentation. Whether it’s using a fluorescent label or examining the impact of various reagents on cellular morphology, the compound light microscope is a piece of equipment that is used daily in lab.
Cells are very small and typically cannot be seen with the naked eye. We must rely on microscopes to magnify our specimen to view cells. Two important terms to know for microscopy is resolution and magnification. Resolution is the ability to see two points as two separate points. A high resolution means we can resolve much more detail, giving us a higher quality image. Magnification is the ability to produce an image at a scale greater than the specimen’s actual size. Although an image can be increased up to 1000X the size of the actual object using higher magnification lenses, the quality of that image is still limited by the resolution. Therefore, as we increase the magnification of our sample, the resolution of the image decreases, decreasing the quality of that image. We’ll demonstrate how to calculate the magnification of the microscope in the procedure below.
First, we introduce the parts of the compound light microscope followed by a short instructional on how to properly operate this foundational piece of equipment.
How to use a Compound Light Microscope
We will be using a binocular compound light microscope. Binocular refers to the fact that there are two ocular lenses through which we view our specimen. Compound refers to the fact that there are multiple lenses involved in the magnification of the image. Light refers to the way in which the image is focused.
1. Always use both hands to carry the microscope: one on the arm and one under the base.
2. Before turning on the microscope, be sure that:
1. The lowest power objective is in place.
i. Important! Check the objective before focusing.
2. The lenses are clean (wipe gently with lens paper only).
3. The stage does not have a slide from previous use.
4. The light source is turned all the way down.
5. The microscope is plugged in.
3. Place the slide on the stage and be sure that it is locked into place. Use the mechanical stage control to center the specimen under objective lens.
4. Look through the ocular lens and begin to focus DOWN with the coarse adjustment knob.
5. When the specimen is nearly focused, the fine adjustment knob can be used to sharpen the image.
6. Most modern microscopes are parfocal. They are also typically paracentral. As we increase the magnification on our microscope, be sure to pay attention when changing the objective – do NOT let the lens touch the slide! We may need to increase the light slightly and fine focus the image.
7. Calculate the Total Magnification at which we are viewing our sample by multiplying the power of the ocular lens (in our case, 10X) by the power of the objective lens.
8. To maximize the resolution of our sample, adjust the condenser lens to the position closest to the stage and open the iris diaphragm completely. We can adjust the contrast of our image by closing the iris diaphragm slowly until the image is optimal.
9. When done with the microscope, be sure to return it to the proper location, in the proper condition (light off and/or turned all the way down, lowest objective, no slides left on the stage, lenses clean, cord wrapped around the base and dust cover in place).
Note: Should we need to bring the microscope closer after setting it on the bench, do not drag it across the bench top. Please pick it up to move it.
Pipettors
Pipettors
I. What is a pipettor?
A. A pipettor is a volumetric instrument that can take up and transfer a precise amount of a small volume of liquid.
1. We will be using variable volume pipettors, each one of which covers a range of volumes.
2. These must be used correctly to take up and dispense the correct volume of liquid.
3. Molecular biology experiments depend on precise concentrations of different reagents. These experiments won't work if you don't have the right amounts of each component. Thus it is very important to establish good technique using pipettors.
4. Pipettors are your best friend in the lab. Take care of them and keep them clean. Wipe off any solution that you get on the shaft or ejector.
II. Anatomy of a Pipettor
A. A pipettor has a narrow tip at one end and a round plunger button on the other end. You will wrap your hand around the blue hand grip and use you thumb to work the plunger. You will put a disposable plastic tip at the narrow end of the shaft. Below the plunger is an ejector button (white) on one side of the pipettor which is used to eject the disposable plastic tip. The volume of liquid to be taken up is visible in a small window when viewing the pipettor from the front. This volume is changed using the volume adjustment knob. Different companies make different brands of pipettors which look a bit different but all use the same basic technique. We will be using VWR brand pipettors.
III. Background info on pipettors
A. There are different pipettors that take up a range of volumes of liquid.
1. Typical sizes of VWR pipettors:
a. 2-20: volume range is 2-20 microliters (mls)
b. 20-200: volume range is 20-200 microliters (mls)
c. 100-1000: volume range is 100-1000 microliters (mls); 1000mls is 1ml
B. There are different size disposable plastic pipet tips for the different pipettors
1. Yellow tips fit the 2-20 and 20-200 VWR pipettors.
2. Blue tips fit the 100-1000 VWR pipettors.
IV. Setting the volume on a pipettor
A. The volume display consists of three numbers and is read from top to bottom.
1. For the 2-20 pipettor, the numbers in black represent microliters and the number is red represents tenths of microliters.
a. example: 12.5mls entered on a 2-20 pipettor
1
2
5
b. Note: A change from black to red indicates the position of a decimal place.
2. For the 20-200 pipettor, the numbers in black represent microliters.
a. example: 75mls entered on a 20-200 pipettor
0
7
5
3. For the 100-1000 pipettor, the number in red represents milliliters and the numbers in black represent hundreds and tenths of milliliters.
a. example: 1.00ml (i.e. 1000mls) entered on a 100-1000 pipettor
1
0
0
b. example: 0.75ml (i.e. 750mls) entered on a 100-1000 pipettor
0
7
5
c. Note: A change from red to black indicates the position of a decimal place.
V. How to use a pipettor
A. Steps in the use of a pipettor
1. Set the correct volume on the pipettor. Turn the volume adjuster knob until the volume is slightly above the desired setting, and then slowly adjust down to the desired setting. Always dial DOWN to the desired volume.
2. Hold the pipettor with your hand around the pipettor and your thumb on the plunger button. The ejector push button should be near where you thumb connects with the rest of your hand.
3. Attach a disposable plastic tip to the shaft of the pipettor by pushing the pipettor tip down on a tip in a tip box.
4. Depress the plunger to the FIRST STOP and keep you thumb at this position. While holding the pipettor vertically, immerse the tip into the liquid of your sample a few millimeters below the surface and then SLOWLY release the plunger.
5. Withdraw the tip from your solution and check the tip to be sure that no air bubbles are present. If any excess liquid is on the outside of the tip, you may blot it with lint free paper, but be sure to avoid the opening. Alternatively, slowly dragging the tip on the side of the container can help eliminate excess liquid on the outside of the tip.
6. Touch the tip against the side wall/bottom of the receiving vessel and slowly depress the plunger to the first stop to dispense. Continue to press the plunger to the SECOND STOP to dispel the remaining liquid. Keeping the plunger depressed, remove the pipettor by drawing the tip along the insider surface of the vessel. Move your arm upward away from the receiving vessel.
7. Slowly release the plunger, allowing it to return to the “up” position.
8. Use the eject button to discard the tip into an appropriate waste container.
B. Things to Avoid when using a pipettor.
1. Don't use a pipettor outside of its volume range.
2. Don't use a pipettor without a tip attached.
3. Don't jam a tip onto the pipettor.
C. Quick Guide for use of a pipettor
1. Select the volume
2. Put on a tip
3. Press and hold the plunger at the first stop
4. Place the tip in the liquid
5. Slowly release the plunger
6. Pause for a second and then remove the tip from the liquid
7. Insert the tip into the delivery vessel
8. Press the plunger to the second stop
9. Pause for 2 seconds
10. Remove the tip from the delivery vessel
11. Release the plunger
12. Eject the tip into the waste container
VI. Practice Pipetting
A. For the following volumes, indicate which pipettor (2-20; 20-200; 100-1000) you would use, write the numbers you would dial in at the three positions (top to bottom), and write the weight of each volume as described in VI.C. below.
volumes: 5 microliters, 156 microliters, 15 microliters, 250 microliters, 27 microliters, 750 microliters.
which pipettor?
numbers (top to bottom)
weight of water (C below)
B. Practice transferring the above volumes using the colored solution supplied by the instructor.
1. Check for drops of liquid on the outside of the pipette tip (if present, wipe the drop off with a kimwipe or by lightly touching the drop to the microcentrifuge tube’s mouth).
2. Make a habit of checking the disposable tip for air bubbles.
3. Try to deposit the solution into the next microcentrifuge tube without creating excessive air bubbles.
4. Check the tip after depositing the solution to be sure that you expelled all liquid from the tip.
C. Check the accuracy of your pipetting using a balance. Use your pipettor to take up water of each of the volumes in the table and dispense it onto a weigh boat on the balance. Write down the weight displayed on the balance. Tare the balance between each measurement. Water has a density of 1g/ml. Thus, for example, 200 ml of water should weigh 0.2g.
VII. Additional Resources
How to read a pipettor:
https://www.youtube.com/watch?v=wlO4zJLR8R8
How to use a pipettor:
https://www.youtube.com/watch?v=cKLzCRCA_N8
https://www.youtube.com/watch?v=uuzY-EgbQZQ
Figure 8. VWR pipettor.
Figure 9. Precision balance (left) and analytical balance (right).
II. Centrifuges and Microfuges
A. A centrifuge is a piece of equipment used to separate biological particles from a solution.
1. Microfuges:
a. In this workshop, we will use small centrifuges called microfuges (Figure 10). They fit tubes that range in size from 0.5-2mls. Typically, we use 1.5ml microfuge tubes. The tubes are placed into a rotor that spins. b. Examples of when to use a microfuge:
• To pellet bacterial cells from a culture before starting a plasmid miniprep
• To precipitate DNA after ligation
• To collect a sample in a tube after a restriction enzyme digest after incubation at 37oC
2. Other common centrifuges found in molecular biology labs
a. high-speed centrifuges (can pellet larger volumes of solution)
b. ultracentrifuges (can spin at very high speeds)
3. Definition of speeds
a. The speed at which the rotor inside the centrifuge spins can be defined in different units.
b. These units are revolutions per minute (RPM) or relative centrifugal force (RCF), which is also sometimes called "x g" (g-force).
4. Basic steps of centrifugation in a microfuge (i.e. How to Spin)
1. Balance the tubes in the rotor. Each tube must have a tube of the same volume across from it. For microfuges, we can balance based on volume. For other centrifuges, the two samples in their respective tubes would need to have the same weight.
2. Put the tubes in the microfuge always in the same orientation. Put the lip on the closure facing out. This way you will know where the pellet in the tube is located (on the lip side).
3. Confirm that each tube has a balance. Put the lid on the rotor and screw it finger tight.
4. Close the lid of the centrifuge.
5. Adjust the setting of the microfuge (time of spin, speed of spin). In some cases, you may be using a refrigerated microfuge and then the temperature would need to be set. This would be done ahead of time so that the rotor and centrifuge are at the correct temp before you begin centrifugation.
6. Wait by the microfuge until it comes to full speed. If there are any problems such as unbalanced tubes (which can result in funny sounds or vibrations), stop the microfuge. This is most important for larger centrifuges.
7. When your spin is done, remove the samples immediately in a careful way so as not to disturb any pellet.
8. Remove the supernatant with a pipettor or pour off the supernatant carefully (i.e. decanting). This decision depends on the sample.
9. If any spills occurred, clean up the rotor and microfuge.
Balances and Centrifuges
Balances and Centrifuges
I. Balances
A. A balance is used to weigh out chemicals when making solutions or to balance tubes that need to be centrifuged.
1. In molecular biology labs, we typically have two types of balances: precision balances (left in Figure 9) and analytical balances (right in Figure 9).
a. precision balance: higher capacity; usually show one or two decimal places
b. analytical balance: use for measuring small amounts; show four decimal places; have a weighing chamber for precise measuring
2. Basic steps in weighing
a. Choose the appropriate balance for the amount of chemical you will be weighing out.
b. Turn on the balance. Make sure the balance is clean; if not, clean the surface.
c. Put an appropriately sized weigh boat on the balance.
d. Tare the balance. The balance should read zero.
e. With a spatula remove some chemical from its container and place it in the weigh boat. Continue until the proper amount of chemical is weighed out.
f. Add the chemical to the beaker with water and stir bar for the solution you are making. Transfer the chemical from the weigh boat to the beaker by grasping the boat by two opposite ends, carefully bend the ends together, and pour the chemical into the beaker. If some of the chemical sticks to the boat, gently flick the boat with your fingers to dislodge the chemical so that it is transferred to the beaker.
g. Clean the balance when you are finished using a kimwipe and water as needed. Also clean the surrounding area if necessary.
Figure 10. Eppendorf Centerifuge 5425.
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