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Included in this lab manual
- Autoclave Instructions (Separate)
- Biosafety Level (BSL) Chart
- Disposal of Biohazardous Materials
- Safety Rules-General and Microbiology specific
- Student Agreement (separate)
Review the following and the handouts. Sign the acknowledgement form.
College of the Canyons
Biological Science Department
Laboratory Safety Guidelines
Each laboratory is a restricted area. Enrolled students may work in a lab only when there are authorized personnel present. Friends of students in lab classes will not be allowed to “visit” inside the laboratory. Students are not permitted into the storage rooms or prep areas unless given specific permission by their instructor or lab personnel.
Ensuring safety in the laboratory is the responsibility of everyone working in the lab. Please follow these guidelines carefully.
USE COMMON SENSE WHEN WORKING IN THE LAB.
Be prepared for your work in the lab. Read all procedures thoroughly before entering the lab. Follow all written and verbal instructions carefully. If you do not understand a direction or part of a procedure, ask the instructor before proceeding.
Do not eat, drink or smoke in the lab. Do not use laboratory glassware as containers for food or beverages.
Always wear close-toed shoes in the lab.
Wear safety goggles whenever working with chemicals or when there is an impact risk.
Long hair should be tied back when working with flames, chemicals or dissections.
Observe good housekeeping practices. Work areas should be kept clean and tidy at all times. Keep aisles clear. Push your chair under the desk when not in use.
No open flames are permitted in the laboratory unless specifically indicated by the instructor. When burners or hot plates are being used, caution should be exercised to avoid thermal burns. If you sustain a thermal burn immediately flush the area with cold water and notify the instructor.
If there is a blood spill, immediately notify the instructor.
ANY ACCIDENTS OR INJURIES THAT OCCUR IN THE LAB MUST BE REPORTED TO THE INSTRUCTOR AT ONCE.
Familiarize yourself with the location of the Fire Extinguisher. There is a telephone in each lab room for EMERGENCY use only. In case of emergency dial 7 to reach the school operator who will contact and direct the emergency personnel.
Broken glass is to be disposed of in the broken glass container and reported to the instructor.
Keep hands away from face, eyes, mouth and body while using chemicals or preserved specimens. Wash your hands with soap and water after performing all experiments. Clean, rinse and wipe dry all work surfaces and apparatus at the end of the experiment. Return all equipment cleaned and to the proper area.
Handle all living organisms used in a lab activity in a humane manner.
Never use mouth suction to fill a pipet. Use a rubber bulb or pipet pump.
When removing an electrical plug from its socket, grasp the plug, not the electrical cord. Hands must be completely dry before touching an electrical switch, plug or outlet.
Wear safety goggles whenever working with chemicals.
Chemicals and biological stains should be used with caution. Follow specific directions regarding all chemicals used during lab. Check the label on chemical bottles twice before removing any of the contents. Take only as much chemical as you need.
If any chemical comes into contact with your skin, immediately flush the area with water for several minutes and notify the instructor. A strong base feels soapy on your skin but will still cause a severe burn.
Familiarize yourself with the location of the chemical eyewash. If any chemical is splashed into your eyes, hold eyelids open and flush with water for 15 minutes. Notify the instructor.
Dispose of all chemical waste properly. Do not pour chemicals down the sink unless told to do so by your instructor. Check the label of all waste containers twice before adding your chemical waste to the container.
Students should consult with the instructor regarding the pros and cons of wearing contact lenses during dissections.
Safety glasses or other protective eyewear is recommended for all students performing dissections.
Protective gloves should be worn during dissections. If your skin comes in contact with a chemical preservative, immediately run water over the area and notify the instructor.
Do not remove preserved specimens from the laboratory.
Preserved biological materials are to be treated with respect.
When using scalpels and other sharp instruments, always carry with tips and points pointing down and away. Notify instructor of any cuts or other injuries.
When working with microorganisms, lab coats must be worn at all times.
The laboratory benches must be cleaned with Sanisol (a disinfectant) before and after all lab work.
If cultures are spilled in the lab, they must be disinfected. Pour Sanisol over the spill, let stand for 5 minutes, then wipe up and dispose of towels in a biohazard receptacle.
Never remove cultures from the laboratory.
When finished working with a specific culture place it in the biohazard receptacle provided.
Agreement: (You will be given a separate sheet to sign and turn in, do not turn this in)
I have read and agree to follow the above safety rules for the Biological Science Laboratories.
I realize that I must obey these rules to ensure my own safety and that of my fellow students and instructors.
Student SignatureStudent Name (print)
Microbiology Specific Guidelines:
These procedures are specific to Microbiology (based on the 2012 ASM Guidelines for BSL 2 teaching labs) and are in addition to the General Safety Guidelines.
- Safety goggles or safety glasses with side shields are required for laboratory procedures involving bacterial cultures.
- Wear closed-toe shoes that cover the top of the foot, and heel. You will not be allowed to participate if you don’t have on the proper shoes.
- Wear gloves when handling BSL 2 microorganisms or hazardous chemicals, including stains.*
- Wear laboratory coats. Coats should cover to the knees. Long pants are recommended. You will not be allowed to participate if you don’t have on the proper attire.
- Wash hands after entering and before exiting the laboratory.
- Tie back long hair.
- Do not wear dangling jewelry, scarfs, etc.
- No food, gum, or water in the lab.
- Do not touch face, including eyes, or use cosmetics in lab.
- Keep hands, pens, pencils, etc., away from mouth (no nail biting!).
- Do not use personal pens, pencils, calculators, etc. in lab. These will be provided for you.
- Cell phones, laptops, etc. are not allowed in lab. If they become contaminated there is no way to disinfect them, and they are a distraction. No exceptions whatsoever. Computers are available in lab when needed.
- Sanitize lab bench before and after lab activities.
- Use test tube racks when moving cultures and store plates and test tubes as instructed.
- Note the location of biohazardous waste and sharps receptacles. Review the section on biohazardous waste disposal.
- Notify instructor of all spills or injuries. Clean up spills as instructed.
- If you are pregnant, immune-compromised, or live or work with someone who is, inform your physician that you are taking this class.
*Special instructions for wearing gloves:
Wearing gloves often makes the wearer feel safe, and this is one reason for wearing them. However, one must also be aware that contamination that gets on gloves can then be transferred to other surfaces, people, pens, pencils, microscopes, etc. So it is very important to follow proper glove protocols.
- Wear gloves when working with BSL 2 organisms and when staining. Your instructor will let you know when this is necessary.
- Nitrile gloves are recommended as some people have latex sensitivity. Gloves should fit snuggly. Loose gloves are hazardous in themselves.
- Gloves are flammable/melt. Use caution when using near a heat source like the Bact-incinerators and hot plates.
- If gloves become contaminated with a bacterial culture or chemical, or there is a hole in the glove, remove them aseptically as instructed, and put on a new pair.
- ALL gloves are discarded in the biohazard waste bin.
- Do not touch other surfaces while wearing gloves, including (but not limited to) things like your face, drawer handles, incubator door, microscope, door handles, sink handles, personal items, pens, pencils, calculators, computer, chairs, etc.
Video on the proper way to glove and un-glove: https://youtu.be/S4gyNAsPCbU
Biosafety Levels (BSL) for Infectious Agents: Summary only
Not known to consistently cause disease in healthy adults. Supervision by a scientist with training in microbiology or related field.
Wash hands after working and before leaving lab.
No eating, drinking, handling contact lenses, applying cosmetics, storing food.
No mouth pipetting.
Sharps/broken glassware precautions and disposal.
Decontaminate work surfaces, materials, and contaminated waste.
Lab coats recommended,
Gloves, as needed.
Associated with human disease which pose a moderate hazard.
Biohazard signage with BSL-2
Demonstrated proficiency with BSL-1.
Biosafety lab manual provided.
Lab coats recommended,
Protective eyewear, face shields (as needed),
Biosafety Cabinets used when splashes/aerosols may occur.
Indigenous or exotic agents that may cause serious or lethal disease via inhalation.
Respiratory protection as needed.
Self-closing double doors.
Ducted ventilation systems with directional airflow, air not recirculated to other areas.
Specially designed lab facilities, clothing areas, etc.
Dangerous and exotic agents, high individual risk of aerosol transmission, life threatening, frequently fatal disease, for which there are no vaccines or treatments, or agent with unknown risk.
Training in extremely hazardous infectious agents.
Clothing change, shower on exit.
Class III cabinets or Positive pressure supplied air protective suit.
Specially designed facilities to prevent dissemination.
HEPA filtered ventilation systems.
Summarized from; for more detail please visit: www.cdc.gov/biosafety/publications/bmbl5/BMBL5_sect_IV.pdf
Disposal of Hazardous Materials:
NEVER MIX CONTAMINATED REUSABLE AND DISPOSABLE ITEMS TOGETHER
NEVER PLACE USED ITEMS WITH STERILE ITEMS
NEVER DISPOSE OF ANY CONTAMINATED MATERIALS IN THE REGUALR TRASH
NEVER REMOVE ANY CONTAMINATED ITEM OR CULTURE FROM THE LABORATORY
A discard cart is provided in the lab for the disposal of biohazardous materials.
- Small biohazard sharps container--for any sharp item (broken loop, glassware, slide, etc.) that has media in it, or that is contaminated with bacteria.
- Test tube racks—contaminated test tubes. Sort the tubes according to size and place in the correct sized rack to prevent spills. Fill racks from back to front, not randomly. Remove ALL tape from caps and tubes before discarding. Test tubes are NEVER discarded in the biohazard trashcan on the floor.
- The cart may sometimes have containers for pipette disposal, flasks, etc.
If a spill occurs on the cart, thoroughly spray the rack, area, etc., with Sanisol, wait 5 minutes and then with gloves on, wipe up area with paper towels (paper towels and gloves are discarded in the biohazard trashcan on the floor).
The biohazard trashcan located on the floor next to the cart should always have a biohazard plastic liner. Do not discard anything if there isn’t a liner—let your instructor know. DO NOT overfill the biohazard trash can; inform your instructor when it is ¾ full. This trashcan is for contaminated disposable items—NO GLASS ITEMS, ever. For example,
- Plastic petri dishes
- Contaminated paper towels
- Contaminated swabs, etc.
Specifics on different materials:
Glass Test Tubes: These tubes are autoclave, washed, and re-used. Remove all tape and place upright in the appropriate sized rack on the discard cart. For broken tubes—remove cap if possible and place cap on the cart; place the broken tube in the small biohazard sharps container on the cart (clean, dry broken tubes may be discarded in the cardboard sharps box).
Plastic Petri Dishes: DO NOT remove markings; it is not necessary since the plates will be autoclaved and discarded. These are placed in the biohazard trashcan.
Contaminated paper towels and gloves: Place in the biohazard trashcan. Remove gloves carefully as instructed.
Uncontaminated paper towels: should be thrown away in a regular trashcan, not the biohazard trashcan. All gloves are discarded in the biohazard trash can.
Pipettes: All serological pipettes should be discarded in pipette trays, on each lab bench, containing Sanisol. A small biohazard container is usually provided at each station for swabs, microfuge tubes, and transfer pipettes.
Scalpels, forceps, spatulas, etc.: These items are usually placed in the pipette trays. Follow directions given by your instructor.
Glass Slides: All microscope slides once stained, will be discarded in dishes of sanisol on each lab bench. Broken contaminated slides are disposed of in the small biohazard sharps container on the discard cart (broken clean slides may be disposed of in the cardboard sharps box).
Flasks, weigh boats, blenders, etc.: When these types of additional materials are used, you will be instructed on how and where to discard them. Usually, there will be a separate cart with tubs for flasks; weigh boats are usually washed and placed on the drain board, etc. Pay careful attention to instructions.
1.3: Materials and Procedures - Biology
At the conclusion of the lab, the student should be able to:
- define the following terms: metabolism, reactant, product, substrate, enzyme, denature
- describe what the active site of an enzyme is (be sure to include information regarding the relationship of the active site to the substrate)
- describe the specific action of the enzyme catalase, include the substrate and products of the reaction
- list what organelle catalase can be found in every plant or animal cell
- list the factors that can affect the rate of a chemical reaction and enzyme activity
- explain why enzymes have an optimal pH and temperature to ensure greatest activity (greatest functioning) of the enzyme (be sure to consider how virtually all enzymes are proteins and the impact that temperature and pH may have on protein function)
- explain why the same type of chemical reaction performed at different temperatures revealed different results/enzyme activity
- explain why warm temperatures (but not boiling) typically promote enzyme activity but cold temperature typically
- decreases enzyme activity
- explain why increasing enzyme concentration promotes enzyme activity
- explain why the optimal pH of a particular enzyme promotes its activity
- if given the optimal conditions for a particular enzyme, indicate which experimental conditions using that particular enzyme would show the greatest and least enzyme activity
1.3: Materials and Procedures - Biology
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DonnaYoung.org Printables and Resources for Home, Home School, and Classrooms
Scientists know that lab reports are a very important part of every experiment. The purpose of an experiment is to answer a question by testing a hypothesis. During an experiment you may collect a lot of information, or data. But that data is not very useful unless it is organized. The purpose of a lab report is to organize and communicate what you did in your experiment. A good lab report explains exactly what you have done. It can be used to repeat the experiment or to test other hypotheses in new experiments.
Lab Report Form
Title: ______(the name of the lab or experiment)
The purpose or problem states the reason(s) why you are doing the experiment. Write down exactly the problem that will be investigated or experimented. Purposes can be stated as a question.
In a few words tell what you already know or have found about the problem that will let you make an educated guess. This is your background information from the text, teacher, or other sources. It gives the reader an understanding of underlying principles and content information of the laboratory.
What do you expect to find? The hypothesis can be stated as an "If. then. " statement. The 'If' part of the statement is based on related facts that you know to be true. The 'then' part of the statement is an educated guess on the outcome of the experiment. The hypothesis does not have to guess the correct outcome, but the experiment must be set up to test the hypothesis.
This is a list of all equipment and chemicals used to do the experiment. Please include quantities (amounts).
The procedure tells exactly what you did. Make statements in the past tense. Be specific. The procedure you use affects the results. So, it is important to be accurate in explaining what you did. The procedure is written in paragraph form.
Observations and Data:
The observations tell exactly what happened when you did the lab. An observation is measurable information that comes to you through your senses. Results include experimental (raw) data in the form of well-labeled tables, graphs, drawings and other observations. Place your observations and data in this section without discussion or comment. This is where you include any calculations made during the experiment. Answer any questions here.
Conclusions explain your observations and describe how your data relates to the problem. It is written in paragraph/essay form and should include why you did this experiment (restate the purpose/problem). You should explain in your own words what you found out or discovered. Your conclusion should state whether or not the data confirms or rejects your hypothesis. Discuss any errors as well as any patterns you see. Part of the conclusion may be a new hypothesis based on your findings and suggestions for testing the new hypothesis in a different experiment. You may also make any predictions you would expect based on what you discovered.
Tyler Fleegenshneeze August 11, 2001
Title: Making a Seismograph
Purpose: How does the magnitude of vibrations affect the amplitude of a seismograph?
Hypothesis: An increase in the magnitude of vibrations will result in an increase in amplitude of the seismograph.
Materials: clamp, metal bar, piece of string, rubber bands (2), table, pencil, two people
Procedure: I laced a piece of paper directly beneath the pen and the clamp stand. One person slowly moved the paper past the pen, as the other hit the end of the table. The first trial represented a medium magnitude movement. The second trial was the soft movement, and the third was the hardest, or the most forceful magnitude. While looking at each individual seismograph, the greatest magnitude was observed and identified. Measuring the distance from the top and bottom of the spike represented the amplitude. The data from each seismograph was recorded in the table on the following page.
a little jagged, the dots are the darkest of all the trials, the pen a little wild
the smoothest line, more dots and dots are closer together, the pen was not out of control
the most jagged, doesn't really have a certain path, the least amount of dots, pen way out of control
Conclusion: This lab investigated how the magnitude of vibrations affects the amplitude of a seismograph. In order to study the problem we created three magnitudes of movement and measured the amplitude of each with a seismograph. My results showed the trial with the greatest amplitude was trial three because the table was being hit with the most force, making the table and the pen move more than the other three trials. The trial with the least amplitude was trial two because the table was hit with the least amount of pressure. While observing the experiment, I noticed that the more vibrations or higher magnitude resulted in a higher amplitude on the seismograph. The harder the table was being hit, the higher the amplitude rose. This proves my hypothesis was correct.
I believe the results are accurate because while the experiment was in progress, the frame moved at the same rate as the table. It was proven in trials one through three that the increased magnitude of table movement caused the greatest amplitude differences on the seismograph. It is clear, therefore, that the movement of the frame also corresponds to the amplitude of the seismograph. The bar and marker shared the same relationship with the table and the frame. The more the frame moved, the greater the amplitude on the seismograph.
In order to further investigate this problem, next time I would try the experiment on a different surface and would add additional movements of varying forces for further readings on the seismograph.
This lab (experiment) investigated __________. In order to study the problem we ___________. My results showed ____________, thus proving my hypothesis was ____________. I believe the results are (accurate/inaccurate) because ____________. In order to further investigate this problem, next time I would _____________.
- Do draw a picture of the experiment, if appropriate.
- Dont say that the purpose was accomplished and then say nothing substantially more. You must include data from the lab results to demonstrate that the purpose was accomplished.
- Dont give the procedure again.
- Dont list the data again. It was already listed in the data (chart, table, etc.). You are to discuss and draw conclusions from the data.
- Dont forget to break up your ideas with more than one paragraph, if necessary. (This is referred to as an essay!)
Cheryl also suggests that the basic format suggested by Dr. Wile: Title, Date, Observations/Data, and Conclusion/Summary is adequate for 7th, 8th and 9th graders and the full format for documentation that she suggests above is optional.
Since DNA is the blueprint for life, everything living contains DNA. DNA isolation is one of the most basic and essential techniques in the study of DNA. The extraction of DNA from cells and its purification are of primary importance to the field of biotechnology and forensics. Extraction and purification of DNA are the first steps in the analysis and manipulation of DNA that allow scientists to detect genetic disorders, produce DNA fingerprints of individuals, and even create genetically engineered organisms that can produce beneficial products such as insulin, antibiotics, and hormones.
DNA can be extracted from many types of cells. The first step is to lyse or break open the cell. This can be done by grinding a piece of tissue in a blender. After the cells have broken open, a salt solution such as NaCl and a detergent solution containing the compound SDS (sodiumdodecyl sulfate) is added. These solutions break down and emulsify the fat & proteins that make up a cell membrane. Finally, ethanol is added because DNA is soluble in water. The alcohol causes DNA to precipitate, or settle out of the solution, leaving behind all the cellular components that aren’t soluble in alcohol. The DNA can be spooled (wound) on a stirring rod and pulled from the solution at this point.
Detergent, eNzymes (meat tenderizer), Alcohol
To extract DNA from cells.
Blender, split peas, salt, detergent, water, measuring cup and spoons, strainer, meat tenderizer, alcohol, test tube, glass stirring rod
- First, you need to find something that contains DNA such as split peas, fresh spinach, chicken liver, onion, or broccoli.
- Measure about 100 ml or 1/2 cup of split peas and place them in a blender.
- Add a large pinch of salt (less than 1 ml or about 1/8 teaspoon) to the blender.
- Add about twice as much cold water as the DNA source (about 200 ml or 1 cup) to the peas in the blender.
- Blend on high (lid on) for about 15 seconds.
- The blender separates the pea cells from each other, so you now have a really thin pea-cell soup.
And now, those 3 easy steps:
- Pour your thin pea-cell soup through a strainer into another container like a measuring cup or beaker.
- Estimate how much pea soup you have and add about 1/6 of that amount of liquid detergent (about 30ml or 2 tablespoons). Swirl to mix.
- Pour the mixture into test tubes or other small glass containers, each about 1/3 full.
- Add a pinch of enzymes to each test tube and stir gently. Be careful! If you stir too hard, you’ll break up the DNA, making it harder to see. (Use meat tenderizer for enzymes. If you can’t find tenderizer, try using pineapple juice or contact lens cleaning solution.)
- Tilt your test tube and slowly pour rubbing alcohol (70-95% isopropyl or ethyl alcohol) into the tube down the side so that it forms a layer on top of the pea mixture. Pour until you have about the same amount of alcohol in the tube as pea mixture.
- Alcohol is less dense than water, so it floats on top forming two separate layers.
- All of the grease and the protein that we broke up in the first two steps move to the bottom, watery layer.
- DNA will rise into the alcohol layer from the pea layer. You can use a glass stirring rod or a wooden stick to draw the DNA into the alcohol.
- Slowly turning the stirring rod will spool (wrap) the DNA around the rod so it can be removed from the liquid.
1. Does the DNA have any color?
2. Describe the appearance of the DNA.
3. Do only living things contain DNA? Explain.
Frequently Asked Questions: 1. I’m pretty sure I’m not seeing DNA. What did I do wrong?
First, check one more time for DNA. Look very closely at the alcohol layer for tiny bubbles. Often, clumps of DNA are loosely attached to the bubbles.
If you are sure you don’t see DNA, then the next step is to make sure that you started with enough DNA in the first place. Many food sources of DNA, such as grapes, also contain a lot of water. If the blended cell soup is too watery, there won’t be enough DNA to see. To fix this, go back to the first step and add less water. The cell soup should be opaque, meaning that you can’t see through it. Another possible reason for not seeing any DNA is not allowing enough time for each step to complete. Make sure to stir in the detergent for at least five minutes. If the cell and nuclear membranes are still intact, the DNA will be stuck in the bottom layer. Often, if you let the test tube of pea mixture and alcohol sit for 30-60 minutes, DNA will precipitate into the alcohol layer.
2. Why does the DNA clump together?
Single molecules of DNA are long and stringy. Each cell of your body contains six feet of DNA, but it’s only one-millionth of an inch wide. To fit all of this DNA into your cells, it needs to be packed efficiently. To solve this problem, DNA twists tightly and clumps together inside cells. Even when you extract DNA from cells, it still clumps together, though not as much as it would inside the cell.
Imagine this: the human body contains about 100 trillion cells, each of which contains six feet of DNA. If you do the math, you’ll find that our bodies contain more than a billion miles of DNA!
3. Can I use this DNA as a sample for gel electrophoresis?
Yes, but all you will see is a smear. The DNA you have extracted is genomic, meaning that you have the entire collection of DNA from each cell. Unless you cut the DNA with restriction enzymes, it is too long and stringy to move through the pores of the gel instead, all you will end up seeing is a smear.
4. Isn’t the white, stringy stuff actually a mix of DNA and RNA?
That’s exactly right! The procedure for DNA extraction is really a procedure for nucleic acid extraction. However, much of the RNA is cut by ribonucleases (enzymes that cut RNA) that are released when the cells are broken open.