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Biology LibreTexts

1.3: Materials and Procedures

  • Page ID
    40151
  • Materials

    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)

    Procedures

    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.

    GENERAL GUIDELINES:

    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.

    HANDLING CHEMICALS

    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.

    DISSECTIONS—Special Precautions

    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.

    MICROORGANISMS—Special Precautions

    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)

    _____________________

    Date

    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.

    1. Safety goggles or safety glasses with side shields are required for laboratory procedures involving bacterial cultures.
    2. 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.
    3. Wear gloves when handling BSL 2 microorganisms or hazardous chemicals, including stains.*
    4. 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.
    5. Wash hands after entering and before exiting the laboratory.
    6. Tie back long hair.
    7. Do not wear dangling jewelry, scarfs, etc.
    8. No food, gum, or water in the lab.
    9. Do not touch face, including eyes, or use cosmetics in lab.
    10. Keep hands, pens, pencils, etc., away from mouth (no nail biting!).
    11. Do not use personal pens, pencils, calculators, etc. in lab. These will be provided for you.
    12. 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.
    13. Sanitize lab bench before and after lab activities.
    14. Use test tube racks when moving cultures and store plates and test tubes as instructed.
    15. Note the location of biohazardous waste and sharps receptacles. Review the section on biohazardous waste disposal.
    16. Notify instructor of all spills or injuries. Clean up spills as instructed.
    17. 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.

    1. Wear gloves when working with BSL 2 organisms and when staining. Your instructor will let you know when this is necessary.
    2. Nitrile gloves are recommended as some people have latex sensitivity. Gloves should fit snuggly. Loose gloves are hazardous in themselves.
    3. Gloves are flammable/melt. Use caution when using near a heat source like the Bact-incinerators and hot plates.
    4. 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.
    5. ALL gloves are discarded in the biohazard waste bin.
    6. 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

    BSL

    Agents

    Practices

    Safety Equipment

    (Primary Barriers)

    Facilities

    (Secondary Barriers)

    1

    Not known to consistently cause disease in healthy adults. Supervision by a scientist with training in microbiology or related field.

    Controlled access.

    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.

    Minimize splashes/aerosols.

    Decontaminate work surfaces, materials, and contaminated waste.

    Biohazard posting.

    Pest control.

    Lab coats recommended,

    Protective eyewear,

    Gloves, as needed.

    Limited access.

    Sink.

    2

    Associated with human disease which pose a moderate hazard.

    BSL-1 plus:

    Limited access.

    Biohazard signage with BSL-2

    Demonstrated proficiency with BSL-1.

    Biosafety lab manual provided.

    Lab coats recommended,

    Protective eyewear, face shields (as needed),

    Gloves, required.

    Biosafety Cabinets used when splashes/aerosols may occur.

    BSL-1 Plus:

    Autoclave

    Eyewash station.

    3

    Indigenous or exotic agents that may cause serious or lethal disease via inhalation.

    BSL-2 plus:

    Specific training.

    BSC.

    BSL-2 plus:

    BSC.

    Protective clothing

    Respiratory protection as needed.

    BSL-2 plus:

    Restricted access.

    Physical separation.

    Self-closing double doors.

    Negative airflow.

    Ducted ventilation systems with directional airflow, air not recirculated to other areas.

    Specially designed lab facilities, clothing areas, etc.

    4

    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.

    BSL-3 plus:

    Training in extremely hazardous infectious agents.

    Clothing change, shower on exit.

    BSL-3 plus:

    Class III cabinets or Positive pressure supplied air protective suit.

    Medical surveillance.

    BSL-3 plus:

    Specially designed facilities to prevent dissemination.

    Secured facility.

    Decontamination protocols.

    Redundant systems.

    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

    Discard Cart:

    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).

    Biohazard Trashcan:

    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
    • Gloves
    • 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.

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