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Wellesley College-BISC 209 Microbiology -Spring 2010


[1]| NIH Biosafety Levels

BISC110 Assignments/ Weekly Lab Planner

Agents Biosafety Level Practices________ Safety Equipment________ Facilities
BSL 1 These agents are not generally associated with disease in healthy people Good micro- biological practice
Hand washing
No eating, drinking, or gum chewing in the laboratory
Pipeting devices-mouth pipeting is prohibited
2 Tues. 2/2-
Wed. 2/3

environmental sampling (steubing for pop sticks or Jannsen for corers) and isolation streaking in different conditions, temps, media etc.

NOTE: MEDIA -use Janssen media-dilute broth pg 2392 (Improved culturability of soil bacteria dn isolation: to enrich for soil groups.

ENVIRONMENTAL SAMPLING: (steubing for pop sticks or Jannsen for corers) and isolation streaking in different conditions, temps, media etc.

Sample dilution for single colonies techniques also? (Wagner-NABT paper)

Plan on 4 students per site and each pair sampling from the site (so 6 samplesper lab)- most protocols the samples go into broth (see seifert)

ON own- isolation streak a new plate. Each individual.

DNA isolation- in pairs Isolating genomic DNA from soil samples (mo bio lab protocol): using mobio ultraclean soil dna purification kit Nano dropper procedure (using machine-on screen read out)

dilute dna to proper concentration and freeze for DNA library process (PCR etc see MBL flow chart and diagram lab 12 OR for rDNA library steps)

Discuss habitats and literature searches Freeze DNA ?

3 Tues. 2/9-
Wed. 2/10
DNA diversity: complete prep for pyrosequencing? OR rDNA library steps: (Use soil DNA from lab 2)

DNA library:

Clean and PCR Amplify for rDNA using Irene's universal primers (Irene's notes)- 3 hours

CULTURES: (Primary plates) observe-count, describe, select soil microbes for further culturing (compare anaerobic, aerobic, microaerophillic) - search web for pics of microbes.

techniques: colony morphology, number (see mIcrobial safari= wagner) and problem solve to "discover" 2 isolation methods (serial dilution and pour plates vs streaking for isolation)

Isolation to pure colony step: #1: How will they pick what/where on plate to isolate? (secondary plates) Each student picks different 4-6 colonies and restreaks on same media (4-6 different plates). Incubate room temp.grow 24 hours, move to CR.

Microscopy introduced: select something that looks isolated and stain and compare to stock cultures.

Control stocks for: gram pos, gram neg and capsule, acid fast and endospore

Simple stain Gram stain

What do you learn from this?

4 Tues. 2/23-
Wed. 2/24
Run gel on PCR from last time

Clone 16s rDNA using TOPO dna cloning kit and MBL lab 12 to complete transformation incubate overnight in plates (see mbl protocol)

Cultures: Examine and pick 3 isolated colonies (if needed) additional technniques:

ID from secondary cultures (Steubing protocol): on real unknown Techniques: repeat all stains from week before: at least - simple stain (3 pure colonies) gram stain etc (and be sure all stains from week 3 are performed).

make stocks from (3) isolated cultures- make 2 fresh slants per organism (each person)


stains and microscopy: on control stock orgs. -negative stain (for capsules) -acid fast -endospore

5 Tues. 3/2-
Wed. 3/3
Select 2 clones and put in 96 well plates (how? protocol ends here) and send them out? where? How long?

UNKNOWN - using stock (1-2 or 3 made) make 2 more stock slants

Techniques: all stains: on unknowns (2-3) capsule endospore acid fast etc

6 Tues.3/9. -
Wed. 3/10


Sterbing lab (9-15) soft agar deeps for motility wet mounts for motility oxygen requirements Thioglyocallate and TSA Selective and differential medial Temp optimum osmotic effect and ph optimum

7 Tues. 3/16-
Wed. 3/17
Metabolic tests (Steubing 16-27) Homework:
8 Tues. 3/30-
Wed. 3/31
Metabolic tests

Presentation, writing FAQ's

Introduce paper, practical, poster etc

9 Tues. 4/6-
Wed. 4/7
analysis of rDNA:

practice exercise in lab on own for their data

Optional complete/redo any tests not completed

10 Tues. 4/13-
Wed. 4/14
lab practical Homework: .
11 Tues. 4/20-
Wed. 4/22
in groups by habitat sampled Homework:
12 Tues. 5/4-
Wed. 5/5
Presentation, writing FAQ's

Introduce paper, practical, poster etc

End of lab

<td valign="top" align="left">BSL-1</td> <td valign="top" align="left"> </td> <td valign="top" align="left">

  • y
    • </td>

      <td valign="top" align="left">

    • Pipeting devices-mouth pipeting is prohibited
    • </td>

      <td> :</td></tr> <tr> <td valign="top" align="left">BSL-2</td> <td valign="top" align="left">These agents are associated with human disease </td> <td valign="top" align="left">

    • Limited lab access
    • Most work may be performed on a bench top
    • Biohazard warning signs
    • "Sharps" precautions
    • Biosafety manual defining any needed waste decontamination or medical surveillance policies
    • </td>

      <td valign="top" align="left">

    • Class I or II Biological Safety Cabinets (BSCs) or other physical containment devices
    • Lab coats, gloves, face protection, as needed
    • </td>

      <td valign="top" align="left">

    • Open bench-top
    • sink for hand washing is required
    • Autoclave available
    • </td></tr>

      <tr> <td valign="top" align="left">BSL-3</td> <td valign="top" align="left">

      These agents:

    • Are associated with human disease and cause illness by spreading through the air (aerosol)
    • Cause diseases that may have serious or lethal consequences
    • </td>

      <td valign="top" align="left">

      BSL-2 practice plus

    • Controlled access
    • Decontamination of all waste
    • Decontamination of lab clothing before laundering
      • </td>

        <td valign="top" align="left">

      • Class I or II Biological Safety Cabinets (BSCs) or other physical containment devices
      • Protective lab clothing, gloves, respiratory protection as needed
      • </td>

        <td valign="top" align="left">

        BSL-2 plus

      • Physical separation from access corridors
      • Self-closing, double-door access
      • Exhaust air is not recirculated
      • Negative airflow into laboratory
      • Design includes back up/redundant systems
      • </td></tr>

        <tr> <td valign="top" align="left">BSL-4</td>

        <td valign="top" align="left">

        These agents:

      • Are associated with human disease and cause illness by spreading through the air (aerosol) or have an unknown cause of transmission
      • Cause diseases that are usually life-threatening
        • </td>

          <td valign="top" align="left">

          BSL-3 practices plus

        • Clothing change before entering
        • Shower on exit
        • All material decontaminated on exit from facility
          • </td>

            <td valign="top" align="left">Class ll procedures conducted in Class III BSCs or Class I or II BSCs in combination with full-body, air-supplied, positive- pressure personnel suit  </td> <td valign="top" align="left">

            BSL-3 plus

          • Separate building or isolated zone
          • Dedicated supply and exhaust, vacuum, and decontamination systems
          • Design includes back-up/redundant systems
          • Other requirements outlined in NIH/CDC publication Biosafety in Microbiological and Biomedical Laboratories
          • </td></tr></tbody></table>


            <a class="back" href="#">back to top</a>


            </tr> </table>

            Wellesley College LABORATORY SAFETY RULES

            Laboratory safety is the responsibility of everyone working in the laboratory. The following laboratory rules are provided to help ensure your safety and the safety of others during the BISC 110 lab sessions. You should also be sure to read the Wellesley College Lab Safety Manual available at

            1. There is NO eating, drinking, chewing gum or smoking permitted in the laboratory at anytime. Smoking is not permitted anywhere in the Science Center.
            2. To avoid possible damage, coats should be hung on the hooks provided in the corridor outside the laboratory.
            3. Unneeded books may be placed in the lockers outside the laboratory or stored in your book bag under the lab bench. This reduces clutter in the lab and protects others against tripping over books or bags in the aisles.
            4. Each laboratory is equipped with fire alarms, fire extinguishers, a fire blanket, a safety shower, an eyewash and a first aid kit. Your instructor will indicate the location of each during the first laboratory period.
            5. If you have long hair, it is advisable to pin or tie it out of your face. Loose hair can contaminate an experiment or catch on fire when using open flame.
            6. Should your hair or clothing catch on fire: DO NOT RUN. Shout for help and someone will give you the fire blanket. Wrap yourself in the blanket, then fall to the floor and roll slowly back and forth to put out the flames.
            7. All accidents such as burns, cuts, eye contamination, spills, etc., should be reported to your instructor immediately.
            8. Keep your hands away from your face to prevent undesirable material being transferred to your face. Always wash your hands before leaving the laboratory.
            9. Chemical and Biological materials must be disposed of in the proper manner. Pay attention to “Clean-Up” instructions that indicate how each material should be discarded or stored. It is the responsibility of each student to follow these directions. When in doubt, double-check with your instructor.
            10. Non-toxic paper products can be placed in the wastebaskets. Disposable glassware should be put in the large blue and white recycle containers at the front of the lab. Non-disposable glassware should be rinsed out and placed either on your bench to dry or in baskets by the sink at the front of the lab. Broken glass and Pasteur pipettes should be discarded in the red “sharps” container at the front of the lab. Wet trash, such as wet leaves, can be discarded in the small garbage pail lined with a plastic bag.

            11. It is the responsibility of all Science Center personnel working in laboratories to know and to follow the provisions of the Chemical Hygiene Plan. The purpose of the Chemical Hygiene Plan is to describe proper practices, procedures, equipment and facilities for faculty, students, staff, and visitors in order to protect them from potential health hazards presented by chemicals used in the laboratory workplace, and to keep exposures below specified limits. You may view the Chemical Hygiene Plan at the Science Center Lab Safety web site at

            The following rules must be observed for the safety and convenience of everyone working in the laboratory.

            1. No mouth pipetting.

            2. Coats and sweaters must be left outside the laboratory on the clothes rack provided.

            3. Pencils, labels, or other materials should never be placed in your mouth.

            4. Transfer needles and loops are sterilized by heating the entire length of the wire to redness before and after using. Spattering is avoided by first holding the needle above the flame.

            5. If a culture is spilled, cover the area with towels and soak them with disinfectant. Notify your lab instructor immediately. Let the towels stand for half an hour. Place the contaminated materials in a container with disinfectant or in a plastic autoclave bag which can be sealed.

            6. Cultures are never to be taken from the laboratory.

            7. Inoculated media placed in the incubator must be properly labeled (i.e. with your name, date, and the nature of the specimen), and put on the assigned shelf.

            8. Gas burners must be turned down or off when not in use during the laboratory period. Be sure that gas burners are turned off at the end of the laboratory period.

            9. Eyepiece, lenses and objectives, as well as the microscope stage, should be clean before and after use. Lenses of the microscope must be wiped off with lens paper only (NO PAPER TOWELS OR KIMWIPES). Return microscopes to lockers at the end of lab.

            10. All reagents and equipment should be returned to their proper place at the end of each laboratory period.

            11. All used tubes, petri dishes, pipettes, etc. should be placed in designated receptacles at the end of the lab period. Please remove all labeling tape.

            12. All non-contaminated waste material should be placed in wastebaskets, not left on benchtops or on the floor.

            13. The laboratory desk top should be cleaned with a disinfectant at the beginning and at the end of each laboratory period.

            14. Personal accidents, such as cuts and burns, must be reported immediately to your instructor - this is for your own safety.

            15. Every student must wash her hands, with disinfectant if necessary, before leaving the laboratory.

            16. Please wear a protective lab coat or apron when you are working with cultures. If possible, avoid wearing clothing with long, full sleeves to the laboratory.

            17. Tie long hair back or put it up. If hair hangs loose, it becomes both a contamination and a fire hazard.

            18. Decontaminate work surfaces at the beginning and end of each laboratory. Use the disinfectant provided in the laboratory.

            19. Mix liquid cultures gently to avoid foaming and splashing, either of which may produce an aerosol of the bacterial culture.

            20. Open cuts should be covered with bandages. If cuts are on the hands, it is advisable to wear disposable gloves.

            21. Students who have suppressed immunity are urged to discuss your relevant medical history with your instructor, privately, before beginning this laboratory course.