Introductory Microbiology Laboratory Manual

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    General MicrobiologyLaboratory Manual

    BIOL 142

    By Paul W. Lepp

    Second Edition

    Biol 142 General Microbiology Spring 2010

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    T ABLE OF CONTENTS

    ...............................................................................LABORATORY SAFETY RULES 4

    ..........................................................................................................MICROSCOPY 11

    ..............................................................THIS PAGE INTENTIONAL LEFT BLANK 17

    ...........................................................ASEPTIC TECHNIQUE AND CULTIVATION 18

    .................................................................................................................STAINING 24

    ..............................................................THIS PAGE INTENTIONAL LEFT BLANK 29

    ...........................................................................ISOLATION OF NORMAL FLORA 30

    ..............................................................THIS PAGE INTENTIONAL LEFT BLANK 35

    ......................................................................................................EPIDEMIOLOGY 36

    ............................................................................................BACTERIAL GROWTH 43

    .................................................................EFFECTIVENESS OF HAND WASHING 51

    ..............................................................THIS PAGE INTENTIONAL LEFT BLANK 55

    ....................................................................CONTROL OF MICROBIAL GROWTH 56

    ..............................................................THIS PAGE INTENTIONAL LEFT BLANK 61

    ...............................................IDENTIFICATION OF AN UNKNOWN BACTERIUM 62

    ..............................................................THIS PAGE INTENTIONAL LEFT BLANK 71

    ............................................................................................KIRBY-BAUER ASSAY 72

    ..............................................................THIS PAGE INTENTIONAL LEFT BLANK 78

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    4

    L ABORATORY S AFETY R ULES

    BIOSAFETY IN THE LABORATORY

    Before beginning to work with microorganisms it is important to understandthe risks of working with potentially dangerous living organisms and working in alaboratory setting in general.

    The primary responsibility for your safety rests with you. You mustunderstand a nd follow the rules outlined below, as well as, tho se provided by theinstructor. The most important rule for staying safe in the laboratory is to useyour common sense.

    You should become familiar with the location and use of the safety equipmentin the laboratory which the instructor will point out. This includes a fire extinguisher,

    fire blanket, eyewash station, safety shower and gas cutoff. In addition, you shouldbe aware of all exits from the rooom.

    During this course you will be working with microorganisms that are classifedas biosafety level I and biosafety level II organisms. Biosafety levels are establishedby the U.S. Center for Disease Control and Prevention. These biosafety levels aredescribed in the section below and summarized in table 1. Bear in mind that you willbe working with biosafety level II agents that have the potential to cause disease.The simplest and most effective way to prevent transmission of potentially harmfulmicroorganisms is handwashing, as we will see in one of the following laboratory

    exercises. Hands must be washed whenever you leave the laboratory.

    BIOSAFETY LEVELS

    From: Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th

    Edition Center for Disease Control and Prevention (CDC) www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4s3.htm

    BMBL Section III

    Laboratory Biosafety Level Criteria

    The essential elements of the four biosafety levels for activities involvinginfectious microorganisms and laboratory animals are summarized in Table 1 of thissection and Table 1. Section IV (see pages 52 and 75). The levels are designated inascending order, by degree of protection provided to personnel, the environment,and the community.

    Biol 142 General Microbiology Spring 2010

    http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4s3.htmhttp://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4s3.htmhttp://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4s3.htm
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    B IOSAFETY LEVEL 1 (BSL-1)

    Biosafety Level 1 is suitable for work involving well-characterized agents notknown to consistently cause disease in healthy adult humans, and of minimalpotential hazard to laboratory personnel and the environment. The laboratory is not

    necessarily separated from the general traffic patterns in the building. Work isgenerally conducted on open bench tops using standard microbiological practices.Special containment equipment or facility design is neither required nor generallyused. Laboratory personnel have specific training in the procedures conducted in thelaboratory and are supervised by a scientist with general training in microbiology or arelated science.

    The following standard and special practices, safety equipment andfacilities apply to agents assigned to Biosafety Level 1:

    A. Standard Microbiological Practices

    1. Access to the laboratory is limited or restricted at the discretion ofthe laboratory director when experiments or work with cultures andspecimens are in progress.

    2. Persons wash their hands after they handle viable materials, after

    removing gloves, and before leaving the laboratory.

    3. Eating, drinking, smoking, handling contact lenses, applyingcosmetics, and storing food for human use are not permitted in the workareas. Persons who wear contact lenses in laboratories should also weargoggles or a face shield. Food is stored outside the work area in cabinets orrefrigerators designated and used for this purpose only.

    4. Mouth pipetting is prohibited; mechanical pipetting devices areused.

    5. Policies for the safe handling of sharps are instituted.

    6. All procedures are performed carefully to minimize the creation ofsplashes or aerosols.

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    7. Work surfaces are decontaminated at least once a day and after anyspill of viable material.

    8. All cultures, stocks, and other regulated wastes are decontaminatedbefore disposal by an approved decontamination method such asautoclaving. Materials to be decontaminated outside of the immediatelaboratory are to be placed in a durable, leakproof container and closed fortransport from the laboratory. Materials to be decontaminated outside of theimmediate laboratory are packaged in accordance with applicable local,state, and federal regulations before removal from the facility.

    9. A biohazard sign must be posted at the entrance to the laboratory whenever infectious agents are present. The sign must include the name ofthe agent(s) in use and the name and phone number of the investigator.

    10. An insect and rodent control program is in effect .

    B. Special Practices None

    C. Safety Equipment (Primary Barriers)

    1. Special containment devices or equipment such as a biologicalsafety cabinet are generally not required for manipulations of agentsassigned to Biosafety Level 1.

    2. It is recommended that laboratory coats, gowns, or uniforms be worn to prevent contamination or soiling of street clothes.

    3. Gloves should be worn if the skin on the hands is broken or if arash is present. Alternatives to powdered latex gloves should be available.

    4. Protective eyewear should be worn for conduct of procedures in which splashes of microorganisms or other hazardous materials isanticipated.

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    D. Laboratory Facilities (Secondary Barriers)

    1. Laboratories should have doors for access control.

    2. Each laboratory contains a sink for handwashing.

    3. The laboratory is designed so that it can be easily cleaned. Carpetsand rugs in laboratories are not appropriate.

    4. Bench tops are impervious to water and are resistant to moderateheat and the organic solvents, acids, alkalis, and chemicals used todecontaminate the work surface and equipment.

    5. Laboratory furniture is capable of supporting anticipated loadingand uses. Spaces between benches, cabinets, and equipment are accessiblefor cleaning.

    6. If the laboratory has windows that open to the exterior, they aretted with y screens.

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    Table 1. Biosafety levels

    BSL Agents PracticesSafety Equipment (Primary Barriers)

    Facilities(Secondary Barriers)

    1 Not known to consistentlycause disease in healthyadults

    Standard MicrobiologicalPractices None required Open bench top sinkrequired

    2 Associated with humandisease, hazard =percutaneous injury,ingestion, mucousmembrane exposure

    BSL-1 practice plus: Limited access Biohazard warning signs "Sharps" precautions Biosafety manual dening

    any needed wastedecontamination ormedical surveillance

    policies

    Primary barriers = Class Ior II