laboratory manual - .2 1. instructions to the microbiology laboratory and good laboratory practices

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    FIRST SEMESTER

    DEPARTMENT OF MICROBIOLOGY

    ST ALOYSIUS COLLEGE (AUTONOMOUS), MANGALURU

    LABORATORY

    MANUAL

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    CONTENTS

    SL.NO EXPERIMENT

    PAGE

    NO

    1

    INSTRUCTIONS TO THE MICROBIOLOGY LABORATORY AND GOOD

    LABORATORY PRACTICES. 2

    2 STUDY OF GLASSWARE USED IN MICROBIOLOGICAL WORK 4

    3

    STUDY OF MICROBIOLOGY LABORATORY EQUIPMENT

    /INSTRUMENTS 7

    4 MICROSCOPY 17

    5 PREPARATION OF BACTERIAL SMEARS 24

    6 SIMPLE STAINING 27

    7 GRAM STAIN 29

    8

    CAPSULE STAINING WET INDIA INK METHOD AND DRY INDIA INK

    METHOD 34

    9 ENDOSPORE STAINING METHOD- SCHAEFFER FULTON METHOD 36

    10

    DEMONSTRATION OF BACTERIAL MOTILITY BY HANGING DROP

    METHOD 39

    11

    PREPARATION OF CULTURE MEDIA: NUTRIENT BROTH AND

    NUTRIENT AGAR 41

    12

    SERIAL DILUTION TECHNIQUE. VIABLE COUNT BY STANDARD PLATE

    COUNT METHOD 46

    13 ISOLATION BY STREAK PLATE METHOD 49

    14 STUDY OF COLONY MORPHOLOGY (CULTURAL CHARACTERISTICS). 50

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    1. INSTRUCTIONS TO THE MICROBIOLOGY LABORATORY AND GOOD LABORATORY PRACTICES.

    1. Wash your hands with disinfectant soap when you arrive at the lab and again before you leave.

    2. Absolutely no food, drinks, chewing gum, or smoking is allowed in the laboratory.

    3.Do not put anything in your mouth such as pencils, pens, labels, or fingers.

    4.Do not store food in areas where microorganisms are stored.

    5. Purchase a lab coat and safety glasses, bring them to class, and use them. Alternatively, a long sleeved

    shirt that buttons or snaps closed is acceptable protective clothing.

    6. Avoid loose fitting items of clothing. Wear appropriate shoes (sandals are not allowed) in the

    laboratory.

    7. Keep your workspace free of all unnecessary materials.

    8. Disinfect work areas before and after use with 70% ethanol or fresh 10% bleach. Laboratory equipment

    and work surfaces should be decontaminated with an appropriate disinfectant on a routine basis, and

    especially after spills, splashes, or other contamination.

    9. Label everything clearly. 8. Replace caps on reagents, solution bottles, and bacterial cultures.

    10. Do not open Petri dishes in the lab unless absolutely necessary.

    11. Inoculating loops and needles should be flame sterilized in a Bunsen burner before you lay them down.

    12.. Turn off Bunsen burners when not in use.

    13.Long hair must be restrained if Bunsen burners are in use.

    14. Treat all microorganisms as potential pathogens.

    Use appropriate care and do not take cultures out of the laboratory.

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    15. Wear disposable gloves when working with potentially infectious microbes or samples (e.g., sewage).

    If you are working with a sample that may contain a pathogen, then be extremely careful to use good

    bacteriological technique.

    16. Sterilize equipment and materials.

    17. Never pipette by mouth. Use a pipetting aid or adjustable volume pipettors.

    18. Do not lick labels. Use only self-stick labels for the identification of experimental cultures.

    19. Consider everything a biohazard. Do not pour anything down the sink. Autoclave liquids and broth

    cultures to sterilize them before discarding.

    20. Dispose of all solid waste material in a biohazard bag and autoclave it before discarding in the regular

    trash.

    21.. Familiarize yourself with the location of safety equipment in the .

    22. Dispose of broken glass in the broken glass container.

    23.Dispose of razor blades, syringe needles, and sharp metal objects in the sharps container.

    24. Report spills and accidents immediately to your instructor. Clean small spills with care .

    25. Report all injuries or accidents immediately to the instructor, no matter how small they seem.

    &&&&&&&&&&&&

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    2. STUDY OF GLASSWARE USED IN MICROBIOLOGICAL WORK

    1)CULTURE TUBES AND PETRI DISHES

    Glass test tubes and glass or plastic Petri dishes are used to

    cultivate microorganisms. A suitable nutrient medium in the form

    of broth or agar may be added to the tubes, while only a solid

    medium is used in Petri dishes. A sterile environment is

    maintained in culture tubes by various types of closures.

    Historically, the first type, a cotton plug, was developed by

    Schreder and von Dusch in the nineteenth century. Today most

    laboratories use sleevelike caps (Morton closures) made of metal,

    such as stainless steel, or heat-resistant plastics. The advantage of

    these closures over the cotton plug is that they are labor-saving

    and, most of all, slip on and off the test tubes easily.

    2) Petri dishes provide a larger surface area for growth and

    cultivation. They consist of a bottom dish portion that contains the medium and a larger top portion that

    serves as a loose cover.

    For routine purposes, dishes approximately 15 cm in diameter are used.

    The sterile agar medium is dispensed to previously sterilized dishes from

    molten agar deep tubes containing 15 to 20 ml of medium, or from a

    molten sterile medium prepared in bulk and contained in 250-, 500-, and

    1000-ml flasks, depending on the volume of medium required. When

    cooled to 40C, the medium will solidify. Remember that after inoculation, Petri dishes are incubated in an

    inverted position (top down) to prevent condensation formed on the cover during solidification from

    dropping down onto the surface of the hardened agar.

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    3) Pipettes: A pipette is another instrument used for aseptic transfers. Pipettes are similar in function to

    straws; that is, they draw up liquids. They are made of glass or plastic drawn out to a tip at one end and

    with a mouthpiece forming the other end. They are calibrated to deliver different volumes depending on

    requirements. Pipettes may be sterilized in bulk inside canisters, or they may be wrapped individually in

    brown paper and sterilized in an autoclave or dry-heat oven.

    4). Slides: A microscope slide is a thin flat piece of glass, typically 75 by 26 mm (3 by 1 inches) and about

    1 mm thick, used to hold objects for examination under a microscope.

    Cavity Slide: These have a depression in the slide. These slides are useful as moist

    chambers for observing hanging drops.

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    5) Cover slips: These are a thin flat piece of transparent material,usually square or rectangular about 20

    mm in wide a nd fraction of mm thick that is placed over objects for viewing with microscope.

    6) Measuring cylinders,7) Conical Flask,8) Volumetric Flask, 9) Beaker,10) Durham tubes .11)Bent

    glass rod( Spreader), 12) Glass rod.

    Transfer Instruments: Microorganisms must be transferred from one vessel to

    another or from stock cultures to various media for maintenance and study. Such a

    transfer is called subculturing and must be carried out under aseptic conditions to

    prevent possible contamination.

    Wire loops and needles are made from inert metals such as Nichrome or platinum

    and are inserted into metal shafts that serve as handles. They are extremely durable

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    instruments and are easily sterilized by incineration in the blue(hottest) portion of the Bunsen burner

    flame

    &&&&&&&&&&&&&&&&&

    3. STUDY OF MICROBIOLOGY LABORATORY EQUIPMENT /INSTRUMENTS

    Autoclave: Autoclave:

    Autoclave is the nucleus of a microbiology laboratory. It is used not only to sterilize liquid substances such

    as prepared media and saline (diluents) solutions, but also to sterilize glasswares, when required.

    It has the same working principle

    as a domestic pressure cooker.

    The maximum temperature that

    can be obtained by boiling water

    in an open container is 100C

    (boiling point of water). This

    temperature is sufficient to kill

    only the non-spore formers, but it

    is difficult to kill the spore-

    forming bacteria at this

    temperature, as they escape by

    forming heat resistant spores. It

    takes very long time to kill the

    spores at this temperature.

    On the other hand, when water is

    boiled in a closed container, due to increased pressure inside it, the boiling point elevates and steam

    temperature much beyond 100C can be obtained. This high temperature is required to kill all the bacteria

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    including the heat resistant spore-formers. Steam temperature increases with increase in steam pressure

    In operating a standard vertical autoclave, sufficient water is poured into it. If water is too less, the bottom

    of the autoclave gets dried during heating and further heating damages it. The materials to be sterilized

    are covered with craft paper and arranged on an aluminium or wooden frame kept on the bottom of the

    autoclave, otherwise if the materials remain half-submerged or floating, they tumble during boiling and

    water may enter. The autoclave is closed perfectly airtight only keeping the steam release valve open.

    Then, it is heated over flame or by the in-built heating element. Air inside the autoclave should be allowed

    to escape completely through this valve. When water vapour is seen to escape through the valve, it is

    closed.

    Temperature and pressure

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