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    Experiment No. 2: Preparation of Basic Solid Media

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    mir shifayat
    This document provides instructions for preparing basic solid media (agar slants and plates) for growing microorganisms. It describes mixing nutrient ingredients with agar, autoclaving the mixture, cooling and pouring it into petri dishes or allowing it to solidify in test tubes at a slant. The prepared media can be used to culture microbes by streaking or spotting them on the surface and incubating the plates upside down to retain moisture and prevent contamination. Questions are also provided about allowing agar to solidify in slanted test tubes and flaming inoculation loops between streaks.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd

    Experiment No. 2: Preparation of Basic Solid Media

    Uploaded by

    mir shifayat
    59 views3 pages
    This document provides instructions for preparing basic solid media (agar slants and plates) for growing microorganisms. It describes mixing nutrient ingredients with agar, autoclaving the mixture, cooling and pouring it into petri dishes or allowing it to solidify in test tubes at a slant. The prepared media can be used to culture microbes by streaking or spotting them on the surface and incubating the plates upside down to retain moisture and prevent contamination. Questions are also provided about allowing agar to solidify in slanted test tubes and flaming inoculation loops between streaks.

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    experiment noor salam

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    12_10_2018

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    This document provides instructions for preparing basic solid media (agar slants and plates) for growing microorganisms. It describes mixing nutrient ingredients with agar, autoclaving the mixture, cooling and pouring it into petri dishes or allowing it to solidify in test tubes at a slant. The prepared media can be used to culture microbes by streaking or spotting them on the surface and incubating the plates upside down to retain moisture and prevent contamination. Questions are also provided about allowing agar to solidify in slanted test tubes and flaming inoculation loops between streaks.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    59 views3 pages

    Experiment No. 2: Preparation of Basic Solid Media

    Uploaded by

    mir shifayat
    This document provides instructions for preparing basic solid media (agar slants and plates) for growing microorganisms. It describes mixing nutrient ingredients with agar, autoclaving the mixture, cooling and pouring it into petri dishes or allowing it to solidify in test tubes at a slant. The prepared media can be used to culture microbes by streaking or spotting them on the surface and incubating the plates upside down to retain moisture and prevent contamination. Questions are also provided about allowing agar to solidify in slanted test tubes and flaming inoculation loops between streaks.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 3

    Dept. of Chem. Engg. Biochem. Eng. Lab. N.I.T.

    Srinagar

    EXPERIMENT No. 2
    Preparation of Basic Solid Media

    Table of Contents
     Objectives
     Introduction
     List of Reagents and Instruments
     Procedures
     Discussions
     Questions
     Comments

    Objectives
    To prepare basic solid media as agar slants and agar plates

    Introduction
    Liquid solid media containing nutrient (broths) are usually solidified by the addition of agar.
    Agar-agar (often called simply agar) is a complex polysaccharide (carbohydrate) consisting of 3,
    6-anhydro-L galactose and D galacto-pyranose, free of nitrogen, produced from various red algae
    belonging to Geladium, Gracilaria and other genera. It liquefies on heating to 96 0C and hardens
    into a jelly on cooling to 40-45 0C. The solidified medium kept in a Petri dish provides an
    artificial environment suitable for rapid growth of microorganisms. While in the liquefied state,
    solid media can be added in test tubes, which are either allowed to cool and harden in a slanted
    position producing agar slants or allowed to harden in a upright position producing agar deep
    tubes.

    List of Reagents and Instruments

    A. Equipment

     Erlenmeyer flasks
     Culture tubes
     Petri dishes, 100*15mm
     Bunsen burner
     Autoclave
     Balance
     Magnetic Stirrer or stirring rod
     Incubator, 37ºC
     Refrigerator

    B.Tech. 8th Sem. March -2019


    1
    Dept. of Chem. Engg. Biochem. Eng. Lab. N.I.T. Srinagar

    B. Reagents

     Nutrient media:
    o Yeast extract
    o Peptone
    o Agar
    o Glucose
     HCl, 0.1 N solution
     KOH, 0.1 N solution

    Procedures

    1. Petri dish and agar slant preparation

    Mix the following nutrient ingredients in proportion to the amount required. Note that a Petri
    dish pour requires about 12 ml and a slant needs 6 ml. Adjust the pH to the desired value
    (pH=7.0) with 0.1N HCl or 0.1N KOH. If a large number (more than 5) of plates are to be
    poured, mix the nutrient in 1-2 liter flasks. It is difficult to handle flasks larger than 2 liters with
    one hand. If only a few (less than 5) are needed, the nutrient can be divided and poured into test
    tubes, each holding enough media for one Petri dish. The advantage of using test tubes is that
    they can be autoclaved separately and may later be heated to melt the agar in a beaker of boiling
    water. The liquefied agar may then be poured into a Petri dish directly from the test tube.
    However, the use of test tubes is not practical when making large quantities of agar plates.

    YPG (east extract- Peptone- Glucose) Agar


    o Yeast extract 5g
    o Peptone 10g
    o Glucose 5g
    o Agar 15g
    o Add water to make 1 liter

    Cover the flask with a beaker or a piece of aluminum foil. Autoclave the media for 20 minutes at
    121 0C temperature and 15 psi pressure. The heat of sterilization will dissolve the agar. Set the
    media on a bench top and cool until the flask can be handled with your bare hands. Wrap paper
    towel around the neck of the flask when pouring agar into Petri dishes just in case the media
    reheats the glassware as the hot liquid passes through the neck in the process of pouring. Of
    course, one cannot pour out a block of solidified agar if the flask is cooled excessively. The agar
    will solidify at approximately 42ºC; thus, the temperature window for handling the liquefied agar
    is quite narrow. If the agar starts to solidify at the bottom as a result of not pouring fast enough,
    the flask can be gently heated with a flame to raise the temperature and melt the agar.

    Stack sterile Petri dishes in a row, three or four per stack, on a disinfected lab bench. The number
    of dishes per stack can be adjusted according to the size of the student's hand. With the right
    hand holding the neck of the flask, open the cover of the lowest Petri dish with the left hand just
    wide enough to pour the media. (The top cover is the larger dish of the two; the actual dish is the

    B.Tech. 8th Sem. March -2019


    2
    Dept. of Chem. Engg. Biochem. Eng. Lab. N.I.T. Srinagar

    smaller one.) Pour about 12 ml into each Petri dish. If too little agar is poured, there may not be
    enough to cover the dish or the agar plate will dry up easily. If too much is poured, the cover dish
    will come in contact with the nutrient agar, leaving no room for microbial growth. The plates are
    rendered useless either way. After pouring the bottom dish, pour the second one from the bottom,
    and so on until the entire stack is poured. Proceed to the next stack until all dishes are poured.

    Leave the plates undisturbed until the agar solidifies. The plates may now be streaked.

    Un-inoculated agar plates may be stacked and stored upside down in a refrigerator for about 3
    months if not used immediately. The inverted position ensures that water does not condense on
    the bottom of the cover plate and retards the loss of moisture. An inoculated agar plate is also
    incubated and stored upside down to retain moisture. In addition, the water condensate can act as
    a medium for transport of cells from one location on an agar plate to another. The slants may be
    stored in a refrigerator for over a year if culture tubes with screw caps are used.

    Incubation of the inoculated plates should last for about 48 hours. They are stored in a
    refrigerator, and the culture can stay viable for over one month in agar plates or for one year in
    sealed slants. Prolonged storage of the plates in the incubator dries up the agar and kills the
    culture. The severe loss of moisture can be visually detected by the appearance of macroscopic
    cracks that develop in the agar plate.

    The used agar plates and other biological wastes should be autoclaved in a pan to destroy all
    biological activities before being disposed.

    Questions

    1. Why is the agar in test tubes allowed to solidify in a slanted position?


    2. During the preparation of a streak plate, why flame the loop between different sets of
    strokes?

    Observe and report

    Task

    1. Cough and thumb some of the agar plates and leave others untouched. Incubate them at
    37 0C. Comment your observation after 2 – 3 days.

    B.Tech. 8th Sem. March -2019


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