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    Home > Chemicals Industry > Chemical Technology > Microbiological analysis of water (3)

    Microbiological analysis of water (3)

    • Last Update: 2021-09-21
    • Source: Internet
    • Author: User
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    3.


    (1) The scope of application


    (2) Instruments


    (3) Medium


    1) Lactose peptone culture solution


    a.


    · Peptone , 10g


    ·Beef extract, 3g


    ·Lactose, 5g


    · Sodium chloride , 5g


    · 1.


    ·Distilled water, 1000ml


    b.


    2) Three times concentrated lactose peptone culture solution


    3) Fuchsin sodium sulfite medium (for multi-tube fermentation)


    a.


    ·Peptone, 10g
    .

    ·Lactose, 10g
    .

    · Dipotassium hydrogen phosphate , 3.
    5g
    .

    ·Agar, 15~30g
    .

    ·Distilled water, 1000ml
    .

    · Anhydrous sodium sulfite , about 5g
    .

    ·Preparation of 5% basic fuchsin ethanol base, 20ml
    .

    b.
    Preparation of stock medium
    .
    First add the agar to 900ml of distilled water, heat to dissolve, then add dipotassium hydrogen phosphate and peptone, mix to dissolve, then make up to 1000ml with distilled water, adjust the pH to 7.
    2~7.
    4, filter with absorbent cotton or flannel while it is hot, Then add lactose, mix and dispense quantitatively into flasks, sterilize in an autoclave at 115°C for 20 minutes, and store in a cool and dark place for later use
    .

    c.
    Preparation of plate culture medium
    .
    Heat and melt the stock medium prepared by the above method.
    According to the capacity of the medium in the flask, use a sterile straw to draw a certain amount of 5% alkaline fuchsin ethanol solution in proportion and place it in a sterile empty test tube
    .
    Then weigh the required anhydrous sodium sulfite in another sterile empty test tube, add a little sterile water to dissolve it, and place it in a boiling water bath to boil for 10 minutes for sterilization
    .

    Use a sterile straw to suck up the sterilized sodium sulfite solution and add it dropwise to the alkaline fuchsin ethanol solution until the dark red color fades to light pink
    .
    Add this mixture of sulphurous acid and basic fuchsin to the melted stock medium and mix it thoroughly (to prevent air bubbles), and pour this medium into a sterilized empty petri dish immediately.
    After cooling and solidifying, put it in the refrigerator for later use
    .
    This kind of prepared medium should not be stored in the refrigerator for more than two weeks.
    If the medium has changed from light red to dark red, it cannot be used again
    .

    4) Eosin Methylene Blue Medium
    .

    a.
    Ingredients
    .

    ·Chen Chen, 10g
    .

    ·Lactose, 10g
    .

    · Dipotassium hydrogen phosphate , 2g
    .

    ·Agar, 20~30g
    .

    ·Distilled water, 1000ml
    .

    ·2% Eosin aqueous solution, 20ml
    .

    ·0.
    5% Methylene Blue aqueous solution, 13ml
    .

    b.
    Preparation of stock medium
    .
    Add agar to 900ml of distilled water, heat to dissolve, then add dipotassium hydrogen phosphate and peptone, mix well to dissolve, and then make up to 1000ml with distilled water, adjust the pH to 7.
    2-7
    .
    Filter it with absorbent cotton or flannel while it is hot, add lactose, mix and dispense quantitatively into flasks, sterilize in an autoclave at 115°C for 20 minutes, and store in a cool and dark place for later use
    .

    c.
    Preparation of plate culture medium
    .
    Heat and melt the stock medium prepared by the above method
    .
    According to the capacity of the culture medium in the flask, use a sterile straw to draw a certain amount of sterilized 2% eosin solution and a certain amount of sterilized 0.
    5% methylene blue aqueous solution respectively, and add them to the melted reserve agar.
    Mix well (to prevent bubbles), immediately pour this medium into a sterilized empty petri dish, and place it in the refrigerator for later use after it has cooled and solidified
    .

    (4) Detection steps
    .

    1) Initial fermentation test
    .
    In 2 large test tubes or flasks (with an inverted tube inside) each containing 50ml of sterilized three-fold concentrated lactose peptone culture solution, add 100ml of water sample by aseptic operation; each contains 5ml of sterilized 5ml In the test tube (with an inverted tube inside) cultured with triple-concentrated lactose peptone, add 10 ml of each water sample aseptically, mix well, and place it in a constant temperature at 37°C for 24 hours
    .

    2) The plate is separated
    .
    After culturing for 24 hours, inoculate the acid-producing and gas-producing and acid-producing fermentation tubes respectively on fuchsin sodium sulfite medium or eosin blue medium, and then place them at 37°C for 18-24 hours, and select those that meet the following requirements For characteristic colonies, take a small part of the colonies for smear, Gram stain, and microscopic examination
    .

    Colonies on magenta sodium sulfite medium: purple-red colonies with metallic luster; dark red colonies with no or slightly metallic luster; pale red colonies with darker center color; colonies on eosin blue medium ; Dark purple black, colonies with metallic luster; purple black, colonies with no or slightly metallic luster; lavender red, colonies with darker center color
    .

    3) Double fermentation test
    .
    If the colony examined by the smear microscopy is Gram-negative abacillus, pick another part of the colony and inoculate it in a normal concentration lactose peptone culture solution (with an inverted tube inside); each tube can be inoculated and isolated from the same initial The most typical colony of the fermentation tube is 1 to 3, and then placed in a 37 ℃ constant temperature box for 24 hours, there are acid gas production (no matter how much gas in the inverted tube is based on acid production and gas production theory), it is confirmed that there are total coliform bacteria The group exists
    .

    4) Check Table 4.
    1.
    2 based on the number of positive tubes (bottles) that have confirmed the presence of total coliforms, and report the total coliforms per liter of water sample
    .

    Table 4.
    1.
    2 Total coliform count checklist

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