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    Purification Bacteria Culture

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    Wan Nabil
    The document describes an experiment to purify bacterial cultures using streak plate methods. Nutrient broth cultures of E. coli and Serratia marcescens were streaked in parallel lines on nutrient agar plates with a sterilized inoculating loop. The streaking was done in sectors to separate individual bacterial cells and allow discrete colonies to form. After incubation, pure cultures of each bacterium were obtained as single colonies grew separately on the plate without mixing. The document also discusses other methods for pure culture isolation like pour plating and spread plating.

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    Purification Bacteria Culture

    Uploaded by

    Wan Nabil
    3K views5 pages
    The document describes an experiment to purify bacterial cultures using streak plate methods. Nutrient broth cultures of E. coli and Serratia marcescens were streaked in parallel lines on nutrient agar plates with a sterilized inoculating loop. The streaking was done in sectors to separate individual bacterial cells and allow discrete colonies to form. After incubation, pure cultures of each bacterium were obtained as single colonies grew separately on the plate without mixing. The document also discusses other methods for pure culture isolation like pour plating and spread plating.

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    The document describes an experiment to purify bacterial cultures using streak plate methods. Nutrient broth cultures of E. coli and Serratia marcescens were streaked in parallel lines on nutrient agar plates with a sterilized inoculating loop. The streaking was done in sectors to separate individual bacterial cells and allow discrete colonies to form. After incubation, pure cultures of each bacterium were obtained as single colonies grew separately on the plate without mixing. The document also discusses other methods for pure culture isolation like pour plating and spread plating.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    3K views5 pages

    Purification Bacteria Culture

    Uploaded by

    Wan Nabil
    The document describes an experiment to purify bacterial cultures using streak plate methods. Nutrient broth cultures of E. coli and Serratia marcescens were streaked in parallel lines on nutrient agar plates with a sterilized inoculating loop. The streaking was done in sectors to separate individual bacterial cells and allow discrete colonies to form. After incubation, pure cultures of each bacterium were obtained as single colonies grew separately on the plate without mixing. The document also discusses other methods for pure culture isolation like pour plating and spread plating.

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    of 5

    PRACTICAL 10

    TITLE: THE PURIFICATION OF BACTERIAL CULTURES PURE CULTURE


    TECHNIQUES

    OBJECTIVES:
    Pure culture of bacteria can be determined by using pure culture methods that are streak
    plate, pour plate and spread plate method.

    MATERIALS/CHEMICALS/EQUIPMENT:
    Nutrient broth culture of E. coli, nutrient broth culture of Serratia marcescens, nutrient
    agar plates, sterile petri dishes, tube containing 10mL of sterile saline, 4 tubes containing
    12-15mL of nutrient agar (melted and in water bath at 45C-50C)

    METHODS/PROCEDURES:
    A. THE STREAK PLATE METHOD
    Firstly, three nutrient agar plates are prepared. One nutrient agar plate is used as the
    control. The other two plates are marked on the underside into four sectors, A, B, C and
    D. Then an inoculating loop is sterilised and a drop of E. coli is transferred to sector A. A
    series of parallel streaks are drawn over the surface of the nutrient agar by using the
    inoculating loop. The inoculating loop is then re-sterilised and is allowed to cool and a
    series of parallel streaks is drawn in sector B cutting across the end of the first streak in
    sector A. After that, the loop is re-sterilised again and is allowed to cool and a series of
    parallel streaks is drawn in sector C cutting across the ends of the streaks in sector B. The
    loop is re-sterilised again and is allowed to cool and a series of parallel streaks is drawn
    in sector D cutting across the ends of the streaks in sector C but not touching the original
    streaks in sector A. The steps above are repeated by using Serratia marcescens culture.
    After all the streaks plates are prepared, all the plates are incubated at 37C for two days
    and then Serratia marcescens is allowed to develop its characteristic red pigment at 30C.
    The inoculated cultures are examined after two days. Lastly, the streaking effectiveness in
    producing pure colonies is evaluated.

    DISCUSSION:
    In this practical, the experiment is about the purification of bacterial cultures by using
    pure culture techniques. The bacteria that are used in this experiment are Serratia
    marcescens ,E.Coli, Bacillus subtilis Streptococcus Aureus,Pseudomonas Fluorescens
    Pure culture, in microbiology is a laboratory culture containing a single species of
    organism. A pure culture is usually derived from a mixed culture, the culture that
    containing many species, by transferring a small sample into new, sterile growth
    medium in such a manner as to disperse the individual cells across the medium surface or
    by thinning the sample many fold before inoculating the new medium.(Collins,1995).
    Both methods separate the individual cells so that, when they multiply, each will form a
    discrete colony, which may then be used to inoculate more medium, with the assurance
    that only one type of organism will be present. Isolation of a pure culture may be
    enhanced by providing a mixed inoculums with a medium favouring the growth of one
    organism to the exclusion of others. There are three ways to isolate bacterial culture
    which are spread plating, streak plating and pour plating but in this experiment, streak
    plate method was used. The purpose of spread plating, streak plating and pour plating is
    to isolate individual bacterial cells (colony-forming units) on a nutrient medium
    (Delost,1997). All of these procedures (spread plating, streak plating and pour plating)
    require understanding of the aseptic technique. Asepsis can be defined as the absence of
    infectious microorganisms. However, the term is usually applied to any technique
    designed to keep unwanted microorganisms from contaminating sterile materials. After
    isolating the bacterial culture, the next step is to count the number of the bacterial
    colonies formed on or in the agar plate. In the study of microbiology, there are numerous
    occasions when it is necessary to either estimate or determine the number of bacterial
    cells in a broth culture or liquid medium. Determination of cell numbers can be
    accomplished by a number of direct or indirect methods. The methods include standard
    plate counts, turbidimetric measurements, visual comparison of turbidity with a known
    standard, direct microscopic counts, cell mass determination, and measurement of cellular
    activity(Collins,1995). From this experiment after 24 hours the result showed that there

    are no formation of single colony of each bacteria that been observed. These are due to
    some mistakes that have been done during this experiment. This mistakes lead to get
    some error in the result. The errors that have been identified which are the time taken for
    the loop to cool down were too short and during the streaking process the position of
    inoculating loop are not inappropriate. This is because, the correct technique the loop
    must in straight position. The precaution that need to be taken during the experiments
    such as the loop is hold at 60 and pass through the flame until the wire becomes red-hot
    along entire length and then cool it for 10 seconds before use it. Other than that, during
    the streaking process was been done near the heat sources.

    CONCLUSION:
    As the conclusion, the pure culture of bacteria can be obtained by using streak plate, pour
    plate and spread plate method.

    QUESTIONS:
    1.

    At what temperature does the agar dissolve? At what temperature does it solidify?
    The agar dissolve at 85C to 100C and the agar solidify at 32C to 40C.

    2.

    Why is the agar cooled to approximately 45C before preparing pour plates?
    The agar is cooled to approximately 45C before preparing pour plates because to
    reduced condensation and provide easier handling of liquid medium. Other than that, it is
    necessary to let the agar cooled so that the bacteria will not die as the bacteria are
    transferred into the agar.

    3.

    Why are the Petri dishes inverted during incubation?


    Petri dishes are inverted during incubation to prevent contamination. It is also to prevent
    condensation from forming at the top of the lid that can contaminate the specimen
    therefore by inverting the specimen will remain unaffected.

    4.

    How can you prevent splattering when flaming a loop which has just been used to
    transfer a culture?
    To prevent the splattering when flaming the loop which has just been used to transfer a
    culture, the loop should be introduced into the cooler part of the flame so that it heats up
    gently before to the hottest part of the flame.

    REFERENCES:
    Collins.1995.Chapter 6 in Collins and Lynes Microbiological Methods,7th Ed.ButterworthHeineiman.

    Delost.1997.Chapter 1 in Introduction to Diagnostic Microbiology.Mosby,Inc.,St.Louis.

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