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    Pure Culture Techniques (Lab Exercise 9)

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    1. Pure culture techniques are used to isolate a single species of bacteria from a mixed population for laboratory study. 2. There are several methods for obtaining a pure culture, including the streak plate method and pour plate method, which involve diluting and spreading a sample on an agar plate to isolate individual colonies. 3. To grow microorganisms, an appropriate culture medium must be selected to provide necessary nutrients, and a suitable environment with proper temperature, pH, and oxygen levels must be maintained in the incubator.

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    Pure Culture Techniques (Lab Exercise 9)

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    1. Pure culture techniques are used to isolate a single species of bacteria from a mixed population for laboratory study. 2. There are several methods for obtaining a pure culture, including the streak plate method and pour plate method, which involve diluting and spreading a sample on an agar plate to isolate individual colonies. 3. To grow microorganisms, an appropriate culture medium must be selected to provide necessary nutrients, and a suitable environment with proper temperature, pH, and oxygen levels must be maintained in the incubator.

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    1. Pure culture techniques are used to isolate a single species of bacteria from a mixed population for laboratory study. 2. There are several methods for obtaining a pure culture, including the streak plate method and pour plate method, which involve diluting and spreading a sample on an agar plate to isolate individual colonies. 3. To grow microorganisms, an appropriate culture medium must be selected to provide necessary nutrients, and a suitable environment with proper temperature, pH, and oxygen levels must be maintained in the incubator.

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    187 views26 pages

    Pure Culture Techniques (Lab Exercise 9)

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    1. Pure culture techniques are used to isolate a single species of bacteria from a mixed population for laboratory study. 2. There are several methods for obtaining a pure culture, including the streak plate method and pour plate method, which involve diluting and spreading a sample on an agar plate to isolate individual colonies. 3. To grow microorganisms, an appropriate culture medium must be selected to provide necessary nutrients, and a suitable environment with proper temperature, pH, and oxygen levels must be maintained in the incubator.

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    Pure Culture Techniques

    (Lab Exercise 9)
    Culturing Microorganisms
    Microorganisms are ideal lab subjects
    - grow rapidly
    -are small in size
    -many generations can be
    observed
    -results can be applied to other
    organisms
    Pure Culture
    Consists of only a single type of
    organism.
    Rarely exist in nature. Naturally
    exist as mixed cultures.
    Pure cultures must be obtained
    artificially
    Pure Culture techniques

    Goal: To isolate a single species of bacteria form


    a mixed population.
    Isolation
    • Microorganisms occur in huge numbers.
    • *Isolation of single species (pure culture)
    is done by :
    • Streak plate method
    • Pour plate method

    *Based on diluting sample out to a point where


    a single cell will give rise to a single colony
    Streak Plate
    (Quadrant Streak)
    Pour Plate
    * We will do loop dilution
    technique (see fig 9.5)
    Growing a Pure Culture
    • To grow microorganisms:
    – nutrients must be supplied in the
    culture medium
    (Agar is the solidifying material used in solid media. It is
    an extract of seaweed that melts at 100 C and solidifies at
    o

    about 42 C)
    o

    – A suitable environment must be


    provided
    Culture Medium
    • Enriched Media- is formulated with all the
    necessary ingredients for a wide variety of organisms
    and grows a multitude of bacterial species (example:
    Trypticase Soy Agar –TSA)
    • Selective Media- are formulated with
    ingredients that inhibit the growth of some bacteria,
    such as an antibiotic, but enhance growth of the target
    organism (example: Blood Agar –BA)
    • Differential Media- includes ingredients,
    such as chemical indicators, that produce observable
    differences between species of bacteria. (example:
    MacConkeys agar –MAC)
    Suitable Environment
    • Temperature
    • pH
    • Oxygen
    Temperature
    • Microbes grow over a range of
    temperatures ( nat. environ)
    • Temperatures maintained in
    –Incubators
    –Waterbaths
    pH
    • Optimal pH varies
    • Bacteria neutral pH 6.5-7.5
    • Buffers are added to neutralize the
    by-products of bacteria
    metabolism
    Oxygen
    • microbes respond differently
    –Strict aerobes
    –Strict anaerobes
    –facultative aerobes
    –facultative anaerobes
    –Microaerophiles
    O2 must be provided , limited or
    excluded
    Colony morphology
    Colony morphology
    Enumeration of Bacteria
    (Lab Exercise 20)
    Methods for Determining Bacteria
    Numbers
    Goal: To estimate the number of bacteria in a sample

    Possible Methods (We will not do these):


    1)Microscopic Counts
    - Uses special slides with a grid (Petroff-Hauser slides)
    - Counts dead and living cells
    - Must know exact volume
    2) Most Probable Number Counts
    - Used in large scale applications
    - tracks metabolic by-products (e.g. acid/gas in coliform
    bacteria) and compares results to established statistical
    tables to estimate probable numbers of bacteria in sample
    - Counts Dead and living
    Microscopic Counts
    (Petroff-Hauser slides)
    Methods for Determining Bacteria
    Numbers continued
    3) Standard Plate Count
    - Sample serially diluted and
    dilutions plated
    - Count multiplied by dilution
    factor
    - Technique assumes that 1 cell
    will give rise to 1 colony
    (colony forming unit (CFU)
    - Counts ONLY living (viable)
    organisms!

    (We will do this method)


    Standard Plate Count

    Chapter 6
    Chapter 6
    Determination of Growth by Optical
    Density
    • Turbidity (cloudiness)
    – Measured by a spectrophotometer at wavelength 686nm
    – Scale
    • %Transmittance (amount of light passing through)(measured)
    • Optical Density or Absorbance(calculated)

    To convert between the optical density (OD)and transmittance(T)


    scales, use the equation:

    OD = 2 – Log (%T)

    Chapter 6
    Chapter 6
    Chapter 6

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