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    Maru Mengesha Worku 18BBT0285
    Bacteria can be cultured using various methods including streak culture, lawn culture, stab culture, pour plate culture, and broth culture. Key steps in bacterial culture include maintaining aseptic technique and providing appropriate conditions for growth such as aerobic or anaerobic environments. Viruses require living host cells for replication and are cultured using animals, embryonated eggs, and tissue culture techniques. Detection of viral growth involves monitoring for cytopathic effects, hemadsorption, interference effects, and other indicators.

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    Reference Material I 19

    Uploaded by

    Maru Mengesha Worku 18BBT0285
    9 views40 pages
    Bacteria can be cultured using various methods including streak culture, lawn culture, stab culture, pour plate culture, and broth culture. Key steps in bacterial culture include maintaining aseptic technique and providing appropriate conditions for growth such as aerobic or anaerobic environments. Viruses require living host cells for replication and are cultured using animals, embryonated eggs, and tissue culture techniques. Detection of viral growth involves monitoring for cytopathic effects, hemadsorption, interference effects, and other indicators.

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    Bacteria can be cultured using various methods including streak culture, lawn culture, stab culture, pour plate culture, and broth culture. Key steps in bacterial culture include maintaining aseptic technique and providing appropriate conditions for growth such as aerobic or anaerobic environments. Viruses require living host cells for replication and are cultured using animals, embryonated eggs, and tissue culture techniques. Detection of viral growth involves monitoring for cytopathic effects, hemadsorption, interference effects, and other indicators.

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    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
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    9 views40 pages

    Reference Material I 19

    Uploaded by

    Maru Mengesha Worku 18BBT0285
    Bacteria can be cultured using various methods including streak culture, lawn culture, stab culture, pour plate culture, and broth culture. Key steps in bacterial culture include maintaining aseptic technique and providing appropriate conditions for growth such as aerobic or anaerobic environments. Viruses require living host cells for replication and are cultured using animals, embryonated eggs, and tissue culture techniques. Detection of viral growth involves monitoring for cytopathic effects, hemadsorption, interference effects, and other indicators.

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    of 40

    Culture Methods-

    Bacteria
    Dr. Pragasam Viswanathan
    Professor, SBST
    Culture
    Methods
    Indications for culture -

     Isolate bacteria in pure cultures.

     Demonstrate their properties.

     Obtain sufficient growth for preparation of


    antigens & for other tests.

     Typing bacterial isolates.

     Antibiotic sensitivity.

     Estimate viable counts.


    Types of culture methods
     Streak culture or surface plating
     Lawn or carpet culture
     Stroke culture
     Stab culture
     Pour plate method

     Anaerobic methods of culturing


    Aseptic technique
    Streak Culture

     Routinely employed for isolation

     Platinum / Nichrome loops


    Dr Ekta, Microbiology
    Lawn or Carpet Culture

     Uniform surface growth

     Bacteriophage typing

     Antibiotic sensitivity testing

     Preparation of bacterial antigens &


    vaccines
    Stroke Culture
     Tubes containing agar slopes
     F o r slide agglutination & other
    diagnostic tests.
    Stab Culture
     B y puncturing a
    suitable medium
    with a long,
    straight charged
    wire.

     For gelatin
    liquefaction, stock
    cultures &
    motility
    Pour Plate Method
     1 ml of appropriately
    diluted inoculum is added
    to 15 ml of molten
    agar and poured on
    petridish.

     Colonies appear through


    out the depth of
    medium.

     Used to
    estimate viable
    count,
    recommended
    method for quantitative
    urine cultures.
    Broth Culture
     Inoculated by a
    charged loop,
    pipette or
    syringes.

     For blood cultures


    & sterility
    testing.
    Anaerobic Culture Methods

    Anaerobic condition can


    be achieved by
    1. displacement of O2 with
    other gases.
    2. absorption of O2 by
    chemical or biological means
    3. reduction of O2
    1. Displacement Method
     Displacement of O2
    with gases like H2 , N2 ,
    He or CO2 .

     Rarely produces
    complete anaerobiosis.
    e.g. Candle jar
    2. Chemical or Biological Method
     Alkaline
    pyrogallol ( pyrogallic acid
    in NaOH) absorbs O2.
     Yellow phosphorous.
     Rosenthalmethod - Mixture of
    chromium & sulphuric acid.
     McIntosh - Fildes’ Jar.
     Gaspak
    McIntosh - Fildes’ Jar

     Most reliable &


    widely used anaerobic
    method

     Complete
    anaerobiosis

     Catalyst –
    palladinised asbestos
    Gaspak
     Method of choice for preparing
    anaerobic jars.

     Commercially available as
    disposable envelope, containing
    chemicals which generate H2 ,
    CO2 with the addition of
    water.

     Reduced methylene blue is used


    as indicator. Remains colorless
    anaerobically but turns blue on
    exposure to O2
    3. Reduction of Oxygen
     Usingreducing agents like 1% glucose,
    0.1% thioglycollate, 0.1% ascorbic
    acid.

    Robertson’s Cooked Meat Medium


     most widely used fluid medium
     Fat free minced cooked meat in
    broth, with a layer of sterile vaseline
    over it.
    Robertson’s Cooked Meat Medium
    05.10.08 Dr Ekta, Microbiology
    Automated Methods
     Bactec - blood culture method
    ◦ The sample to be tested is
    inoculated into one or more
    vials which are inserted into
    the BACTEC fluorescent
    series instrument for
    incubation and periodic
    reading.

    ◦ Each vial contains a chemical


    sensor which can detect
    increases in CO2 produced by
    the growth of
    microorganisms.
    Automated Methods

    ◦ The sensor is monitored


    by the instrument
    every
    ten minutes for an
    increase in its
    fluorescence, which is
    proportional to the amount
    of CO2 present.

    ◦ A positive reading
    indicates the presumptive
    presence of viable
    microorganisms in the vial.
    Virus culturing and
    assaying methods
     Viruses are obligate intracellular
    parasites
     They multiply only inside the living host cells
     Animals, plants, humans, bacteria, fungus,
    protozoa and algae are the natural hosts of
    viruses
    Viruses are host specific and grow only in
    selective hosts. Virologists use only a suitable
    host system for cultivation of a virus

    Viruses cannot grow in artificial media


    .They cannot grown in non-living culture
    or on agar plates alone , they must require
    living cells to support their replication

    There is no universal cell that will


    support all viruses.
    Main purpose of virus cultivation

     To isolate and identify viruses in clinical


    samples.
     To prepare viruses for
    vaccine production.
     To do research on viral structure, replication,
    geneticsand effects on host cells.
    METHODS FOR CULTIVATION OF VIRUSES

    Inoculation of virus
    into animals

    Inoculation of virus into


    embryonated eggs

    Tissue culture
    1. Inoculation of Virus in animals

     Viruses which are not cultivated in


    embryonated egg and tissue culture are
    cultivated in laboratory animals. e.g: mice,
    guinea pig, hamester, rabbits and primates are
    used
     The selected animals should be healthy and
    free from any communicable diseases
     Suckling mice (less than 48 hours old) are most
    commonly used
    Advantages and disadvantages of animal
    inoculation

    Advantages :
     Production of antibodies can be identified
     Diagnosis, pathogenesis and
    clinical symptoms are determined
     Primary isolation of certain viruses
     Mice provide a reliable model
    for studying viral replication
     Used for the studyof immune responses,
    epidemology and oncogenesis
    Disadvantages :
     Expensive and difficulties in maintaince of
    animals.
     Difficulty in choosing of animals for particular
    virus.
     Some human viruses cannot be grown in
    animals or can be grown but do not cause
    diseases.
     Mice do not provide models for vaccine
    development.
    2. Inoculation of virus into embryonated
    egg

    The process of cultivation of viruses in


    embryonated eggs depend upon the type of
    egg being used.
    Egg provide a suitable means for :
    i. The primary isolation and identification
    of viruses.
    ii. The production of vaccines.
    iii. The maintainance of stock culture.
    Inoculation of virus into
    embryonated eggs
     Virus growth and multiplication in the
    egg embryo is indicated by the death of the
    embryo , by embryo cell damage , or by the
    formation of typical pocks or lesions on the
    egg membrane.
     Viruses can be cultivated in various
    pats of egg like :
    1) Chorioallantoic membrane (CAM)
    2) Allantoic cavity
    3) Amniotic sac
    4) Yolk sac Pock
    Advantages of inoculation into
    embryonated egg
     Widely used method for the isolation of virus and
    growth.
     Cost effective and maintenance is much easier.
     The embryonated eggs are readily available.
     They are free from contaminating bacteria
    and many latent viruses.
     Ideal substrate for the viral growth and replication.
     less labor is needed.
     Widely used method to grow virus for
    some vaccine production.
     Defense mechanisms are not involved
    in embryonated eggs.
    Disadvantage of inoculation into
    embryonated egg

    • The site of inoculation for varies


    with different virus . That is , each
    virus have different sites for growth
    and replication.
    3) Tissue culture
    Cultivation of bits of tissues and organs
    in vitro had been used by physiologists
    and surgeons for the study of
    morphogenesis and wound healing
     Before the advent of cell culture, animal

    viruses could be propagated only on whole

    animals or embryonated chicken eggs


     Cell cultures have replaced
    Embryonated eggs as
    preferred type of growth
    medium for many viruses.
     Cell culture consists of cells
    grown in culture media in the
    laboratory.
    Advantages of cell culture
     Relative ease, broad spectrum, cheaper and
    sensitivity
    Disadvantage of cell culture
     The process requires trained technicians with
    experience in working on a full time basis.
     State health laboratories and hospital
    laboratories do not isolate and identify
    viruses in clinical work.
     Tissue or serum for analysis is sent to central
    laboratories to identify virus
    Detection of virus growth
    The following methods are available to detect
    the virus growth in the cell or tissue cultures

    a) Cytopathic effect
    b) Haemadsorption
    c) Interference
    d) Transformation
    e) Immunofluorescence
    f) Metabolic inhibition

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