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    LECTURE 4-Microbiological methods-S201920I - NMM

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    NUR ANISAH MOHD ADNAN
    The document outlines 5 basic microbiological techniques: inoculation, incubation, isolation, inspection, and identification. Inoculation involves introducing a microorganism sample into a nutrient medium to initiate growth. Incubation allows microbes to grow by controlling temperature and atmosphere. Isolation produces pure cultures by sub-culturing. Inspection evaluates growth macroscopically and microscopically. Identification determines the microbe through biochemical tests, microscopy, or advanced methods.

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    LECTURE 4-Microbiological methods-S201920I - NMM

    Uploaded by

    NUR ANISAH MOHD ADNAN
    46 views42 pages
    The document outlines 5 basic microbiological techniques: inoculation, incubation, isolation, inspection, and identification. Inoculation involves introducing a microorganism sample into a nutrient medium to initiate growth. Incubation allows microbes to grow by controlling temperature and atmosphere. Isolation produces pure cultures by sub-culturing. Inspection evaluates growth macroscopically and microscopically. Identification determines the microbe through biochemical tests, microscopy, or advanced methods.

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    LECTURE 4-Microbiological methods-S201920I_NMM

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    The document outlines 5 basic microbiological techniques: inoculation, incubation, isolation, inspection, and identification. Inoculation involves introducing a microorganism sample into a nutrient medium to initiate growth. Incubation allows microbes to grow by controlling temperature and atmosphere. Isolation produces pure cultures by sub-culturing. Inspection evaluates growth macroscopically and microscopically. Identification determines the microbe through biochemical tests, microscopy, or advanced methods.

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    © All Rights Reserved
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    Download as pptx, pdf, or txt
    46 views42 pages

    LECTURE 4-Microbiological methods-S201920I - NMM

    Uploaded by

    NUR ANISAH MOHD ADNAN
    The document outlines 5 basic microbiological techniques: inoculation, incubation, isolation, inspection, and identification. Inoculation involves introducing a microorganism sample into a nutrient medium to initiate growth. Incubation allows microbes to grow by controlling temperature and atmosphere. Isolation produces pure cultures by sub-culturing. Inspection evaluates growth macroscopically and microscopically. Identification determines the microbe through biochemical tests, microscopy, or advanced methods.

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    of 42

    MICROBIOLOGICAL

    METHODS
    5 BASIC TECHNIQUES

    INOCULATION IDENTIFICATION

    INCUBATION ISOLATION

    INSPECTION
    INOCULATION

    INOCULATION is introduction of small sample of


    microorganism cells (INOCULUM) into a
    container of nutrient medium to initiate growth

    CLINICAL SAMPLE - blood, urine , CSF, feces, etc


    HABITAT SAMPLE - soil, water, sewage, food, etc

    CONTAINERS
    (individual) test tube, flask, agar plate (petri dish)
    (industry) large scale fermenters
    CULTURE MEDIA

     CULTURE MEDIUM is a nutrient material


    prepared for the growth of microbes in a
    laboratory
     It provides nutritional requirements for
    organisms
     The microbes that grow and multiply in or on
    a culture medium are referred to as a
    CULTURE.
     2 types
    SIMPLE - few inorganic compounds

    COMPLEX - inorganic & organic compounds


    Classification based on:
    1. CHEMICAL COMPOSITION

    SYNTHETIC - Chemically defined media, exact chemical


    composition is known
    Highly pure organic & inorganic compounds

    COMPLEX - (Non synthetic) – complex media, ingredient


    not chemically definable
    from plant, animal or yeast extract
    Synthetic media

    eg: Nutrient broth, N. agar

    - NB consists of peptone,
    meat extract, NaCl

    - NB + 2% agar = Nutrient agar


    Complex Media
    • Luria Broth
    • Made with yeast extract
    • Sometimes chosen based on
    price and sometimes by
    necessity – some
    microorganisms have never been
    cultured on defined media
    2. PHYSICAL STATE OF MEDIA
    • LIQUID – enrichment/large number of
    bacteria need to be grown
    • SOLID – film surface for microbes to
    grow
    • SEMI SOLID – motility test
    LIQUID MEDIA

    • Water based solutions


    • do not solidify at temperatures above freezing,
    • flow freely in containers
    • various solutes dissolved in distilled water

    BROTHS, MILKS, INFUSIONS


    SEMI SOLID MEDIA

    • Such media are soft


    • clot like consistency, contain solidifying agent
    (agar/gelatin - 0.3-0.5%)
    • Used to determine motility, localize reaction at
    specific sites
    SOLID MEDIA
    • Agar is the most commonly used solidifying agent
    • Golden –yellow granular powder
    • Prepared from seaweeds.
    • Not affected by the growth of the bacteria.
    • Melts at 98oC & sets at 42oC
    • firm surface, allows cells to form discrete colonies
    • Advantageous for ISOLATION/SUBCULTURING

    2 Forms:
    1. LIQUEFIABLE : reversible solid, agar, thermoplastic (solid at
    room temp./incubation temp., become liquid at 100oC,
    resolidifies at 42oC

    2. NON LIQUEFIABLE : NOT thermoplastic, cooked


    meat, potato slices, egg media
    If we want to grow E.coli in the
    laboratory, we need
    • E. coli pure culture
    • The proper medium for E.coli to grow
    (nutrient, moisture, pH, oxygen,) & the
    culture must be sterilized first
    • Followed by incubation at the proper
    temperature
    E coli O157:H7
    3. Functional type of media
    • General purpose
    • Enriched
    • Selective
    • Differential
    • Assay
    • Enumeration
    • Specimen transport
    • Anaerobic growth
    a. GENERAL PURPOSE MEDIA

    • Used for a broad spectrum of


    microbes

    • Examples: Brain-heart
    infusion
    Tryptose soy agar
    Tryptose soy broth
    Nutrient agar
    b. ENRICHMENT MEDIUM

    • complex organic substances


    • Substances like blood, serum, egg or growth
    factors are added to the simple medium

    • Used for FASTIDIOUS ORGANISMS


    (organism that has complex or particular
    nutritional requirements)
    Eg: Streptococcus pneumoniae
    Requires blood - sterile horse, sheep or rabbit

    Eg. Blood agar, Chocolate agar


    c. SELECTIVE & DIFFERENTIAL MEDIA

    • designed for isolation & identification of specific groups of


    microbes from mixed populations
    • The inhibitory substance is added to a solid media to inhibit
    commensal or contaminating bacteria such as :
    Antibiotics
    Dyes
    Chemicals
    Alteration of pH

    • SELECTIVE - contains 1 or more inhibitory agents


    DYES, ACID, ANTIMICROBIAL AGENTS

    Example: growth of bact A, B and C are INHIBITED, but selective


    growth of D
    Examples:
    MANNITOL SALT AGAR - 7.5%
    NaCl, inhibitory concentration to
    human pathogen’s

    MACCONKEY
    AGAR/DEOXYCHOLATE
    CITRATE AGAR - High Bile salt
    concentration, inhibitory to
    Gram +ve bacteria

    SABOURAUD’S AGAR (Fungi) -


    pH 5.6 (acid), inhibits bacteria
    d. DIFFERENTIAL MEDIUM
    • Designed in such a way that different bacteria can be recognized on the
    basis of their colony color, allows for growth of several types BUT
    highlights differences

    • Dyes and metabolic substrates are incorporated so that those bacteria


    that utilize them appear as differently colored colonies.

    • DYES (differential agents) - act as pH indicators colour change due


    to production of acid or base

    Colony size, colour, formation of gas, precipitation


    EXAMPLES

    MAcCONKEY AGAR - lactose +


    neutral red

    E. coli produces acid,


    metabolizes lactose
    RED-PINK colonies

    Salmonella sp produce no acid


    OFF WHITE colonies
    E. coli & Salmonella sp. On MacConkey Agar
    XYLOSE LYSINE DEOXYCHOLATE
    AGAR (XLD)

    • contains xylose, lysine, iron,


    thiosulphate, bile + phenol red
    • Used for the recovery of Salmonella and
    Shigella species

    • E.coli acid production


    Yellow colonies

    • Salmonella sp. convert thiosulphate


    to H2S gas (SMELL) forms a black
    precipitation with iron
    E. coli & Salmonella sp. On XLD Agar
    e. OTHER MEDIA

    • REDUCING - thioglycollic acid or cystine absorbs


    oxygen/slows penetration of oxygen thus having reducing
    availability

    • REQUIRED for growing ANAEROBIC BACTERIA

    • CARBOHYDRATE FERMENTATION - sugars for


    fermentation, conversion to acids, pH indicator
    REQUIRED for BIOCHEMICAL/IDENTIFICATION TEST
    • TRANSPORT - required for maintaining and preserving
    specimens for a period of time
    • Delicate organisms may not survive the time taken for
    transporting the specimen without a transport media.

    • Examples: STUART’S + AMIES


    (contains salts, buffers & absorbants)

    • Prevents cell destruction, pH changes, toxic substances


    • NO GROWTH of desired organisms
    Serial Dilutions
    • Stepwise dilutions of substance in a solution
    INCUBATION

    Chamber (INCUBATOR)
    temperature & atmospheric gas controlled

    LAB INCUBATORS : 20 - 40oC


    Aerobic or Anaerobic

    INCUBATION PERIOD : hours-several weeks depending upon the


    organism
    Eg. Staphylococcus aureus, small colonies, required longer incubation
    period ~ 48 hours
    INSPECTION

    • Observable growth on or in the medium (CULTURE) at


    various stages of incubation (EVALUATE GROWTH)

    MACROSCOPICALLY - naked eye


    LIQUID MEDIA - cloudiness, sediment, scum or colour
    change, gas production

    AGAR PLATE - discrete isolated colonies, mass of clinging


    cells (fungi)
    Pseudomonas, Staphylococcus & Serratia on
    TSA plates

    Sarcina lutea Aspergillus niger Pseudomonas flurescence


    • MICROSCOPICALLY
    observe individual cells within a colony

    Evidence of cellular morphology: size, shape, details of


    structure

    allows for IDENTIFICATION

    • SUB-CULTURE common microbiological procedure


    allows for a pure STOCK-CULTURE of organism
    IDENTIFICATION

    MICROSCOPICALLY

    BIOCHEMICAL TEST

    RAPID TEST

    IMMUNOLOGY TEST

    DNA ANALYSIS
    • DISPOSAL OF CULTURES
    most important - if presents a biological hazard

    Autoclaving - steam sterilization


    Incineration - burning
    Radiation - X or  rays
    Disinfection - chemical

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