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Culture Media


In the microbiology laboratory many tests and procedures depend on culture media ... All prepared couture media should be checked for sterility. ... – PowerPoint PPT presentation

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Title: Culture Media

Culture Media
  • Mohammad Rhbar (PhD)
  • Dep. Of Microbiology ,research center of
    reference Laboratories of Iran ,Tehran

  • In the microbiology laboratory many tests and
    procedures depend on culture media being
    consistent and providing reproducible results.
    Several hundreds of formula of dehydrated culture
    media are commercially available and many more
    ,designed for specific purpose, are described in
    literature . ,in laboratories charring out the
    microbiological examination of clinical specimens
    the main objective are growth and rapid detection
    of pathogenic organisms.

Terminology of Culture Medium
  • Lot or batch) Fully traceable unit of a medium
    referring to defined amount of bulk
    ,semi-finished product or end product which is
    consistent in type and quality and which has
    passed the requirements production (in-process
    control) and quality assurance testing and which
    has been produced within one defined production
    period have been assigned the same lot number.

  • Culture media
  • Formulation of substance ,in liquid, semi-solid
    or in solid form, which contains natural and/or
    synthetic constituents intended to support the
    multiplication , or to preserve viability of

Culture media classified by
  • Chemically defined media
  • culture medium consisting of chemically defined
    constituents (i.e. of known molecular structure
    and degree of purity )only
  • Chemically undefined culture media
  • Culture medium consisting entirely or partly of
    natural materials or other wise ,the chemical
    compositions of which are not completely defined.

culture Media classified by consistency
  • Liquid culture medium
  • Colure medium consisting of an aqueous solution
    of one or more constituents ( e.g.., peptone
    water ,nutrient broth) Note1 In some cases
    ,solid particles are added to the liquid medium
  • Note2 Liquid media in tubes, flasks or bottles
    are commonly called broth

Solid culture medium and semi-solid culture
  • Liquid culture medium containing solidify martial
    ( e.g. agar-agar ,gelatin ,et c.,) in different
  • Note1 Due to the world-wide use of culture media
    solidified with agar-agar ,the shortened term
    agar is s often used synonymously for solid
    culture media and therefore is connection with
    nouns, e.g. plate count agar

Culture Media classified by intent use
  • Transport medium
  • Cultured medium designed to preserve and maintain
    the viability of microorganisms for the time
    period between sample collection and laboratory
    processing of sample.
  • Note1- Transport media usually contain substance
    that do not permit multiplication of
    microorganisms but ensure their preservation
    (e.g. ,Stuart's Carry Blair or Amies Transport

Preservation medium
  • Culture medium designed or preserve and maintain
    the viability of microorganisms over an extended
    period, to protect tem against the adverse
    influence with may occur during long-term storage
    and to allow recovery after this period( e.g.
    Dorset egg medium, Skimed Milk)

Enrichment medium
  • Predominantly liquid culture medium which ,due
    to its composition ,provide particularly
    favorable condition for multiplication of

Selective enrichment medium
  • enrichment medium which supports the
    multiplication of specific microorganisms while
    inhibiting other microorganisms (e,g .SF,GN broth

Non selective enrichment medium
  • Enrichment medium is not devised to selectively
    inhibit microorganisms
  • (e.g. nutrient broth)

Isolation medium
  • Solid or semi-solid culture medium which supports
    growth and/or the formation of colonies of

Selective isolation medium
  • Isolation medium which supports growth of
    specific microorganisms ,which inhibiting other
    microorganism (e.g. MacConky agar EMB agar)

non-selective isolation medium
  • Isolation medium which is not devised to
    selectively inhibit microorganisms ( e.g.
    nutrient agar)

Differential medium
  • culture medium which permits the testing of one
    or more physiological/ biochemical character of
    the microorganisms for the their identification (
    e.g Urea medium ,Kiligler Iron agar.LDC.ODC.ADH
    .Cimmon,s Citrate)
  • Note differential media which can be used as
    isolation media are referred to as isolation
    /differential media( e.g. Xylose lysine
    desoxycholate (XLD ) Hekton Enteric agar (HEA)

Identification medium
  • culture media designed to produce a specific
    identification reaction which dose not require a
    further confirmatory tests
  • Note 1-idetification which can be used as
    isolation are referred to as isolation/
    identification media

Media with multiple intents of use
  • certain culture media may be assigned to several
    categories .e.g. Blood agar is a enrichment
    medium ,an isolation medium. and a differential
    medium for detection of hemolysis

Culture media classified to the form
of product
  • ready to-use medium
  • Culture medium which is supplied container s in
    ready form( e.g. Petri dishes or tubes or other

Dehydrate commercially culture medium
  • Culture medium in dry form which is not reedy to
    use (e.g. powder ,granules lyophilsed products)
    rehydration will make one of two kinds of
  • 1- a complete ready to-use medium
  • 2- an incomplete medium to which labile
    components are added at time o use

Practices for quality of culture dehydrated
  • Documentation required from manufacturethe
    following details should be availed from the
    manufacture, some them only on request1-
  • 1--Name of medium and product code
  • 2- Batch code
  • 3-PH value
  • 4-Storage information and expiry date
  • 6-any performance evaluation and control strain
  • 7-Technical data sheet
  • 8-Quality control certificate ( fore ready to
    use media)
  • 9-Safety and /or hazard data where needed

Check list by laboratory
  • Laboratory checks following data at the media's
  • 1--Name of medium and batch code
  • 2--Date of receipt expiry date
  • 3- -condition of packaging and integrity i.e.
    checking of the seal
  • 4- -Container damage if needed

Quality management and control for dehydration
media and supplements
  • Media nowadays are usually purchased from
    commercial manufactures. They are delivered in
    dehydrated powdered or granulated form in sealed
    container and supplements of different selective
    or diagnosis substances are supplied in either
    the lyophilized or liquid state. However
    purchases of should be planned to encourage a
    regular turnover of stock. To maintain an
    effective inventory (i.e. first in first out)
    further checks should include
  • Re-checking
  • Date of first opening
  • Visual assessment of contents of opened container

  • Especially after opening a new container ,the
    quality of the medium may depend on the storage
    environment. Loss of quality of dehydrated media
    is shown by change in flow characteristics of the
    powder ,homogeneity ,caking, color changes etc..
    Any dehydrated medium that has absorbed moisture
    or shows obvious changes in physical appearance
    should be discarded

Laboratory preparation of media
  • The accurate preparation of culture media is one
    of the fundamental steps in microbiological
    examination and it shall be given special care.
  • Good laboratory practice or the manufacture's
    instructions regarding the handling of dehydrated
    media and other components ,particularly those
  • Hazardous material i.e. bile salts or other
    selective agents.

  • When media are prepared from dehydrated
    commercial formulation follow the manufactures
    instructions precisely. Document all relevant
    data i.e. weights ,pH date of preparation
    ,sterilization conditions, operator
  • For media prepared from individual components
    ,follow the recipe precisely and record all
    details ,in addition the full identity ( i.e.
    code and batch number ) of all the components

Preparation of culture Media
  • Dehydrated media are hygroscopic and are
    sensitive to moisture ,heat and light .They are
    adversely affected by drastic changes in
    temperature e.g. hot/cold cycling temperature may
    which may occur between day and night
    laboratory temperature in winter.

  • 1-write on the label the date of receipt in the
  • 2- Store as directed on the label usually below
    25 C in a dry area ,away from direct sunlight
    ,autoclaves, drying ovens or other heat sources,
    where indicated store at 2-8C
  • 3- Check expiry date on the label ,some media
    significantly shorter shelf lives than others.

  • 4- Use stock in lot/batch number order .Do not
    open a new bottle until the previous bottle has
    been emptied .Note on the label the date the
    container is first opened . After use ,make sure
    the container is tightly closed and return it to
    the designed storage area.

  • - order the medium in an appropriate size of
    container and in a quantity which accords to
    normal use requirements .A medium in a large
    container which has been opened many times will
    deteriorate on storage . Discard the medium if
    the powder is not free flowing, if the color has
    changed or if it appears abnormally in any way.

Reconstitution of dehydrated media
  • Weighing out
  • Using a top-pan balance with an accuracy of 0.1
    gram the powder should be spooned to a weighing
    boat or clean beaker . Do not tip the media out
    of container as this will cause excess dust
    which may be irritant and will certainly need
    cleaning up. The components of some formulation
    can irritant so the wearing of a face mask at
    this stage is advisable.

  • Complete instruction for the preparation of
    culture media are given on the label of each
    bottle .As a general rule it is wise to prepare
    one week's requirement only.
  • 1- use water prepared by distillation,
    deionization or reveres osmosis. Toxic metals
    such as copper must be absent. Check the pH of
    water ,if below 5.5 ,heat the water to drive off
    CO2 and re-check .The conductivity of the water
    should be below15 Mir siemens (µS). Rinse
    glassware before use

  • 2-Prepare the medium in a vessel about twice the
    final volume of the medium to allow adequate
    mixing .Follow the instructions given on the
    label of each product
  • 3-Open the culture medium container away from
    draughts and moisture ,Avoid inhaling the powder
    and prolong skin contact .Weigh the powder
    quickly accurately and without creating ,clouds
    of dust . Reclose the container as soon as

  • 4- pour the half the required volume of the water
    in the vessel, then the weighed quantity of
    medium and agitate briskly for a few minutes
    .Pour the rest of the water down the side of
    vessel to wash any adherent medium back into
    solution. This is an important step because dry
    culture media powder above the level of the water
    may not be sterilized in the autoclave and may be
    source of contamination

  • Agar free media will usually dissolve with gentle
  • Media containing agar should be heated to
    dissolve the agar before autoclaving . Bring the
    medium to the boil without scorching or burning .
    Those media which should not be autoclaved will
    be ready to pour into dishes or other containers
    after this amount of heating (e.g XLD, TCBS, SS
    agar). Most culture media will required final
    sterilization an autoclave at 121 .Do not adjust
    pH before sterilization.

  • the pH of the dehydrated medium has been
    adjusted so that the final pH of the prepared
    medium confirm with the label specification when
    the medium has been cooled at 25ºC . Do not
    adjust pH before sterilization.

Sterilization of Culture Media
  • Although sterilization of culture media is best
    carried out in a steam autoclave at temperature
    around 121C it has recognized that damage is
    caused to the medium by the heating process.
  • Heat treatment of culture media which contain
    peptide ,sugars minerals and metals results in
    nutrient destruction ,either by direct thermal
    degradation or by reaction between the medium
    components. Toxic products caused by
    chemo-oxidation can also be formed during heat
    treatment .

  • It is important ,therefore ,to optimize the
    heating process that a medium is sterile after
    heating but minimal damages caused to the
    intergradient of the medium . As a general rule
    it is accepted that shrot duration ,high-
    temperature process are more lethal to organisms
    and less chemically damage than are longer ,lower
    temperature process.

  • A general instruction for sterilization culture
    media in volume up to one liter at 121ºC for 15
    minutes is given on each label . Autoclaves vary
    in performance ,however ,and thermocouple tests
    using different volume of media should be carried
    out to determine the ,heat-up and cool-down times
    ,It will be essential to do this when volume of
    media greater than two liters are prepared . In
    order to avoid overheating large volume units of
    media ,the heat up, and cool-down periods are
    normally integrated into 121º C holding time

Sterilization Cycle
  • the sterilization cycle can be divided into
    four cycle
  • 1- Chamber heat-up
  • 2- Heat penetration
  • 3- Holding time at the prescribed temperature
  • 4- Cool-down time for the chamber to reach 80ºC

Sterilization cycle
  • Sage 1 20-121ºC
  • Stage 2 lt100-121ºC
  • Stage 3 121-121ºC
  • Stage 4 121- 80ºC

Stage 1
  • The chamber heat-up time depends on the
    efficiency of the autoclave (air discharge /steam
    input) and the size of the load in the chamber.
    The time required for this stage is measured with
    a recording probe located in the air-discharge
    valve located in the base of the chamber.

Stage 2
  • The heat penetration time depends mainly on the
    volume of the individual containers, although the
    shape and the heat transfer properties of the
    containers may affect this stage .The time
    required the medium volume to reach 121 is
    measured with thermocouples placed in the center
    of the innermost container

  • Volume (ml) in glass bottles. Time
  • 100 19
  • 500 18
  • 1000 22
  • 2000 20
  • 5000 37

  • These time assume that agar media have been
    dissolved before autoclaving .It is also assumed
    that maximum exposure to steam is possible Thus
    although the single 100ml bottle required
    12minutes to reac121C when placed in a crate with
    other bottles it required 19 minute and when
    placed in the center of staked crates it require
    30 minutes.

  • Stage 3
  • The holding time at 121C depends on (i) The
    number of organism originally present
  • In the medium (ii) The fractional number of an
    organism presumed present after heating .e.g.
    N0.001 equivalent o one bottle in everey1000
    bottle heated becoming contaminated. (iii) the
    thermal death rate constant of the presumed
    organism present at 12ºC

  • Stage 4
  • The cool down time depends on the size of the
    load in the camber and the heat loss rate from
    the autoclave . water-spray are used to
    accelerate cooling in commercial sterilizers but
    very careful control is required to avoid bottle
    fracture and the ingress of the cooling spray
    into the sterilizes medium. The latter problem
    occurs when the vacuum formed in the heat space
    during cooling sucks contaminated cooling fluid
    up the thread of the cap and into the bottle

  • Culture media autoclaves should be untagged and
    of moderate chamber capacity only. Thermal locks
    on the doors should prevent them opening when the
    chamber temperature is above 80 º C but even in
    these circumstances care should be taken to avoid
    thermal shock when removing glass bottles of hot
    liquid from the autoclave.

  • . When screw-capped containers are placed in an
    autoclave the caps should be a half-turn free to
    allow the escape of heated air. When removed from
    the autoclave the caps are screwed down tight
    after the contents have cooled to ambient

  • All autoclaves should be calibrated and checked
    at fixed periods of time to ensure that they are
    functioning efficiently. Physical measurements
    should be made on temperature and pressure
    readings, the quality of the steam should be
    checked ,the efficiency of the steam should be
    checked ,the efficiency of the ,near to-steam
    ,air traps in the base of the autoclave should be
    determined and the safety valves checked.
    Mandatory inspection of autoclaves as pressure
    vessels are normally carried out annually by
    specialist under instructions from insurers of
    such apparatus

Sterilization Checks
  • Biological indicators of sterilization will
    demonstrate the ability of the autoclaves to
    destroy spores .Such tests may be compulsory in
    certain countries. Chemical indicators will show
    the temperature reached or exceed and some will
    indicate the time held at the specified
    temperature . Under autoclaving is usually
    self-evident because failure to destroy all the
    bacterial spores naturally present in dehydrated
    media( the bioburden) will allow grow to take
    place in the stored or incubated medium. Failure
    of sterilization should always be suspected when
    contamination of prepared media occurs with
    sporing organisms.

Faults and Possible Causes in
Media sterilization
  • Fault Wrong pH value.Possible causes.
  • pH test carried out above 25ºC
  • Overheating through prolonged sterilization
  • remeltingor overlong period at 50 ºC
  • Incomplete solution of Medium.
  • Poor quality water or container
  • Dehydrated medium stored incorrectly or beyond
    the stated shelf lif

Turbidity ,Precipitation
  • Possible causesPoor quality water or container
  • Overheating or prolonged storage at 50ºC
  • pH value incorrect
  • Incomplete solution

  • Possible causes
  • Overheating.
  • Incomplete solution
  • pH drift

Soft gel
  • Possible causes
  • Agar not in solution, poor mixing, prolonged
    storage at 50ºC
  • Overheating at low pH value
  • Error in weighing or over dilution with inoculum
    or media supplement.

poor bacteriological growth
  • Prolonged and excessive heating ,incomplete
  • Inhibitory substance in water or container
  • Darkening and pH drift.

Preparation of sterilized Media
  • Liquid media which are sterilized in their final
    concentration should be cooled down room
    temperature as rapidly as possible. Screw caps
    should then tightened.
  • Container of agar media which have been
    sterilized should be placed at 50ºwater bath and
    the medium dispensed as soon as possible as it
    reach this temperature, or within a maximum of 3
    hours in the bath . The medium should be mixed
    thoroughly without bubble formation and
    aseptically dispensed into sterile containers. Do
    not expose dishes of agar media to sunlight it
    causes excessive condensation on the lids and may
    cause the formation of inhibitory substance by

Continue ..
  • Heat labile supplements should be added to the
    medium after it has cooled to 50ºC
  • Allow the sterile supplement to come to room
    temperature before adding it to the agar medium.
    Cold liquids may cause agar to gel or form
    transparent flakes which can easily be seen in
    blood-enriched agar .Mix all supplements into the
    medium gently and thoroughly then distribute into
    the final containers as quickly as possible.

  • Blood used for the preparation of the blood agar
    should be as fresh as possible and should have
    been stored at 2-8º C( blood must not be frozen)
    Warm the blood in an incubator to about 35-37º
    before adding to sterile molten agar base, which
    has been cooled to 40-45 . A adequate mixing in a
    large head space vessel is essential to ensure
    aeration of the blood. Poorly oxygenated blood
    plates are purplish in color whereas properly
    aerated blood agar is cherry-red.

Sterilty Check
  • All prepared couture media should be checked for
    sterility. After preparation incubate 5 of all
    media for 24 hours.

Storage of Prepared Media
  • The recommended shelf-life of prepared culture
    media varies considerably .Screw-capped bottles
    of nutrient broth and agar can be stored for 6
    month at low ambient temperature (12-16C) it is
    important to store all media away from light. .
    Do not freeze the culture media.
  • Loss of moisture from agar plates is a common

Quality assurance for commercially prepared
culture media
  • Testing required by Manufacture
  • The media must be tested by manufactures for
    performance .Use of control strains ,the
    incubation condition are important factors

Source of Control Strains
  • American Type culture Collection (ATCC).
  • Commercial Sources
  • Reference Laboratories
  • Patents isolates

Maintance of control Strains.
  • Trypticase Soy agar( for non-fastidious )
  • Deep freeze
  • Lyophilization.

Test procedure for Culture Media (briefly)
  • 1-Prepare a 0.5 Mac Farland Suspension
  • 2-For testing the nutritive capacity of plate
    media( Blood agar Nutrient agar) dilute the basic
    cell suspension1100
  • 3-Inoculate 10-µL ,the diluted suspension to
    provide 1to 2x104CFU

  • For testing the inhibitory capacity(e.g EMB)
  • 1 -Prepare 1 10 suspension
  • 2-Inoculate 10-µl by streaking inplate to provide
    1 to2 x105CFU
  • For testing the performance of tubed media
    ,inoculate with a 10-µL .

Reporting Quality Assurance Data to the user
  • The manufactures should indicate that performance
    testing has beene established and documented. The
    manufactures should Insert
  • Label
  • Package
  • Technical manual

transport and storage
  • Media shall be shipped to prevent excessive loss
    of moisture and to provide mechanical and thermal
    protection. Whenever possible The number of
    intermediate handlers should be minimized.

  • There is not necessary performance testing by
    user ( campylobacter agar and media for isolation
    Neisseria spp)
  • User of commercialyy prepared media must inspect
    all media in each shipments for any the following

User inspection
  • Cracked Petri dishes
  • Unequal filling of plates
  • Cracked medium in plates
  • Hemolysis (blood agar)Freezing
  • Excessive numbers of bubbles
  • Contamination.
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