Culture Media Mohammad Rhbar (PhD) Dep. Of Microbiology

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Culture Media

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

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Introduction

• 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.

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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.

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Terminology..

• 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
  microorganisms

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Culture media classified by
composition

• 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.

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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

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Solid culture medium and semi-solid culture
medium.

• Liquid culture medium containing solidify martial
  ( e.g. agar-agar ,gelatin ,et c.,) in different
  concentrations
• 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

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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
  medium)

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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)

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Enrichment medium

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

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Selective enrichment medium

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

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Non selective enrichment medium

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

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Isolation medium

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

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Selective isolation medium

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

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non-selective isolation medium

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

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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)

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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

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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

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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
  carriers)

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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
  medium.
• 1- a complete ready to-use medium
• 2- an incomplete medium to which labile
  components are added at time o use

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Practices for quality of culture dehydrated
media

• 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
  used
• 7-Technical data sheet
• 8-Quality control certificate ( fore ready to
  use media)
• 9-Safety and /or hazard data where needed

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Check list by laboratory

• Laboratory checks following data at the media's
  delivery
• 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

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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

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• 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

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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
  containing
• Hazardous material i.e. bile salts or other
  selective agents.

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• 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
  used.

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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.

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• 1-write on the label the date of receipt in the
  laboratory.
• 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.

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• 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.

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• - 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.

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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.

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• 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

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• 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
  possible.

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• 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

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• Agar free media will usually dissolve with gentle
  agitation.
• 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.

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• 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.

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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 .

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• 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.

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• 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

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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

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Sterilization cycle

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

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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.

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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

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

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• 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.

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• 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

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continue.

• 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

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• 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.

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• . 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
  temperature.

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• 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

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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.

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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

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Turbidity ,Precipitation

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

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Darkening

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

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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.

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poor bacteriological growth

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

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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
  photo-oxidation

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• 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.

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• 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.

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Sterilty Check

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

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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

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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

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Source of Control Strains

• American Type culture Collection (ATCC).
• Commercial Sources
• Reference Laboratories
• Patents isolates

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Maintance of control Strains.

• Trypticase Soy agar( for non-fastidious )
• Deep freeze
• Lyophilization.

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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

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• 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 .

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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

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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.

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User

• 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
  conditions

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User inspection

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