Specimen Cultivation

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Specimen Cultivation

• How do you grow these bugs?

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Introductory remarks

• Although the future trends in clinical
  microbiology are clearly pointing in the
  direction of developing rapid, non growth
  dependent means of detecting the presence of
  infectious microorganisms (detection of microbial
  antigens, probing for genes, PCR), the isolation
  and identification of viable pathogens is still
  the major means by which infectious diseases are
  diagnosed today. This is particularly true for
  bacterial infections.

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Introductory remarks

• In order to identify an infectious bacterial
  agent by biochemical methods and to do
  antimicrobic sensitivity testing, it is necessary
  to have a pure culture of the organism. Why?
• The way that we do this in the clinical lab is to
  streak a portion of the original sample out on
  primary isolation media to get isolated colonies.

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Streaking for isolation
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Introductory remarks

• Each isolated colony is a pure culture since it
  is theoretically the progeny of a single cell.
• Each type of bacteria forms a characteristic
  colony in terms of shape, size, color, texture,
  and adherence to the medium.
• These colonial characteristics are often used in
  the clinical lab as a first step in the process
  of identification of bacteria.

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Different bacterial colonial morphologies
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Different bacterial colonial morphologies
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Introductory remarks

• When the term culture media is used, it is
  important to keep in mind that it refers to
  artificial media on which bacteria and fungi are
  grown.
• It is also important to remember that some
  bacteria have never been successfully grown on
  artificial lab media. For example
• Treponema pallidum is usually grown in the
  testes of rabbits

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Introductory remarks

• Mycobacterium leprae is usually grown in the
  nine-banded armadillo or in the foot-pads of
  mice.
• Some bacteria (Rickettsia and Chlamydia) and all
  viruses are obligate intracellular parasites and
  can only be grown in living host cells, such as
  whole organisms or tissue culture cells.
• For viruses grown in tissue culture,
  identification is often based on electron
  microscopic visualization of virus in the cells,
  or
• Cytopathic effects (visible damage to or changes
  in the cells) produced by growth of the virus in
  the cells or by
• Inclusion bodies
• Syncytia formation
• Rounding up of the cells

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Cytopathic effects inclusion bodies
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Cytopathic effects syncytia formation
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Cytopathic effects cells rounding up
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Introductory remarks

• Growth of obligate intracellular parasites is
  very time consuming and expensive
• Diagnosis of these organisms as the causative
  agent of an infection is usually by immunological
  means.
• In a direct assay one looks for antigens from the
  organism. Therefore, one is looking for the
  organism, itself, usually in tissues.
• In an indirect assay, one looks in the host for
  evidence of an immunological response against the
  organism, usually in the form of specific
  antibodies against the organism.

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Introductory remarks

• When trying to diagnose an infection by assaying
  for antibodies against the causative agent, it is
  best to take both an acute (when patient is the
  most ill) and a convalescent (when patient is
  recovering) phase specimen. Typically one looks
  for a 4-fold rise in antibody titer (amount of
  antibody/ml of sera) between acute and
  convalescent specimens. Why?
• Parasites are not easily grown on artificial
  media or in living hosts.
• The diagnosis of parasitic infections is usually
  based on the visual identification of the
  parasite itself or on microscopic identification
  of the egg, ova, or cyst produced by the
  parasite.

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Taenia solium scolex
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Taenia solium proglottids
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Entamoeba histolytica trophozoite
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Entamoeba coli cyst
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Enterobius vermicularis egg
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Ascaris lumbricoides egg
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Plating of specimens and incubation of cultures
for bacteria and fungi

• When should the specimen be plated?
• The specimen should be plated immediately because
  if there is a delay this may result in
• A loss of fastidious or anaerobic organisms or
• There may be an overgrowth by normal flora in the
  specimen resulting in a change not only in the
  total number of microorganisms, but also in the
  relative numbers of microorganisms. Why is this
  a problem?

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Plating of specimens and incubation of cultures
for bacteria and fungi

• What should the specimen be plated on?
• Diagnostic labs vary in their choices of routine
  plating media that is used for growing bacteria
  and fungi typically isolated from different types
  of specimens.
• The choices used by different labs take into
  account what pathogens are anticipated in a given
  type of specimen, their growth requirements (?
  CO2, different temperature requirements, etc.)
  and the peculiarities of the available media.

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Example
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Example
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Plating of specimens and incubation of cultures
for bacteria and fungi

• Types of media that may be used include
• General purpose media this is media that
  supports the growth of most common pathogens and
  is therefore, classified as non-selective.
  However, in plate form, this type of media
  permits the isolation and differentiation of a
  wide variety of bacteria via differences in
  colony size, shape, color, texture, and adherence
  to the culture media. Examples of general purpose
  media include

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Plating of specimens and incubation of cultures
for bacteria and fungi

• Nutrient agar

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Plating of specimens and incubation of cultures
for bacteria and fungi

• Blood agar this media also allows
  differentiation based on the production by
  bacteria of hemolysins that destroy red blood
  cells in the agar.
• Alpha (?) hemolysis is incomplete hemolysis and
  appears as a green halo surrounding the bacterial
  colony
• Beta (?) hemolysis is complete hemolysis and
  appears as a clear area surrounding the bacterial
  colony
• Gamma (?) hemolysis is no hemolysis at all

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Types of hemolysis
Alpha
Beta
Gamma
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Plating of specimens and incubation of cultures
for bacteria and fungi

• Chocolate agar is essentially the same as blood
  agar, except that the RBCs are lysed when added
  to the agar. This releases hemin and NAD for
  utilization by fastidious bacteria. The lysis
  gives the medium its chocolate brown color.

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Plating of specimens and incubation of cultures
for bacteria and fungi

• Some books call blood agar and chocolate agar
  enriched media because the blood that is added
  to the media acts to enrich the media.
• Selective media this type of media contains
  special nutrients that support the growth of
  certain pathogens and/or inhibitors that suppress
  the growth of competing normal flora. For
  example
• Phenyl ethyl alcohol agar (PEA) contains phenyl
  ethyl alcohol to inhibit the growth of Gram
  negative bacteria (Why does it inhibit the growth
  of G bacteria?)

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PEA agar
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Plating of specimens and incubation of cultures
for bacteria and fungi

• Selective media is also usually differential.
  What makes it differential is often the addition
  of a carbohydrate and a pH indicator that
  together allow one to differentiate between
  organisms that ferment the carbohydrate and those
  that do not. For example
• Mannitol salts agar (MSA)- this media contains
• 7.5 NaCl to suppress the growth of organisms
  that are not halophilic (selection)
• Mannitol and the pH indicator phenol (for
  differentiation).
• If an organism that grows on the media ferments
  mannitol, acid is produced and this lowers the
  pH. At a low pH phenol red turns yellow.
  Therefore, organisms that ferment mannitol turn
  the media yellow

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MSA
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Plating of specimens and incubation of cultures
for bacteria and fungi

• MacConkey agar (Mac) this media contains
• Crystal violet to inhibit the growth of G
  bacteria and fungi (selection).
• Lactose and the pH indicator neutral red which is
  red or pink at an acid pH (differentiation).
• Organisms that are able to grow on the media and
  that ferment lactose produce pink colonies on the
  media
• Organisms that dont ferment lactose produce
  colorless colonies.
• Mac plates are an example of what are
  collectively called enteric agar plates because
  they facilitate the isolation and differentiation
  of enteric pathogens. You will be discussing
  other types of enteric agar plates in the
  laboratory.

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Mac plate
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Mac plate
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Plating of specimens and incubation of cultures
for bacteria and fungi

• Reducing media this type of media is used for
  cultivating anaerobes. It contains compounds
  that chemically combine with dissolved oxygen in
  the media to deplete the O2 from the media. For
  example
• Sodium thioglycollate broth contains
• Thioglycolic acid as a reducing agent to create
  an anaerobic atmosphere deeper in the tube and
• Resazurin as an oxygen-reduction indicator. In
  the presence of O2, resazurin turns pink. Where
  do different type of organisms grow in the
  thioglycollate broth?

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Growth of different types of organisms in
thioglycollate broth
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Plating of specimens and incubation of cultures
for bacteria and fungi

• Enrichment culture media is used to prevent
  missing a type of bacteria present in only small
  numbers.
• This media is usually liquid and it provides
  nutrients and environmental conditions that favor
  the growth of one type of organism while being
  unsuitable for the growth of others. For example,
  to enrich a stool culture for enteric pathogens
  that may be found in low numbers relative to the
  normal flora from the intestine
• Gram negative broth
• Tetrathionate broth
• Selenite broth

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Plating of specimens and incubation of cultures
for bacteria and fungi

• Special plating procedures
• Anaerobic cultures
• Reducing media may be used, or
• Plates can be incubated in a special jar or pouch
  in an oxygen free atmosphere. In an anaerobic
  jar the oxygen free atmosphere is generated by
  the following mechanism

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Plating of specimens and incubation of cultures
for bacteria and fungi

• Envelopes containing sodium bicarbonate and
  borohydride are placed in the jar and water is
  added and the chemical reaction that occurs
  generates CO2 and H2.
• The H2 that is generated combines with O2 in the
  jar in the presence of a palladium coated alumina
  pellet that acts as a catalyst for the following
  reaction
• 2H2 O2?2H2O
• Thus O2 is removed from the atmosphere.
• An indicator strip with methylene blue that is
  colorless in the absence of oxygen and blue in
  the presence of oxygen is also placed in the jar.

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An anaerobic jar
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Plating of specimens and incubation of cultures
for bacteria and fungi

• Blood cultures as discussed previously, blood
  cultures are usually inoculated into blood
  culture media directly at the bedside of the
  patient.
• Frequently two bottles of liquid media are
  inoculated, one for aerobic growth (TSB tryptic
  soy broth) and one for anaerobic growth (Thio).
• The bottles are usually examined every day for
  turbidity for 7-14 days.
• If turbidity develops, some of the media is
  removed for Gram staining and subculturing onto
  solid media.
• Depending upon the lab policies, blood cultures
  may routinely be Gram stained and subcultured at
  specific intervals (24 hrs.,48 hrs, etc.) even in
  the absence of turbidity.

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Plating of specimens and incubation of cultures
for bacteria and fungi

• Some labs have machines (Bactec) that
  automatically detect growth in blood cultures by
  ? CO2 production
• Quantitative counts as discussed previously,
  this is often done on urine specimens.
• A known volume of specimen is plated on the agar
  medium, via the use of a calibrated loop, and the
  number of colonies that grow are counted.
• Caution this only represents the number of
  bacteria present at the moment of plating.
• For a clean catch urine specimen gt100,000
  colonies /ml is considered significant and
  indicative of disease.
• For a bladder or kidney specimen gt10,000
  colonies/ml is considered significant and
  indicative of disease. Why?

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Quantitative urine counts
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Plating of specimens and incubation of cultures
for bacteria and fungi

• Plating for unusual bacteria
• Some of the more rarely encountered pathogens
  need unusual media and/or techniques to
  facilitate their isolation.
• If a physician suspects that one of these
  organisms is the causative agent of an infection,
  he/she must notify the lab so that appropriate
  media can be prepared and proper precautions can
  be taken, if necessary. For example
• Brucella species (sp.)
• Bordetella sp.
• Legionella sp.

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Plating of specimens and incubation of cultures
for bacteria and fungi

• Incubation of cultures
• Temperature
• Media inoculated with human specimens is usually
  incubated at 35-370 C, which is the optimum
  growth temperature for most human pathogens.
• Fungi are often grown at room temperature (RT).
• Many fungi are dimorphic and will grow as yeast
  at 370 C and as molds at RT. Candida albicans is
  different in that at RT it grows as a yeast, but
  at 370 C and in the presence of serum, it grows
  as a mold.

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Plating of specimens and incubation of cultures
for bacteria and fungi

• Atmosphere
• Most pathogenic bacteria grow best at 2-10 CO2
  and most clinical labs routinely use 5 CO2
  incubators for their cultures.
• Certain bacteria actually require 5-10 CO2 in
  order to grow or to grow well (Neisseria sp.,
  Streptococcus sp., Haemophilus sp.).
• To facilitate the growth of theses organisms, we
  will grow them in candle jars to provide the
  higher CO2 that they need for growth.

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Candle jar
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Plating of specimens and incubation of cultures
for bacteria and fungi

• Anaerobes have already been discussed
• Length of incubation time
• Most routine cultures are kept for 16-18 hours
  (overnight) before being reported out as negative
• CSF and blood cultures are usually kept 1 week
  before being reported out as negative
• Wound cultures may be kept for 48 hours before
  being reported out as negative

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Plating of specimens and incubation of cultures
for bacteria and fungi

• Fungal cultures are kept for 3-4 weeks before
  reporting out as negative
• Cultures for Mycobacteria are kept for 6-10 weeks
  before reporting out as negative.