Diagnostic Microbiology

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Diagnostic Microbiology

• WT 2006
• Bad Bugs An Introduction to Infectious Disease

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Specimen Collection

• A properly collected specimen is the single most
  important step in the diagnosis of an infection,
  because the results of diagnostic tests for
  infectious diseases depend upon the selection,
  timing, and method of collection of the specimen.

(Brooks, G. F., J. S. Butel, and S. A. Morse.
(2001) Jawetz, Melnick, Adelbergs Medicial
Microbiology, 22nd ed. Lange Medical
Books/McGraw-Hill. New York, NY. p. 597)
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Specimen Collection

• Sufficient quantity of specimen should be
  collected.
• Specimen should be representative of infectious
  process.
• Contamination must be avoided during collection.
• Specimen should be transported promptly and in
  correct manner.
• Specimen should be collected before beginning
  antimicrobial therapy.

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Commonly Used Specimens

• Blood
• Urine
• Sputum
• Tissue biopsies
• Pus
• Cerebrospinal fluid (CSF)
• Swabs
• Feces

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Methods

• Morphologic identification
• Culture isolation and identification
• Immunological detection
• Nucleic acid detection
• Immune response to agent

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Morphologic Identification

• Microscopic examination of stained and unstained
  specimens.
• Requires 100,000 organisms/milliliter.

http//www.cdc.gov/ncidod/EID/vol7no6/jerniganG2.h
tm
Gram stain of cerebrospinal fluid of patient with
anthrax.
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Microscopy Stains

• Gram Stain
• Acid-fast Stain
• Immunofluorescent Antibody Staining
• Darkfield Microscopy

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Gram Stain
http//www.pathguy.com/lectures/pneumococci.jpg
http//www.meddean.luc.edu/lumen/DeptWebs/microbio
/med/gram/slides/slide13.jpg
Gram stain of sputum shows Gram-positive
Streptococcus pneumoniae.
Gram stain of urine shows Gram-negative
Escherichia coli.
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Acid Fast Stain
Mycobacterium tuberculosis. Lewis Tomalty and
Gloria Delisle, authors. Licensed for use, ASM
MicrobeLibrary.
Mycobacterium tuberculosis in sputum sample.
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Immunofluorescent Staining

• Use fluorescently labeled antibodies to detect
  specific organisms.

http//www.cdc.gov/ncidod/eid/vol8no2/images/01-01
98-2b.jpg
Immunofluorescent stain of Borrelia hermsii,
etiologic agent of relapsing fever.
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Darkfield Microscopy

• Used primarily to detect Treponema species.
• Bright objects on dark background
• Advantages
• No staining
• Better resolution

http//depts.washington.edu/nnptc/online_training/
std_handbook/gallery/images/treponemapallidum.jpg
Darkfield image of Treponema pallidum, etiologic
agent of syphillus.
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Culture Systems Type of Media

• Rich medium
• Contains many nutrients to promote growth of
  fastidious organisms.
• Selective medium
• Promotes growth of some organisms while
  inhibiting growth of others.
• Differential medium
• Gives rise to colonies with a distinctive
  appearance.

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Rich Media Blood Agar

• Nonselective
• Contains 5 sheep blood.
• Facilitates identification of hemolytic organisms.

Blood Agar Plate. Rebecca Buxton, author.
Licensed for use, ASM MicrobeLibrary.
Normal upper respiratory flora mixed with
Streptococcus pyogenes on blood agar.
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Rich Media Chocolate Agar

• Contains heated blood which causes release of X
  and V factors from RBC.
• Used to isolate Haemophilis and Neiserria species.

http//medinfo.ufl.edu/year2/mmid/bms5300/images/d
7106.jpg
Haemophilus influenzae on chocolate agar.
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Selective Media
Gram -

• Phenylethanol agar
• Selects for Gram and against Gram - organisms.
• Phenylethanol inhibits DNA synthesis.

http//www.austincc.edu/microbugz/html/phenylethyl
_alcohol_agar.html
Gram
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Differential Media

• MacConkey Agar
• Selective differential
• Bile salts and crystal violet inhibit Gram
  growth.
• Acids produced due to lactose fermentation causes
  neutral red pH indicator to change color.

http//www.mc.maricopa.edu/johnson/labtools/Dbioc
hem/3mac.jpg
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Differential Media

• Eosine Methylene Blue Agar (EMB)
• Selective differential
• Aniline dyes inhibit Gram .
• Facilitates differentiation between lactose
  non-fermenters, weak fermenters, and strong
  fermenters.

http//www.mc.maricopa.edu/johnson/labtools/Dbioc
hem/3emb.jpg
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Immunological Detection

• ELISA
• Enzyme Linked Immunosorbant Assay
• Commonly used to detect Chlamydia, Rotavirus,
  HIV, some bacteria.

http//www.managingdesire.org/ELISA.jpg
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Immunological Detection
- - -

• Western Blot
• Used to detect presence of antibodies in patient.
• Separate antigens on gel and use patients sample
  as antibody.
• Used for diagnosis of Lyme disease HIV.

http//jeeves.mmg.uci.edu/immunology/Assays/HIVWes
tern2b.jpg
HIV Western blot test
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Immunological Detection

• Latex agglutination
• Antigen or antibody is attached to latex beads.
• Antigen-antibody complex formation causes
  agglutination (visible clumping).
• Basis for quick strep test.

- agglutination
agglutination
http//jeeves.mmg.uci.edu/immunology/Assays/LatexA
gglut.jpg
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Nucleic Acid Detection

• PCR
• Polymerase Chain Reaction
• Detects specific DNA target.
• RT-PCR
• Real Time PCR
• Faster than normal PCR.

http//www.clongen.com/Copy20of20Lyme20Disease
20Sensitivity20Illustration.gif
http//www1.qiagen.com/products/pcr/quantitectpcrs
ystems/quantitectmultiplexpcrkit.aspx?ShowInfo1
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Detection of Immune Response

• TB skin test
• Also known as Mantoux test, tuberculin test, or
  PPD test.
• reaction due to hypersensitive cell-mediated
  response.
• test doesnt mean youve got TB--means that
  youve been exposed to TB.

http//www.lung.ca/tb/tbtoday/tbdiagnosis/skin_tes
t.html
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Susceptibility Testing

• Determines what drugs should be used to treat
  disease.