Specimen Processing in the Laboratory

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Specimen Processing in the Laboratory

• MICI 1100

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Specimen Accessioning

• The specimen must be clearly identified
• Patient source
• Site of sampling
• Input of data into the computer
• Clinical information
• Specimen number for tracking
• Request
• Assessed for appropriateness
• Specimen type, handling, methodology

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Specimen Set Up

• Rapid Testing
• Microscopy
• Other Rapid tests
• Culture
• Molecular testing
• Special handling/processing needs
• Special processing requirements. e.g. TB
  specimens
• Referred out tests testing done in others labs

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Rapid Testing

• Point of Care Tests
• Usually detect antigen e.g. Streptococcus
  pyogenes in pharynx
• Sometimes these are done in clinics, offices or
  Emergency Rooms
• Detection of antigens from the pathogen
• Presence of antigen may cause particles to
  agglutinate or a coloured line to appear
• Mistakes in technique can give incorrect results
• Sensitivity is usually sacrificed for speed
• May not be significant loss, but if so,
  confirmation may be required

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Microscopy

• Gram stain is the most commonly used
• Wet prep or acid fast stain are less common
• Used for diagnosis
• Presence of organisms/cells in the specimen
• Gram stain is much less sensitive than culture
• Used to assess the quality of the specimen Q
  scoring
• Types of cells (squamous cells) used to infer
  contamination has occurred (e.g. saliva)

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Culture

• Generally one of the most definitive methods
• Sensitivity high (less for viruses, some rare
  bacteria)
• Specificity high
• Provides isolates for further testing
• Often relatively slow
• May take days (most commonly) to weeks (fungi,
  TB)
• Influenced by
• Types of media
• Incubation conditions
• Identification method

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Media

• May be liquid (broth) or solid (plates)
• Broth is used for
• Detecting very low numbers of organisms
• e.g. blood cultures
• Increasing the number of a type of organism
  (enrichment) in a specimen
• e.g. Used to increase the number of salmonella in
  stool samples.
• Biochemical testing

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Media

• Solid media (usually solidified with agar)
• Used for
• Isolation of colonies of bacteria
• This enables us to work with single organisms
• Can be used to detect low numbers
• Differential colonies have different colours
  based on a biochemical reaction built into the
  plate e.g. red lactose positive vs pale lactose
  negative colonies on MacConkey medium
• Selective species are inhibited from growing on
  the plate by a constituent of the medium
• Many media may be both to some extent

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Common Media

• Blood agar
• Contains about 5 sheep blood
• Useful work-horse medium
• Allows detection of hemolysis (Streptococci)
• Chocolate agar
• Similar to blood agar but the blood is cooked
• More nutritious medium
• MacConkey medium
• Contains bile salts and crystal violet to inhibit
  gram positive organisms
• Lactose fermentation detected
• Useful for Enterobacteriaceae and Pseudomonas
• Often used for urine and stool specimens

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Incubation Conditions

• Determines the types of organism that will be
  cultured
• Atmosphere, usual conditions are
• aerobic (ambient air)
• 5 CO2 in ambient air (18 oxygen)
• Often used for organisms that cause respiratory
  infections
• Microaerobic (2-5 oxygen)
• Anaerobic (lt1 oxygen)
• Temperature
• Most often at 35oC, rarely at 30o or 25oC
• Some organisms can be selected for by growing at
  42oC. (Many gut organisms are inhibited at 42oC)

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Identification Colony

• Colony the growth of bacteria on solid medium
  a pile of bacteria of a single species
• Appearance can be useful for identification
• Size, shape, edge, surface appearance
• Presence of haemolysis (esp. Streptococci)
• Pigment giving a coloured colony or in the medium
  (diffusable pigment)
• Each colony is descended from a single or
  sometimes a group of attached individuals of a
  species
• Referred to as colony forming units (CFU)

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Identification Biochemical

• Based on the enzymes that an organism is actively
  expressing
• Tends to be a profile typical of each species
  with minor intra-species variation
• The most commonly used method for identification
• Automated methods used for some common organisms
• Abbreviated (short) tests can be used to give a
  presumptive identification

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Other Identification Methods

• Include
• Identification of specific antigens on the
  organisms surface
• Agglutination tests
• Immuno-fluorescent microscopy
• Molecular methods
• Nucleic acid probes
• Genome sequencing

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Susceptibility testing

• Determines which antimicrobials are likely to be
  effective against an individual strain
• Limitations include that the testing is done in
  artificial circumstances
• Does not take into account the ability of the
  persons defenses to resist organisms attack
• Guidelines on testing and interpretation do not
  exist for all organisms, or for all agents

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Susceptibility testing methods

• Expose organism to concentration of antimicrobial
  
• Antimicrobial may be in a disc
• Antimicrobial may be in broth
• Antimicrobial may be dissolved in the agar
• Lack of growth indicates inhibition
• If the concentration is measured it is called the
  Minimal Inhibitory Concentration (MIC)
• With discs a zone size correlates with a
  susceptible MIC, if the zone is bigger it is
  Susceptible if smaller it is Resistant.
• Organism may be killed at a higher concentration
• This concentration is rarely measured
• It is called the minimal bactericidal
  concentration (MBC)

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Other Susceptibility Methods

• Detection of enzymes that break down
  antimicrobials e.g. beta lactamase
• Sometimes susceptibility to one agent predicts
  the susceptibility to another
• e.g. oxacillin for penicillin in pneumococci. Or
  tetracycline for doxycycline in E. coli
• Molecular means
• Finding genes that encode for changed targets of
  antimicrobials e.g. mecA for MRSA
• Detecting mutations that make organisms resistant
  e.g. HIV

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Further Testing

• Typing can be useful to determine the
  relationship of organisms
• Antibiogram and biotype (pattern of biochemical
  tests)
• Allow a rough idea of similarity
• Serotyping
• Detects antigens in the cell wall of the organism
• Phagetyping
• Detects susceptibility of organisms to bacterial
  viruses
• Molecular typing
• Pulse field get electrophoresis typing compares
  bacterial genomes

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Types of Report

• Preliminary Report
• First report of findings e.g. gram stain findings
• Further Report
• An update to the preliminary but not the
  completed work
• Final Report
• The complete report. Generally, no more
  information will be given on the specimen
• Amended(Corrected) Report
• Information that was incorrect in the Final
  Report is corrected or supplemented, if
  incomplete (e.g. comment added)
• Supplementary Report
• May be used to give further information to add to
  a final report

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