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Flashcards in MB 5 - Diagnostic Techniques Deck (41):

Learning Outcomes (for general perusal)

Be able to describe with examples

•Direct and indirect diagnostic tests

•Microscopy and specifically the gram stain

•The principles of culture of microbes

•The basics of antibody detection methods for diagnosis of infection

•The basics of molecular detection of infectious agents

•The approach to taking blood cultures 


What can microbiology be used for?



•Infection control



Give examples of some infectious diseases

  • Mumps
  • Chickenpox
  • SARS
  • Tetanus
  • Diphtheria


Microbiological tests can be...

  • Diagnostic - What infectious agent is there? Is this significant?
  • Theronostic (to direct therapy) - more about the bug
    • Viral load
    • serial testing
    • Sensitivity/ resistance testing
  • Immunity Testing - Has the person responded to a vaccine? Does the exposed person need prophylaxis?


  1. Outline Direct testing
  2. Outline Indirect testing

  1. See a germ, Grow a germ, Find a bit of germ (antigen), Find a bit of germ nucleic acid

  2. Find evidence of a specific immune response to a germ (lymphocytes, antibodies)


Give an example of direct testing

See a germ - Microscopy e.g Gram Stain (Gram negative diplococci In PMN in CSF =Meningococcal meningitis)

Grow a germ - eg. Staphylococcus aureus, Allows antibiotic sensitivities to be determined

Find a bit of germ (antigen) - Legionella urinary antigen

Find a bit of germ nucleic acid - PCR


Give an example of indirect testing

Finding evidence of a specific immune response to a germ - in practice this usually means serology

  • Ab Assay Examples
    • CFT (complement fixation test)

    • ELISA (enz-linked immunosobent assay) / EIA (enz immunoassay) - same thing

    • Indirect immunofluorescence (IIF)

  • Measurements

    • IgG / IgM (short lived = recent infection) / IgA Total Antibody or Positive, Negative, Equivocal or Quantity in Units

    • Rising levels (serial sampling)

    • IgG Avidity (estimation of how recent)

    • Immunoblots (reactivity to xple Antigens from a bug)


Diagnostic Process

What is the role of the microbiology lab?

•Clinical history and examination

•Differential diagnosis


–Laboratory investigations

•Microbiology investigations


–Direct detection


Clinical Diagnosis example
Meningococcal meningitis


  1. What would the patient present with?
  2. What simple clinical test can be done?
  3. What microorganism would be seen microscopically?
  4. Describe it's appearance
  5. What microbiological tests are carried out?

  1. Headache, fever, vomiting,drowsiness, Haemorrhagic rash, neck stiffness. Infants = poor feeding, bulging fontanelle

  2. Tumbler Test Positive in meningococcal infection

  3. Neisseria meningitidis

  4. Gram negative diplococci

  5. Microscopy




What determines if a good specimen is taken?




–transport time

–storage conditions

–transport medium


Give examples of some specimens that can be taken?

–Throat swab





–Genital swab



Give the different types of blood samples

  • Clotted blood
    • Serum e.g. serology
  • EDTA blood 
    • Plasma / WBCs / Whole Blood e.g. PCR
  • Blood culture 


EDTA =  Ethylenediaminetetraacetic acid. Anticoagulant, prevents haemolysis,


How will a diagnosis come about in a 

  1. Patient
  2. Specimen

  1. Clinical Diagnosis. Haematology, Biochemistry, Non-microbiological investigations eg. Radiology
  2. Correct specimen, taken correctly, labelled and packaged correctly, appropriate transport and storage of specimen


  1. What is sensitivity?
  2. What is specificity?
  3. What is positive predictive value?
  4. What is negative predictive value?

  1. Measure of the lack of false negatives

  2. Measure of the lack of false positives

  3. TP/TP+FP

  4. TN/TN+FN



  1. What can be seen with an electron microscope?
  2. What can be done with a light microscope?

  1. Herpes Virus, Paramyxovirus
  2. Staining e.g Gram Stain



Outline the process of Gram staining

  1. Crystal Violet - Everything stains
  2. Gram's Iodine - Stain differently fixed
  3. Decolorise (Acetone) - Gram-negatives destain
  4. Counterstain with Methyl Red - Gram-positives appear purple, Gram-negatives appear pink



Name some different shapes and gram reactions

  • Gram positive cocci
  • gram-positive rods
  • gram-negative cocci
  • gram-negative rods


What is the appearance of 

  1. Neisseria meningitidis in CSF

  2. Staphylococcus aureus and E. coli - surgical wound

  1. Gram negative diplococci (intracellular)

  2. Gram positive cocci & gram negative rods



​Describe the appearance of 

  1. Haemophilus influenzae

  2. Streptococcus pyogenes

  1. Pleomorphic
  2. Gram-positive connected in long chains


What can the Ziehl-Neelsen (ZN) stain be used for?

Used for the bacteria that cause TB


Old technique


'Grow a germ'

  1. What is a Bacteriological culture?
  2. What types of atmospheres can the culture be incubated in?
  3. Give an example of colony appearance

  1. Clinical specimen inoculated onto various media (selective, enrichment). Appearance of colonies suggests species present. Further tests to clarify 
  2. Anaerobic, Aerobic, CO2-rich
  3. Haemolysis
    1. Beta-complete, Clear zone, B haemolysis, blood has been lysed = Streptococcus pyogenes

    2. Alpha-incomplete, Green zone, blood hasn't been lysed, made leaky = Streptococcus pneumoniae 



Antimicrobial Sensitivities

Outline these

•Filter paper disc

•Impregnated with antibiotic

•Zone of inhibition

If it can grow right up to the disc = resistant


What is a viral culture conducted in?

Giving this test what characterisitics?

Living cells

Slow and sensitive


Molecular Tests

What do these involve?



•Detection of nucleic acid


•Quantitative or Qualitative


•Amplification methods  e.g. PCR


Molecular Tests


  1. Give an example

  1. How much eg. HIV load (Viral RNA in blood)
    1. Can be done before and during treatment


Polymerase Chain Reaction (PCR)

Outline this process

What can be a limitation, but at the same time a benefit?

  • Specific primers - need to know what you're looking for. BUT lends extra specificity 
    • 20 nucleotides
  • Melt
  • Anneal primers
  • Extend new DNA
  • 25- 35 cycles


  1. Outline Realtime PCR
  2. What can carry out Single Test Realtime?

1. PCR is quatifiable – can work out how long it takes to become positive

Good at saying how much there is in a sample

Going up, going down, staying the same


2. Xpert MTB/RIF -  a test to detect tuberculosis and rifampicin resistance directly from sputum in less than 2 hours


What does Molecular Diagnosis of  Rifampicin Resistance (TB) involve?

DNA Extraction



Valid Results


Indirect Testing

Give examples of methods of serology 

  • Complement fixation test
    • Ag-Ab complex fixes complement
  • Enzyme immunoassays (ELISA)
    • colour change
  • Immunofluorescence
    • labelled antibody



What does the Complement fixation test (CFT) involve?

  • Positive result = no RBC lysis
    • complement 'fixed', can't lyse
      •  complement attached to patient antibody which is attached to antigen
  • Negative Result = RBC lysis
    • When complement is added, cells burst, no antibody to attach to 


If have antibody, will create antigen antibody complex, complement will stick to. Complement sucked out. RBC doesn’t burst



What do Enzyme immunoassays EIA or ELISA involve?

Enzyme mediated colour change

An enzyme - labelled anti-human antibody is added

This will attach to human antibody which is attached to an antigen (eg.HIV)

Enzyme will change from colourless-coloured



What does Immunofluorescence
 serology involve?

Mouse anti-human Ig with a fluorescent label added

Will attach to patient's antibody attached to antigen eg. HSV

Can see if there is a patient antibody there or not

Can do serial diluations of the blood see where it goes from positive to negative.


Examples of Microbiology Testing

Chicken Pox

  1. What is the earliest diagnostic microbiological test?
  2. When will blood tests be effective?

  1. PCR (from day 14)
  2. IgM (day 18) and IgG (day 20) - blood tests will give negative results earlier in the course


Examples of Microbiology Testing

Staphylococci  & Streptococci

  1. What is their appearance on gram stain?
  2. How will Staph.aureus look?
  3. How will Strep.pyogenes look?

  1. Gram positive cocci
  2. In clusters, catalase-positive
  3. In chains, catalase-negative


Examples of Microbiology Testing

Streptococcal pharyngitis

What does diagnosis involve?

  • Throat swab
    • Plate out (Blood agar)
    • Beta-haemolysis (complete haemolysis)
    • latex agglutination typing test (Lancefield grouping of capsular antigen)
    • (Result is Group A)


Examples of Microbiology Testing

Dermatophyte Infections

​How is a diagnosis made?

  • direct microscopic examination of scales dissolved in KOH
  • by culture of scrapings of lesions


Examples of Micriobiology Testing


  1. What microorganism is likely to be present?
  2. What will the patient present with?
  3. How is it diagnosed?
  4. What specimen is used?

  1. RSV - Respiratory syncytial virus
  2. febrile wheeze can progress to respiratory obstruction or exhaustion, requiring hospitalisation

  3. Immunofluoresence or PCR

  4. nasopharyngeal aspirate


Outline the Identification and typing of organisms

  • Antigenic type
    • e.g. Serotype
  • Genotype
    • e.g. RFLP (Restriction Fragment Length Polymorphism), Sequence
  • Others
    • biotype eg API biochemical tests
    • antibiotic resistance pattern


Examples of Microbiology Testing


What does it involve?

  • Biochemical tests
    • Commercial kits commonly used, e.g. API20
  • Antigenic tests
    • Sero-typing agglutination test


Blood Cultures

Outline the principles

  • Sensitivity increases with quantity of blood
    • Take 10-20 ml per set
  • THREE blood culture sets usually taken except in  suspected endocarditis when up to SIX are taken.
  • Samples can be taken through lines if line infection suspected


Blood Cultures

  1. How should they be stored?

  • After blood cultures have been taken they are placed in an incubator immediately for maximum speed & sensitivity
  • never put blood cultures in the fridge!!!
  • Incubate at 37°C for up to 5 days
    • longer in endocarditis, PUO (pyrexia of unknown origin)