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POM > Diagnostic bacteriology and diagnostic virology > Flashcards

Diagnostic bacteriology and diagnostic virology Flashcards

1
Q

Why is diagnostics important?

A

Prevent misuse of treatment - reduce use of antibiotics when they’re not necessary. Identify and prevent outbreaks.

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2
Q

What are the 3 main types of testing for diagnostic bacteriology?

A

Microscopy, culture and sensitivities.

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3
Q

What microscopy tests are done and why are they done?

A

Gram stain tells us what type of bacteria.

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4
Q

Why is a bacteria culture test done?

A

To work out species of bacteria.

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5
Q

What bacteria culture tests are done?

A

Hemolysis test. Lactose test. Catalase test. Coagulase test.

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6
Q

Why is a sensitivity test done?

A

To work out what is the most effective treatment for that bacterium.

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7
Q

What does the minimum inhibitory concentration mean?

A

The lowest concentration of an antibiotic that inhibits the growth of a given strain of bacteria.

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8
Q

What is the antimicrobial breakpoint?

A

An antimicrobial breakpoint is the agreed concentration of an agent at which bacteria can, and cannot, be treated with the antimicrobial agent in question.

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9
Q

What antibiotic sensitivity test is done?

A

Disk diffusion test.

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10
Q

Structure of cell wall in gram positive bacteria?

A

One outer membrane with thick peptidoglycan.

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11
Q

Structure of cell wall in gram negative bacteria?

A

Two outer membranes with thin peptidoglycan.

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12
Q

What colour do gram negative bacteria stain?

A

Pink.

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13
Q

What colour do gram positive bacteria stain?

A

Purple.

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14
Q

How is a gram stain performed?

A

Bacteria culture spread on glass slide. Heat to fix bacteria to surface. Application of crystal violet solution to slide. Application of iodine to slide. Alcohol wash performed on slide. Counter stain performed by application of safranin.

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15
Q

What colour is crystal violet?

A

Purple.

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16
Q

What colour is safranin?

A

Pink.

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17
Q

What occurs to gram positive and gram negative bacteria after alcohol wash?

A

Gram positive bacteria stay purple as iodine and crysal violet can’t be washed out of cell wall. Gram negative bacteria lose purple colour.

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18
Q

What are the basic bacteria morphology?

A

Cocci and bacilli.

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19
Q

What shape are coccus bacteria?

A

Spheres.

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20
Q

What shapes are bacillus bacteria?

A

Round ended cylinders.

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21
Q

What are hemolysins?

A

Enzymes that damage red blood cells.

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22
Q

Three types of hemolysis in diagnostic bacteriology?

A

Gamma hemolysis, Alpha hemolysis, Beta hemolysis.

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23
Q

What is gamma hemolysis?

A

No hemolysis.

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24
Q

What is alpha hemolysis?

A

Partial hemolysis.

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25
Q

What is beta hemolysis?

A

Full hemolysis.

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26
Q

What would you see in gamma hemolysis?

A

No zone.

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27
Q

What would you see in alpha hemolysis?

A

Opaque green zone.

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28
Q

What would you see in beta hemolysis?

A

Transparent zone.

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29
Q

How is a hemolysis test performed?

A

Streak out bacterial colony on blood agar. Assess hemolysis after overnight incubation.

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30
Q

What is the hemolysis test useful for differentiating?

A

Differentiating members of the genera Staphylococcus, Streptococcus and Enterococcus.

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31
Q

What is the lactose fermentation test useful for differentiating?

A

Useful for differentiating gram negative bacteria.

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32
Q

What do lactose fermenting bacteria contain allowing them to produce lactic acid from lactose?

A

Lactase.

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33
Q

What type of agar is used in lactose fermentation test?

A

MacConkey agar.

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34
Q

What can’t grow on MacConkey agar? When is MacConkey agar used?

A

Gram positive bacteria as they are inhibited by bile salts. MacConkey agar is used for differentiating gram negative bacteria.

35
Q

How is a lactose fermentation test performed?

A

Bacterial colony is taken from agar plate and streaked onto MacConkey agar that has a pH indicator called neutral red. Assess after overnight incubation.

36
Q

Colour change in lactose fermentation test if bacteria is lactose fermenting? Why?

A

Red to pink. If the bacteria ferment lactose, then acid is produced as a by-product which decreases the pH of the media. Neutral red changes to pink when pH becomes acidic.

37
Q

Colour change in lactose fermentation test if bacteria is lactose non-fermenting? Why?

A

Red to yellow. If the bacteria are lactose non-fermenters then they use the peptone instead, then ammonia is formed as a by-product which increases the pH of the media. Neutral red turns yellow when pH becomes basic.

38
Q

What type of bacteria is the hemolysis test used for?

A

Gram positive bacteria.

39
Q

What type of bacteria is the catalase test used for?

A

Gram positive bacteria.

40
Q

What genus of bacteria produce catalase?

A

Staphylococci.

41
Q

What genus of bacteria don’t produce catalase?

A

Streptococci.

42
Q

What does catalase do?

A

Breaks down hydrogen peroxide into water and oxygen (bubbles).

43
Q

How is a catalase test performed?

A

Application of bacteria to glass slide. Application of hydrogen peroxide to slide. Observation for generation of bubbles.

44
Q

What is the coagulase test useful for differentiating?

A

Members of the staphylococci genus.

45
Q

What does coagulase do?

A

Enzyme produced that converts prothrombin to thrombin. Thrombin cleaves fibrinogen into fibrin. Factor VIIIa crosslinks fibrin to form fibrin clots.

46
Q

How to perform coagulase test?

A

Application of bacteria to glass slide. Application of plasma to slide. Incubate for 15 seconds and gently rotate. Observe for clumps.

47
Q

What bacterium produces coagulase?

A

S.aureus

48
Q

What test is used to determine minimum Inhibitory concentration for an antibiotic?

A

E test.

49
Q

What does HTLV-I stand for?

A

Human T-cell leukaemia virus type I.

50
Q

What does HTLV-1 cause?

A

Adult T- cell leukemia (ATL). Adult T-cell leukaemia/lymphoma (ATLL).

51
Q

How is HTLV-1 transmitted?

A

Mother to infant (breastfeeding). Sexual contact. Blood transfusion.

52
Q

What is HTLV-1 structure?

A

Enveloped virus. Single strand RNA virus. Contains reverse transcriptase.

53
Q

HTLV-1 replication cycle?

A

HTLV-1 enters T-cell
ssRNA released into host cell cytosol
ssRNA reverse transcribed (RT-enzyme) to ssDNA
ssDNA converted to dsDNA
dsDNA enters nucleus and integrates into host genome
viral genome can replicate as part of the host chromosome

54
Q

What triggers oncogenesis in T cell infected with HTLV?

A

The HTLV-1 Tax oncoprotein triggers oncogenesis and induces the production of its own RNAs.

55
Q

What gene is found in HTLV and not other viruses?

A

Tax gene

56
Q

How is a polymerase chain reaction done?

A

DNA sample is denatured by heating to 94 degrees celsius. Cooled down to 54 degrees to allow forward and reverse primers to anneal. Sample is heated to 72 degrees and new DNA chain is extended.

57
Q

How many cycles is a typical PCR?

A

30-40.

58
Q

What are the 5 key reagents of a PCR?

A

DNA template, Primers, DNA polymerase, deoxynucleotide triphosphates and reaction buffer.

59
Q

How is DNA sample for HTLV taken?

A

Blood is taken from patient and mixed with seperation medium. It is then centrifuged. Peripheral blood mononuclear cell fraction is extracted and DNA is isolated.

60
Q

How many PCR reactions would need to be setup if testing 4 patients for htlv?

A
  1. 4 patients PCR reactions. One positive control (HLTV). One negative control (no HLTV).
61
Q

What anode does DNA move towards in gel electrophoresis?

A

Positive anode as DNA is negatively charged.

62
Q

What does electrophoresis seperate DNA based on?

A

Size of DNA.

63
Q

What is used to visualise the DNA in electrophoresis?

A

Intercalating DNA stain.

64
Q

Why is a DNA loading dye used?

A

Increase weight of DNA sample. See which wells contain a sample. Indicate how far the DNA fragments have migrated during run.

65
Q

Why is a DNA ladder used?

A

To estimate size of DNA fragment.

66
Q

What is the point of a buffer in PCR?

A

Provides suitable pH for DNA polymerase.

67
Q

Why is the western blot method used for a HTLV patient ?

A

To assess if patients have antibodies specific to HLTV-1 proteins.

68
Q

How is the western blot method performed for HTLV?

A

Different HLTV viral proteins are separated based on their size on a protein gel using electrophoresis. The proteins will be transferred using an electro-transfer system onto a PVDF membrane that the viral proteins will stick to, and this will basically create an image of the gel. Incubate membrane with patient serum so antibodies can bind to viral proteins. Wash membrane to remove all unbound antibodies. Incubate membrane with secondary antibody that is conjugated with an enzyme. Secondary antibody binds to fc region of patient’s antibodies. Wash again to remove any unbound secondary antibodies. Substrate added, enzyme produces colour.

69
Q

For a positive HTLV diagnosis what antibodies need to be present on western blot method?

A

MTA-1, p53, p24, p19 and gd21(recombinant glycoprotein).

70
Q

What is the advantage of doing a quantitative real time PCR?

A

Provides information on amount of viral DNA present in sample. Can help predict severity of disease and how likely transmission is to occur.

71
Q

What are the two ways in which quantitative real time PCR is done?

A

Fluorescence dye-based method and DNA probe-based method.

72
Q

How do you know if someone is positive for HTLV in QPCR?

A

Increase in fluorescence above a certain threshold. Negative HTLV patients the fluorescence doesn’t increase above threshold.

73
Q

What is the CT value in QPCR?

A

Number of PCR cycles needed for the sample to reach threshold (positive diagnosis).

74
Q

How will the CT value in QPCR change in a patient with a high viral load?

A

Low CT value.

75
Q

Pros and cons of use of DNA probe based method (taqman method)?

A

High specificity. Cost.

76
Q

How is DNA probe based method done?

A

DNA Oligonucleotide probe added. Probe binds to specific gene that is amplified on template strand. Probe contains fluorophore at 5 prime and quencher at 3 prime sides. Fluorescence increases when fluorophore and quencher are seperated which happens when DNA polymerase synthesises new DNA stand and degrades probe. Fluorescence is proportional to amount of the specific gene in sample.

77
Q

Explain primer design?

A

The forward primer synthesizes the upper strand using the bottom strand as a template. Whereas Reverse primer uses the upper strand as a template and synthesizes the lower strand.

78
Q

Relationship with ct value and tax gene copy number?

A

Linear with the log10 of tax gene copy number.

79
Q

S.aureus lab diagnosis?

A

Gram +, Catalase +, Coagulase +.

80
Q

S.pneumoniae diagnosis?

A

Gram +, Catalase -, Alpha hemolytic.

81
Q

S.pyogenes lab diagnosis?

A

Gram +, Catalase -, Beta hemolytic.

82
Q

E.Coli lab diagnosis?

A

Gram -, Lactose fermenting.

83
Q

Salmonella lab diagnosis?

A

Gram -, Lactose non fermenting.

POM (41 decks)

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