4. Diagnostic Techniques

Question 1

Size of VIRUS + what microscope used?

Answer 1

Virus: 0.03-0.3 microns, use electron & light microscope

Question 2

Size of BACTERIA + what microscope used?

Answer 2

Bacteria: 0.1-10 microns, light microscope

Question 3

Size of MICROSCOPIC PROTOZOA & FUNGI + what microscope used?

Answer 3

Microscopic protozoa & fungi: 4-40 microns, light microscope

Question 4

Membrane pore size that filters out bacteria:

Answer 4

0.4 microns (μm)

Question 5

Resolution of microscopes of 1. Electron 2. Light 3. Human eye

Answer 5

Electron: 0.003 microns
Light: 0.2 microns
Human eye: 40 microns

Question 6

Why do we use microscopy?

Answer 6

1. Microscopy (of original sample) is faster than culturing the bacteria (culturing takes time)
2. Some microbes cannot be cultured
3. Make ‘best guess’ to guide therapy

Question 7

Stains used in microscopy:

Answer 7

1. Gram stain (for most bacteria)
2. Ziehl Neelsen (usually to detect mycobacteria, stained reddish)
3. Immunoflorescence

Question 8

What are the 5 bacteriological investigations commonly used?

Answer 8

1. Microscopy
2. Culture
3. Serology (antibody)
4. Antigen detection
5. PCR: polymerase chain reaction

Question 9

Culture gives more information than microscopy:

Answer 9

1. Definitive identification
2. Antibiotic sensitivity testing
3. Typing

Question 10

What is MALDI-TOF

Answer 10

MALDI-TOF is a mass spectrometry method used to identify most bacteria in the clinical laboratory (via peptides/proteins)

Question 11

What is Ziehl Neelson Stain usually used for?

Answer 11

• used to detect Mycobacterium species (aka acid fast bacilli) => it causes TB
• Mycolic acid in its cell wall => acid-fastness (resistance to decolourisation by acids => retains stain)
• doesn’t let any gram stain in
• reddish on histo slide

Question 12

Steps to using Ziehl Neelson Stain:

Answer 12

1. Bacteria stained w hot carbol fuchsin
2. Decolourisation using acid-alcohol + wash
   - most bacteria lose stain BUT cell walls of Mycobacteria retain stain (APPEARS PINKISH/REDDISH)
3. Counterstain (to see other bacteria)

ZN stain used to identify acid-fast bacilli (AFB)

Question 13

IgG and IgM in primary and secondary infection.

Answer 13

IgM: prominent in primary infection, less common in secondary infection

IgG: takes times to appear in primary infection, much more prominent in secondary infection

Question 14

Serodiagnosis is used:

Answer 14

• for a minority of bacterial infections when organism is difficult or impossible to culture

Serodiagnosis: a diagnosis involving tests on blood serum or other serous fluid of the body. (E.g. identifying IgG/IgM levels, antibody levels, antigen levels, immunofluorescence)

Question 15

Pros and cons of PCR:

Answer 15

Pros: sensitive & specific
Cons: prone to contamination by DNA, limited to targeted pathogen, test inhibited by substances in specimen

Question 16

Steps for Gram Staining

Answer 16

1. Bacteria are fixed to glass slide
2. Apply crystal violet + wash
3. Apply Lugol’s Iodine + wash
4. Decolourisation w ethanol (gram -ve cells lose the gram complex but gram +ve cells retain it)
   - gram +ve appear purple
5. Apply Saffranin counterstain + wash + dry
   - gram -ve coloured by counterstain => appear red
6. Examine under oil immersion (w x100 lens)

Question 17

Which bacteria does Gram stain not work on:

Answer 17

1. Too thin: treponema pallidum
2. Dont retain stains: mycoplasma spp
3. Dont let any stains in: mycobacterium spp

Question 18

What does MALDI-TOF detect?

Answer 18

Protein & peptides

Question 19

MacConkey agar is used for:

Answer 19

Test gram -ve bacteria

• cuz it inhibits growth of gram +ve bacteria
• lactose fermenters turn agar pink (due pH change)
  e.g. E.coli

Question 20

Gram stain: what step differentiates bacteria w a thick and thin peptidoglycan cell wall?

Answer 20

Decolourisation using acetone and alcohol