Lecture 3: Microbial Diagnostic

Question 1

What are the three steps in determining the organism that caused disease?

Answer 1

1. find the culprit (swabs, blood, urine)
2. grow the culprit (agar plates, liquid medium)
3. identify the culprit (diagnostic tools)

Question 2

What is key in diagnostics when obtaining a bacterial sample

Answer 2

important to get single colonies as multiple bacteria/colonies will warp test results, want a pure sample - derives from one single cell

Question 3

What are the 5 diagnostic methods

Answer 3

• based on structural features (colony, morphology, staining)
• based on biochemical properties (O2 dependence, chemical resistance, antiobiotic resistance)
• based on antibody binding (serological tests, immunological assays ELISA)
• based on infecion by phage (phage typing)
• based on DNA/RNA (DNa hybridisation, PCR)

Question 4

How can agar plates aid in diagnostics

Answer 4

colonies often have characteristics such as
1. shape
2. appearance
3. color
4. odor
that can give clues but arent sufiecient for conclusive identification

Question 5

Explain the process of gram staining and the difference between gram postive and gram negative bacteria

Answer 5

Crystal violet – penetrate both cell types

Iodine – precipitate crystal violet, crystals – get trapped in thick peptidoglycan layer (+ve)

**Decolorizer **– decolorize everything in -ve, because crystals not trapped, outer membrane damaged too, +ve will retain crystal violet
(Can see +ve in microscope at this stage but not -ve)

• This is why you need to do a counterstain

Safranin – orange dye visible = -ve (E.coli should have been orange)

Question 6

Explain what yeast budding is

Answer 6

“mother cell” grows protrustions i.e buds that grow larger and eventualy separate from the mother cell

Question 7

What is the difference between hypae and pseudohyphae and state one fungi that exhibits pseudohyphae

Answer 7

The main difference between hyphae and pseudohyphae is that the hyphae are the elongated, thread-like filaments whereas the pseudohyphae are the newly-divided cells through budding

candida albicans when cultured in serum (yeast)

Question 8

Explain what Ziel-Neelsen (acid fast) staining is used for and why it is necessary

Answer 8

• used to inderify Mycobacterium tuberculosis
• mycobacteril have a way-like coat made up of mycolic acid - gram is water based, so cant use gram stain

Question 9

Explain the process of a Ziel-Neelsen (acid fast) stain

Answer 9

1. stain using carbolfuchsin and heat
2. destain using ethanol, hydrochloric acid
3. counter stain using methylene blue

mycobacteria will show up red under the microscope as they retain the orange dye (rest takes up the blue)

Question 10

What is Lactophenol Cotton Blue Stain used for

Answer 10

visualisazion of moulds

Question 11

Explain how Lactophenol Cotton Blue Stain works

Answer 11

1. lactophenol serves as mounting fluid
2. phenol kills the mould and prevents cell lysis
3. cotton blue stains the chtin in the cell wall
4. visual hypha, fruiting bodies and spores

Question 12

Explain the difference between hypha, fruiting bodies and spores

Answer 12

1. hypha - branching filament that make up mycelium of fungus
2. fruiting bodies - contain spores
3. spores- fruiting body produces spores

Question 13

What is the role of fruiting bodies and spores?

Answer 13

to reproduce and help spread spore so they can colonise elsewhere

Question 14

What is an assay test for?

Answer 14

find and measure the amount of a specific substance

Question 15

What are the three assay tests based on specific enzymes?

Answer 15

1. catalase test
2. coagulase test
3. oxidase test

Question 16

What is a catalse test used for?

Answer 16

used to differentiate between staphylococci and streptococci

Question 17

Explain how a catalase test works

Answer 17

staph - contains enzyme that will break down peroxide into water and oxygen producing bubbles
strep - no bubbles

bactera + H2O2 –> (catalase) H2O + O2 (bubbles)

Question 18

What is a coagulase test used for?

Answer 18

• used to indetify Staphylococcus aureus specifically (from other staph strains)
• cell surface-bound protein that mediates fibirn polymerisation

Question 19

Explain how a coagulase test works

Answer 19

For staph aureus, have specific enzyme

• Tube with serum and bacteria, clot fomration after 1-4h (fibrin polymerisation)
• plasma has fibrinogen (causes blood clots)
• see clotting = staph aureus (
• no clotting = other staph strain

Question 20

What is an oxidase test used for?

Answer 20

determine whether bacteria is aerobic or anaerobic

Question 21

Explain how an oxidase works

Answer 21

• esentially testing if the bacteria has an electron transport chain for respiration
• Use reagent that reacts with cytochrome c oxidase – final enzyme in chain
• Aerobic bacteria turns substrate blue (oxidase positive)
• Anaerobic bacteria doesn’t turn it blue (oxidase negative eg. E. coli)

Question 22

what assay is used that is based on metabolic reactions, explain how it works

Answer 22

• microbact strips
• Different enzymatic reactions in one strip, each hole has different enzyme/substrate and is an independent test
  example:
• PH indicator for glucose fermentation (acidic)
• Bacteria can ferment glucose –ph drops, bromothymol blue changes to yellow
• Not able to ferment glucose, stays blue

Question 23

Describe the difference between selective agar and differential agar

Answer 23

selective: contains inhibitors to prevent growth of certain organisms
differential: contains indicators to differentiate organisms

Question 24

What is Sabouraud agar

Answer 24

• selective for fungi, non differential
• low ph supresses growth of most bacteria

Question 25

What is Eosin-methylen blue agar (EMB)

Answer 25

* selective for Gram-negative bacteria * aniline dyes are toxic for gram-positive bacteria * differentiates lactose fermenters: pink - weak/modertate, gree - strong/rapid

Question 26

What is MacConkey agar

Answer 26

* selective for intestinal pathogens * bile salts inhibit non-enteric bacteria * differentiates lactose fermentation (pink)

Question 27

What is blood agar

Answer 27

* growth of many fastidious bacteri * differentiates between hemolytic reactions * Alpha – can partially break down RBC but not completely * Beta – complete hemolysis, all RBC gone, white area all RBC gone * Gamma – not hemolytic, bacteria grow but not hemolytic

Question 28

What is Mannitol Salt agar?

Answer 28

* selective for haloduric bacteria (Staphylococci) * differentiates mannitol fermenters (yellow) * S. aureus is the only staph that ferments mannitol | used to identify staph aureus, srep cant grow in high salt conditions ## Footnote selective for staph genus, differental for staph species

Question 29

What is Bile-Esculine agar

Answer 29

* selective for enteric bacteria * oxgall inhibits non-enteric bacteria * esculine hydrolysis gives a dark brown color | differential for esculine hydrolysis

Question 30

Explain how an antibiotic susceptibilty test on an agar plate would be interpreted

Answer 30

* create bacterial lawn * Filter soaked with AB, onto agar plate and then incubate * Bacteria will grow as lawn, may not grow around AB disk * Bigger halo diameter more susceptible bacteria are to AB * Grow all the way to disk, resistant to AB | used to differentiate between species

Question 31

List the methods that are. based on antibody-antigen interaction

Answer 31

1. cell agglutination 2. latex bead agglutination 3. western blot 4. ELISA 5. Immunofluorescence microscopy

Question 32

Explain how Cell agglutination works

Answer 32

* AB recognises component on cell surface * serum can be used to distinguish between serotypes based on specific antibodes to a variable molecule

Question 33

Explain the term agglutination

Answer 33

the action of an antibody when it cross-links multiple antigens producing clumps of antigens

Question 34

Explain how latex bead agglutination works

Answer 34

* similar to cell agglutination except latex beads used instead of cell * the bead allows to visualise the AB-antigen interaction

Question 35

Explain how a western blot works

Answer 35

* identification of a protein with a specific antibody * or identification of antibody in serum using specifc proteins * separattion of pfotein based on size, gel, blot on membrane, immobalise membrane * wash with primary antibody * wash with secondary antibody - conjugated to enzyme, add substrate (2 antibody binds to first antibody) – all present –get a color, protein is recognized by first antibody you used

Question 36

Explain how HIV was detected with a western blot

Answer 36

aim: Have a protein, want to see if antibody is in serum 1. Separate proteins of virus (HIV) blot on western blot 2. Add patient serum 3. Antibody present that recongises protein - get colour reaction HIV patinet - day 3 no reaction - day 7 strong reaction to protein p24 - 30 days, clear streak at p24 = HIV positive ## Footnote Protein find antibody or antibody find protein works both ways

Question 37

Explain how ELISA works

Answer 37

* similar to western blot but reaction is in solution * easier to quantify amount of protein or antibody by intensity of color change

Question 38

What is the main difference between a western blot and ELISA

Answer 38

western blot is qualitative ELISA is quantative

Question 39

Explain how Imminofluorescence miscroscopy works

Answer 39

* primary antibody conjugated to fluorophpre * Fluorophore instead of enzyme, add to antibody, emits ligth in certain color range * can mix different fluorphores for different character recognition (eg. carbs, protein, depends on what antibody binds to)

Question 40

List the three DNA based assays

Answer 40

1. Restriction fragment length polymerisation (RFLP) 2. PCR 3. FISH

Question 41

Explain how RFLP (DNA fingerprinting) works

Answer 41

* any genomic DNA can be differentiated according to the presence or absence of restriction enzyme sites. * used mainly for epidemiological studies * DNA sample is digested into fragments by one or more restriction enzymes, and the resulting restriction fragments are then separated by gel electrophoresis according to their size.

Question 42

Explain how PCR works

Answer 42

* uses specific oligo DNA primers and a heat-stavle DNA polymerase (usually Taq) * important to identify organisms that cannot easily be grown in culture -> amplifiation 1. denaure 2. annealing 3. elongation - run on agarose gel for fragment sizing