Lecture 3: Microbial Diagnostic Flashcards
What are the three steps in determining the organism that caused disease?
- find the culprit (swabs, blood, urine)
- grow the culprit (agar plates, liquid medium)
- identify the culprit (diagnostic tools)
What is key in diagnostics when obtaining a bacterial sample
important to get single colonies as multiple bacteria/colonies will warp test results, want a pure sample - derives from one single cell
What are the 5 diagnostic methods
- 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)
How can agar plates aid in diagnostics
colonies often have characteristics such as
1. shape
2. appearance
3. color
4. odor
that can give clues but arent sufiecient for conclusive identification
Explain the process of gram staining and the difference between gram postive and gram negative bacteria
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)
Explain what yeast budding is
“mother cell” grows protrustions i.e buds that grow larger and eventualy separate from the mother cell
What is the difference between hypae and pseudohyphae and state one fungi that exhibits pseudohyphae
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)
Explain what Ziel-Neelsen (acid fast) staining is used for and why it is necessary
- 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
Explain the process of a Ziel-Neelsen (acid fast) stain
- stain using carbolfuchsin and heat
- destain using ethanol, hydrochloric acid
- counter stain using methylene blue
mycobacteria will show up red under the microscope as they retain the orange dye (rest takes up the blue)
What is Lactophenol Cotton Blue Stain used for
visualisazion of moulds
Explain how Lactophenol Cotton Blue Stain works
- lactophenol serves as mounting fluid
- phenol kills the mould and prevents cell lysis
- cotton blue stains the chtin in the cell wall
- visual hypha, fruiting bodies and spores
Explain the difference between hypha, fruiting bodies and spores
- hypha - branching filament that make up mycelium of fungus
- fruiting bodies - contain spores
- spores- fruiting body produces spores
What is the role of fruiting bodies and spores?
to reproduce and help spread spore so they can colonise elsewhere
What is an assay test for?
find and measure the amount of a specific substance
What are the three assay tests based on specific enzymes?
- catalase test
- coagulase test
- oxidase test
What is a catalse test used for?
used to differentiate between staphylococci and streptococci
Explain how a catalase test works
staph - contains enzyme that will break down peroxide into water and oxygen producing bubbles
strep - no bubbles
bactera + H2O2 –> (catalase) H2O + O2 (bubbles)
What is a coagulase test used for?
- used to indetify Staphylococcus aureus specifically (from other staph strains)
- cell surface-bound protein that mediates fibirn polymerisation
Explain how a coagulase test works
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
What is an oxidase test used for?
determine whether bacteria is aerobic or anaerobic
Explain how an oxidase works
- 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)
what assay is used that is based on metabolic reactions, explain how it works
- 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
Describe the difference between selective agar and differential agar
selective: contains inhibitors to prevent growth of certain organisms
differential: contains indicators to differentiate organisms
What is Sabouraud agar
- selective for fungi, non differential
- low ph supresses growth of most bacteria
What is Eosin-methylen blue agar (EMB)
- selective for Gram-negative bacteria
- aniline dyes are toxic for gram-positive bacteria
- differentiates lactose fermenters: pink - weak/modertate, gree - strong/rapid
What is MacConkey agar
- selective for intestinal pathogens
- bile salts inhibit non-enteric bacteria
- differentiates lactose fermentation (pink)
What is blood agar
- 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
What is Mannitol Salt agar?
- 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
selective for staph genus, differental for staph species
What is Bile-Esculine agar
- selective for enteric bacteria
- oxgall inhibits non-enteric bacteria
- esculine hydrolysis gives a dark brown color
differential for esculine hydrolysis
Explain how an antibiotic susceptibilty test on an agar plate would be interpreted
- 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
List the methods that are. based on antibody-antigen interaction
- cell agglutination
- latex bead agglutination
- western blot
- ELISA
- Immunofluorescence microscopy
Explain how Cell agglutination works
- AB recognises component on cell surface
- serum can be used to distinguish between serotypes based on specific antibodes to a variable molecule
Explain the term agglutination
the action of an antibody when it cross-links multiple antigens producing clumps of antigens
Explain how latex bead agglutination works
- similar to cell agglutination except latex beads used instead of cell
- the bead allows to visualise the AB-antigen interaction
Explain how a western blot works
- 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
Explain how HIV was detected with a western blot
aim: Have a protein, want to see if antibody is in serum
- Separate proteins of virus (HIV) blot on western blot
- Add patient serum
- 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
Protein find antibody or antibody find protein works both ways
Explain how ELISA works
- similar to western blot but reaction is in solution
- easier to quantify amount of protein or antibody by intensity of color change
What is the main difference between a western blot and ELISA
western blot is qualitative
ELISA is quantative
Explain how Imminofluorescence miscroscopy works
- 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)
List the three DNA based assays
- Restriction fragment length polymerisation (RFLP)
- PCR
- FISH
Explain how RFLP (DNA fingerprinting) works
- 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.
Explain how PCR works
- 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
