Bacteriology Final Lab Exam Flashcards
What are the features of Microsporum spp.?
Macroconidia: Spindle shaped
Microconidia: Tear shaped & few or absent
What are the features of Trichophyton spp.?
Macroconidia: Cigar shaped & few or absent.
Microconidia: Grape like clusters & Numerous
What are the features of Microsporum canis?
Infects dogs, cats, humans.
6-15 cells within MACROconidia.
Spindle shaped.
What is this?
Microsporum canis
~remember spindle shaped & 6-15 cells for MACROconidia.
What are the features of Microsporum gypseum?
Infects: rodents, horses and dogs.
4-6 cells within MACROconidia.
Boat shaped.
What is this?
Microsporum gypseum
~remember 4-6 cells within MACROconidia, this is fewer than M. canis.
~Gypseum=gypsy, frizzy gypsy hair fringed around edges.
What are the features of Microsporum audouinii?
Most plates are sterile.
Think houdini=audouinii, they are never around.
Macro- and Microconidia are rare.
Most closely resembles M. canis.
What are the features of Trichophyton mentagrophytes?
Infects: rodents, dogs and horses.
3-7 celled MACROconidia.
Cigar shaped.
2 colony types: granular (animal pathogen) and downy.
What is this?
Trichophyton mentagrophytes
What are the features of Trichophyton equinum?
Infects: horses.
3-5 celled MACROconidia.
MACROs are rare.
Can see chlamydospores with old cultures.
What is this?
Trichophyton equinum
~since MACRO’s are rare should be looking more at the hyphae.
What are the features of Trichophyton rubrum?
- **Abundant **clavate to pyriform MICROconidia.
- Moderate to abundant cigar-shaped MACROconidia.
What is this?
Trichophyton rubrum
~resembles a very skinny cigar
What are the features of Trichophyton tonsurans?
- Broad hyphae (think tonsurans toned or big/broad)
- **Abundant ** MICROconidia forming right angles to hyphae. Think about having to do bicep curls, you are forming right angles with your arm and since it is so toned…that’s why it has the right angles.
- Occasional MACROconidia.
What is this?
Trichophyton tonsurans
~remember broad hyphae and cigar shaped MACROcondidia
~ MICROconidia forming right angles.
What are the features of Epidermophyton floccosum?
- On Sab’s agar is slow growing.
- Smooth thin walled MACROconidia
- Clusters of MACRO’s growing directly from the hyphae.
What is this?
Epidermophyton floccosum
What are the features of Geotrichum candidum?
-Fragmented hyphae, **rectangular one-celled arthrospores **in chains
What is this?
Geotrichum candidum
~Geo: think geometric shapes as in rectangular shaped arthrospores.
What are the defining features of Sprothrix schenckii?
-Flowerettes of conidia
What is this?
Sporothrix schenckii
~shank you very much for the “flowers”>>I know, corny as hell.
What is the fungus on the left?
What is the fungus on the right?
Left: Penicillium ~ Paint brush-illium
Right: Aspergillus~ Afro-gillus
What are the features of Aspergillus fumigatus?
- Afro shaped
- Chains of bluish green conidia
- Stem= conidiophore
- Have FOOT at base.
What is this?
Aspergillus fumigatus
~remember: Afrogillus fumigatus
What are the features of Aspergillus flavus?
- Dandelion shaped
- Round vesicles
- Round conidia
What is this?
Aspergillus flavus
~Flava flava dandy dandelion shaped
What are the features of Aspergillus niger?
-Small black balls that are rough
What is this?
Aspergillus niger
~the word niger in Latin means black, these are your little black balls.
What are the features of Penicillium spp.?
- Paint brushed, Paint-brushillium
- Blue green
What is this?
Penicllium spp.
~remember: Paint-brushillium
What are the features of Bipolaris spp.?
- Pale brown
- Condia rounded at ends
- Acorn shaped
What is this?
Bipolaris spp.
~The bipolar squirrels eat their acorns, I get it…I’m crazy.
What are the features of Curvularis?
- Brown
- 3 or more transverse septa
- Central ball is larger
- Curvy
What is this?
Curvularis
-The curvier acorn, hence it has 3 or more transverse septa.
What are the features of Cryptococcus neoformans?
- Found in pigeon droppings due to high contrast of creatinine.
- Creatinine will inhibit other organisms
- Can survive a year in pigeon droppings
- Budding yeast with a capsule.
What is this?
Cryptococcus neoformans
~Remember: Crypto have to stay in their “crypts” aka the capsule. They also take a long time to die, like a crypt keeper, survive 1 year in feces.
What are the features of Candida albicans?
-Budding yeast cells in Sab’s or blood agar
What is this?
Candida albicans
~Remember:My old “buds” from “Canada”, yeast buds are Candida.
What are the features of Fusarium?
-Banana shaped MACROconidia
What is this?
Fusarium spp.
~Remember: I’m going to blow a “fuse” if I can’t eat my banana for a snack. Yeah I get it…that one is a stretch haha.
What are the steps in performing a Gram Stain?
- With plastic loop, add a small amount of bacteria to slide mixed with water.
- Heat and dry slide
- Primary stain: with Crystal violet~ 1 min
- Mordant: Iodine~ 2 min
- Decolorizer: Alcohol or Acetone~ seconds
- Counter stain: Safranin~ 1 min
(Make sure to rinse in between each step)
What is TSA?
Tryptic soy agar
- General purpose medium
- Useful for : culture storage, enumeration, isolation of pure culture and general cultures.
What are the components of Tryptic Soy Agar?
- 15 g Tryptone
- 5 g Soytone
- 5 g NaCl
- 15 g agar
What is blood agar?
- General purpose enriched medium
- Grows fastidious organisms
- Differentiates bacteria based on their hemolytic properties.
-
What type of hemolysis is this?
What type of toxin produces this?
Beta hemolysis or (complete hemolysis)
Alpha toxin produces this.
What type of hemolysis is this?
What type of toxin produces this?
Alpha hemolysis or (incomplete hemolysis)
Beta toxin produces this.
Remember: don’t be fooled by “colors”, it is whether or not you can see all the way through, because although this is “yellow” which is similar to beta hemolysis you will notice you cannot see all the way through it. There may be hints of green if you want to go by color.
What type of hemolysis is this?
Gamma hemolysis or (no lysis)
What is an example of the type of bacteria that would produce this type of hemolysis?
What type of hemolysis is this?
Staphylococcus aureus
Beta-hemolysis
What is PEA?
Phenylethyl Alchohol Agar with 5% sheep’s blood
-Selective medium used to isolate most Gram (+) bacteria
How does PEA inhibit growth of most Gram (-) bacteria?
It disrupts the lipid structure of Gram (-) resulting in stunted or no growth.
What is MacConkey Agar?
- Agar designed to grow Gram (-) bacteria.
- Used to: differentiate lactose fermenters from non-fermenters.
Remember: Mac sounds like Lac, MacConkey used for Lactose differentiation.
What is in MacConkey Agar that prevents Gram (+) bacteria from growing?
Bile salts (most Gram +)
Crystal Violet dye (some Gram +)
What is present in MacConkey Agar that stains microbes that ferment lactose?
Neutral red dye
What other 2 components are included in MacConkey Agar?
Lactose and Peptone
What are the features of a lactose fermenter?
Produce red/pink colored colonies
Produce acid which lowers the pH of the medium.
E. coli, Enterobacter and Klebsiella: all are Gram (-)
Remember: since they produce red colonies and red is the color of blood think “eek blood!” E. coli, Enterobacter and Klebsiella.
What are the features of a non-lactose fermenter?
Produce white/or colorless colonies.
Utilize peptones instead of lactose and this raises the pH.
Proteus spp., Pseudomonas aeruginosa, Salmonella, Shigella
Remember: they are NON-lactose fermenters so “ppss I hate milk” Proteus, Pseudomonas, Salmonella, Shigella, white colonies like milk.
The plate on the left displays what type of lactose fermentation? Examples of bacteria that would do this?
The plate on the right displays what type of lactose fermentation? Examples of bacteria that would do this?
Left: Non-Lactose fermenters (white colonies)
ex: “ppss I hate milk” Proteus, Pseudomonas, Salmonella, Shigella
Right: Lactose fermenters (red colonies)
ex: “eek blood” E. coli, Enterobacter, Klebsiella
What is Hektoen enteric agar?
A differential selective medium for isolation of Salmonella and Shigella.
On Hektoen Enteric Agar, Shigella will give what type of apperance?
Green colonies
On Hektoen Enteric Agar, Salmonella will give what type of apperance?
Blue/green colonies, with or without black centers.
Hektoen Enteric Agar includes indicators for what?
Lactose fermentation and H2S production
What are the 2 things used to indicate H2S production on Hektoen Enteric Agar?
Thiosulfate and ferric ammonium citrate
Hektoen Enteric Agar contains inhibitors for what?
Gram (+) bacteria
What are the 2 things used to prevent growth of Gram (+) bacteria on Hektoen Enteric Agar?
Bile salts and Bromothymol Blue
What other things does Hektoen Enteric Agar contain?
Lactose, Sucrose and Salicin
Since Hektoen Enteric Agar contains indicators for lactose fermentation, how would something that does ferment lactose appear on HEA?
Orange/Red
ex: E. coli
Remember: same color as with MacConkey’s. If you ferment lactose you will be red/pink/orange in color. Those that do you’ll think “eek blood” E. coli, Enterobacter, and Klebsiella.
What does Brilliant Green (BG) Agar look for?
If certain bacteria are able to ferment sugars: lactose and sucrose.
On Brilliant Green Agar how would non lactose/sucrose fermenting organisms appear?
What are some examples of bacteria that do this?
Red/pink/white colonies
ex: Salmonella
Remember: Brilliant Green Agar is brilliant, it’s different from MacConkey’s and Hektoen in that the non-lactose/sucrose fermenters appear pink _this is opposite to the others._ It’s so brilliant though it’s able to do this.
On Brilliant Green how would lactose/sucrose fermenters appear?
What are examples of bacteria that do this?
Yellow to greenish color
ex: E. coli, Enterobacter, Klebsiella
Remember: our “eek blood” doesn’t hold true here, although these bacteria STILL ferment these sugars they are not red, they are yellow-green.
What is the pH indicator for Brilliant Green Agar?
Phenol Red
~**Red **at pH of 8.2 (alkaline)
~Yellow at pH of 6.4 (acidic)
What is the inhibitor in Brilliant Green (BG) Agar?
What is it inhibiting?
The inhibitor is brilliant green dye.
Inhibits the growth of most enterobacteria (all Gram (-) organisms) **except: Salmonella.
What is XLD agar?
Stands for: Xylose Lysine Deoxycholate agar.
It is used to see if certain bacteria can ferment these different sugars: lactose, sucrose, and xylose.
What is the pH indicator used for XLD agar?
Phenol red
~**Red **at pH 8.2 (alkaline)
~Yellow at pH 6.4 (acidic)
What are the inhibitors present in XLD agar?
What are they inhibiting?
Bile salts
Inhibiting Gram (+) bacteria
What substrates are used to detect H2S with XLD agar?
Lysine
Salmonella will appear to have what color colonies on XLD agar?
Red with a black center (checkers)
Why do Salmonella have this appearance (red with black center) on XLD agar?
They will first ferment the xylose.
This creates a temporary acid reaction.
Reversed by subsequent decarboxylation of lysine.
Alkaline metabolic products.
Superimposed on the red (alkalkine) colonies is the production of H2S.
The alternative to Salmonella having (red with black centered) colonies on XLD is what?
Yellow colonies
Ex: E. coli, Enterobacter, and Klebsiella
Remember: “eek blood” doesn’t hold true here, that only held true for MacConkey’s and Hektoen Enteric Agar. However even though they don’t appear red they still ferment lactose.
What is Eosin Methylene Blue Agar (EMBA)?
A selective stain for Gram (-) organisms.
What is both the pH indicator and selective agents of Eosin Methylene Blue Agar?
Eosin Y and Methylene Blue
What is the amino acid contained within Eosin Methylene Blue Agar?
What is the purpose of this amino acid?
Peptone
It may be deaminated (forming an alkaline reaction.)
What is the fermentable sugar in Eosin Methylene Blue Agar?
Lactose 1%
On Eosin Methylene Blue Agar, something that ferments lactose will have what type of colony apperance?
What is an example of bacteria that do this?
Metallic green/with dark centers
Net producers of acid.
ex: E. coli
On Eosin Methylene Blue Agar, what would something look like that does not ferment lactose?
What is an example of bacteria that display this?
Lightly colored
Net alkaline
ex: Salmonella and Shigella
T/F: With the Gram Stain, Crystal violet only penetrates Gram (+) cell walls.
**False: **Crystal violet dissociates in aqueous solution to CV+ and Cl- (chloride ions), these ions penetrate the cell wall and membranes of both Gram (+) and Gram (-) bacteria.
T/F: CV+ ions interact with positively charged components of bacterial cells.
**False: **CV+ ions interact with negatively charged components of bacterial cells and stain them purple.
T/F: Iodine (I- or I3-) interacts with CV+ and forms large complexes of crystal violet and iodine (CV-1) within the inner and outer layers of the cell.
True!!!
T/F: When a decolorizer such as alcohol or acetone is added, it interacts with the protein of the cell membrane.
**False: **It interacts with the lipid of the cell membrane.
T/F: A Gram (-) cell will lose it’s outer membrane and the peptidoglycan layer is left exposed
True!!!
T/F: The CV-1 complexes stay intact with the Gram (-) cell wall while the rest is washed away.
**False: **The CV-1 complexes are washed away as well.
T/F: In contrast, a Gram (+) cell wall becomes dehydrated from decolorizing treatment and the large CV-1 complexes become trapped within the Gram (+) cell due to the multi-layered nature of the peptidoglycan.
True!!!
T/F: The counterstaining is the critical step of the Gram Stain process.
**False: **the decolorizing step is the critical step and must be timed correctly!!!
Why is it important that the decolorization be timed precisely?
Because, if you leave the decolorizer on for too long, the Crystal Violet will eventually be washed away from Gram (+) in addition to Gram (-) bacteria.
T/F: At the end of the process, Gram (+) cells appear pink.
False: Gram (+) cells will be purple!!
T/F: At the end of the Gram Stain process, Gram (-) bacteria will appear pink.
True!!!
T/F: Counterstain, which is usually positively charged safranin, or basic fuschin, is applied last to to give decolorized Gram (-) bacteria a pink or red color.
True!!!
What is this?
Gram (+) bacteria: Streptococcus
~Gram (+) we know because it’s purple
~Strep=chains
~Coccus/cocci= circles
What is this?
Gram (-) bacteria: Staphylobacilli
~Gram (-) because of the pink color.
~Staph=groups/clusters
~Bacillus/bacilli=rods
How does the catalase test work?
Detects the enzyme catalase which converts H2O2 (hydrogen peroxide) into H2O and O2.
What type of catalase result is this?
What type of bacteria produce this type of result?
Positive result
Most aerobic bacteria
“Staph species” like Staphylococcus aureus
What type of catalase test result is this?
What type of bacteria would produce this result?
Negative result
Obligate anaerobes, mainly produce this
“Strep species” like Streptococcus equi
The Oxidase Test is detecting the presence of what?
**Cytochrome C Oxidase **in the bacterial cell wall.
What type of Oxidase test result is this?
What type of bacteria would produce this result?
Oxidase negative
Anaerobic bacteria
Staphylococcus aureus
What type of Oxidase test result is this?
What type of bacteria would produce this result?
Oxidase positive
Aerobic bacteria
Pseudomonas aeruginosa
What is added to the medium of motility test to detect the presence of motility in a bacteria?
Tetrazolium salts
Tetrazolium salts are colored/colorless?
Colorless
As the bacterium grows the dye is incorporated into the bacterial _____ ______where it is reduced to an insoluble red pigment.
Cell wall
What is the name of the insoluble red pigment in the motility test?
Formazan
What type of motility test result is this?
What type of bacteria produce this result?
Positive motility test
Proteus mirabilis
What type of motility test result is this?
What type of bacteria would give this result?
Negative motility test
Klebsiella pneumoniae
What is the Urease Test looking for?
If the bacteria contains urease and is able to split urea with water to form ammonia and CO2.
What is the medium present with the Urease Test?
Urea broth (contains ~2% urea)
What is the indicator in the Urease Test?
Phenol red
What type of Urease Test result is this?
What type of bacteria would produce this result?
Negative result
Produces a yellow-orange color
Salmonella spp.
What type of Urease Test result is this?
What type of bacteria would result in this?
Positive test result
Hot pink color
Proteus spp.
Alkaline
What does IMViC stand for?
I-Indole
M-Methyl
V- Voges Proskauer
C- Citrate
What is the IMViC test, testing for?
It is a group of individual tests use to differentiate the Family Enterobacteriaceae.
What is the Indole Test, testing for?
To see if the bacteria can convert **Tryptophan to indole. **
What is the indicator used for the Indole Test?
Kovac’s Reagant
What type of Indole Test result is this?
What type of bacteria would produce this type of result?
Negative indole test
Enterobacter spp.
Yellow color
What type of Indole test result is this?
What type of bacteria would produce this type of result?
Positive indole test
E. coli
Red color
What is the Methyl Red (MR) test used for?
Testing for low molecular weight acids: such as* lactic, formic and acetic acid.*
What is the medium used with Methyl Red (MR) test?
Peptone-glucose broth
What is the indicator used for the Methyl Red (MR) test?
Methyl Red….duhhhh……
What type of Methyl Red test result is this?
What type of bacteria would produce this type of result?
Positive methyl red test
E. coli
Red color (the test is the Methyl Red test~ of course a postiive will be considered Red)
Red, red, red, red, red, red, ++++++++
What type of test result is this for the Methyl Red test?
What type of bacteria would produce this result?
Negative methyl red test
Enterobacter spp.
Yellow color
What is the Voges-Proskauer Test, testing for?
To see if bacteria can convert glucose to acetoin.
What is the medium used for the Voges-Proskauer test?
Peptone-Glucose broth
What is the indicator used for the Voges-Proskauer test?
40% KOH (Potassium hydroxide) and α-naphthol
What type of Voges-Proskauer test result is this?
What type of bacteria would produce this result?
Negative test result
E. coli
Reagant layer yellow
What type of Voges-Proskauer test result is this?
What type of bacteria would produce this type of result?
Positive test result
Enterobacter spp.
Reagant layer red
What is the Citrate Test, testing for?
The ability of the bacteria to use citrate as it’s sole carbon source.
What is the medium present in the Citrate test?
Simmons citrate agar
What is the indicator present with the Citrate Test?
Bromothymol Blue
What type of Citrate test result is this?
What type of bacteria would produce this result?
Negative test result
Green color
E. coli
What type of Citrate test result is this?
What type of bacteria would produce this type of result?
Positive test result
Blue color
Salmonella spp.
What are the 3 methods of antimicrobial susceptibility in the lab?
- Kirby Bauer (disc diffusion)
- E-test (MIC)
- Sensitire broth microdiliution (MIC)
When you test for susceptibility in the lab, how many ways possible are there and what are they called?
This is debatable: he has told classes 2 different things. It seems like the general consensus is that there are 2 ways to test for susceptibility:
1. Diffusion methods
2. Dilution methods
~Also you have a **combo **of both but this is not counted as a 3rd.
The Kirby Bauer Disc Diffusion method is a qualitative or quantitative interpretation?
According to a student who emailed him: it is** qualitative **(his notes which say quantitative are apparenlty wrong; however in my book it would be quantitative due to the fact you have to measure the zones of inhibiton.) It is still quite unclear but I’d go with _qualitative_ at this point.
For the Kirby Bauer Disc Diffusion what are the 3 possible results you can get?
~Susceptible
~Intermediate
~Resistant
What are the discs that are used in the Kirby Bauer test?
Can be: **Antibiotic impregnated paper discs (several suppliers) **or tablets (Rosco)~ neosensitabs
What are the steps you’d use in performing the Kirby Bauer Disc Diffusion test?
Prepare a pure culture (18-24 hrs) of the sample on a non-selective medium.
Adjust turbidity until it is equivalent to 0.5 McFarland Standard.
Dip sterile cotton tipped applicator into the sample.
Streak a lawn on a dry Mueller-Hinton agar plate.
Apply impregnated discs to lawn.
Do not attempt to pick up disc and move it once you drop it.
Incubate at 18-24 hrs.
What is the zone of inhibition?
The area around an antibiotic disc that contains no bacterial growth.
Measured in mm’s
For bacteria that have a fuzzy edge instead of a sharp border around the zone of inhibition and you are already at the widest point where you are deciding on whether to call something susceptible or resistant, what would you do?
If it has a fuzzy edge: Report as susceptible.
If it has a sharp edge: Report as resistant.
What is an example of a bacteria and an antibiotic where you may need to decipher fuzzy from sharp zone of inhibition borders?
*Staphylococcus aureaus *with Benzylpenicillin
What are factors that influence the zone of inhibition?
- Susceptibility of the bacterium
- Antibiotic disc (whether it’s paper or tablets)
- Diffusion speed of antibiotic
- Medium
- Incubation circumstances (ex: CO2 or temperature)
- Growth rate of the bacterium
- Inoculum density (BSAC: semi-confluent vs CLSI/Eucast:confluent)
What is the breakpoint?
A level of minimum inhibitory concentration (MIC) at which a bacterium is deemed either susceptible or resistant to an antibiotic being used.
What is the MIC?
Minimum inhibitory concentration
Lowest concentration of an antibiotic by which there is no visible growth anymore in vitro.
mg/L or μg/mL
The point where bacteria stop growing.
What is the MBC?
Minimum bactericidal concentration
The concentration of the antibiotic by which the strain does not grown anymore after subculture.
The E-test is a combination of _______ and _______.
Dilution and Diffusion
When reading an E-test what does <1 correspond with?
Susceptible
When reading an E-test what does 1-4 correspond with?
Intermediate
When reading an E-test what does >4 correspond with?
Resistant
What is the upside to using an E-test?
It’s a good and fairly accurate test.
Few methodological problems.
What does the E in E-test stand for?
Ellipsoid
What does the E-test give you?
Your MIC which is quantitative data.
Can also be used in a specific research setting when a single antibiotic is involved.
What are 2 dilution tests used?
Agar dilution
Broth dilution (Macro-broth dilution **and **Micro-broth dilution.)
Between the 2 dilution tests which one is the best one?
Agar dilution
How is the agar dilution test performed?
Two-fold dilutions of the antibiotic in agar medium.
Dilutions should be made according to a specific protocol.
Care: solubility of the antibiotic.
Deposit of a well defined quantity of bacteria on the agar plates.
If you’re doing a broth macro-dilution which of these tubes would be considered your MIC?
It would be the next most concentrated tube but with no growth.
Which of these tubes would be your MIC?
How is micro broth dilution performed?
In small tubes or in 96 well plates with lyophilized antibiotics.
Prepare 0.5 McFarland suspension of bacterium.
Transfer 6 μL of 0.5 McFarland standard in 6 mL Mueller Hinton broth.
Pour in pipette basin.
Using a multi-channel pipette dispense 50 μL into each well of the 96 well plate.
Seal with a plastic cover.
Incubate overnight at 35 C.
Micro broth dilution is most suitable for what?
Surveillances
~microbiological criterion
Who is the founder of modern microbiology?
Robert Koch
What are Koch’s Postulates?
Cause and effect relationship in infectious disease.
- The organism **or parts thereof (e.g. PAMPs) **must always be present in disease.*
- The organism must be isolated from a diseased host and grown in pure culture. Although this is not always possible, it can be difficult to culture some organisms, PAMP’s and prions.*
- The cultured microbe should cause disease when transferred to a healthy animal. Emphasis on should and not will–there is also asymptomatic carriers.*
- The same type of microorganism **or parts thereof that cause disease **should be isolated from the newly infected animal.*
What are 2 types of PCR?
RT-PCR (Real Time quantitative PCR)
End-point PCR (convential)
What is considered the Gold Standard diagnostic test?
Culture and biochemical identification
What is gene cloning?
It is where DNA can be isolated and manipulated (cut and joined) by enzymes
What is nucleic acid hybridization?
Single-stranded nucleic acids will form a double-stranded complex if the two strands are complementary to one another.
Single stranded DNA can be used as a template to make complementary DNA using ________ _________.
Polymerase enzymes
Polymerases require a ______ that create regions of double-stranded DNA to initiate DNA synthesis.
Primer
What are the basic reagants you need for PCR?
Template DNA (must be an isolated template)
Target gene specific primers
Taq polymerase (thermostable)
Nucleotides
Divalent cation (Mg2+)
Buffer (pH)
Thermocycler
What are the steps of PCR?
- Heating to denature the original DNA (~95 C)
- Annealing [glueing] of the primers to the template DNA. (~50-60 C)
-
Taq product synthesis and subsequent primer extension (72 C)
* Repeat process: thermocycling*
What does PCR stand for and what is it?
Polymerase chain reaction
Exponential amplification of a segment of DNA
Doubling with each cycle.
What are some things to take into consideration when performing PCR?
- Contamination
- Primer set design
- Choice of target genes
- Product detection
- End point PCR
Describe what can happen with contamination when performing PCR?
False positives (amplicon build-up)~ *Possibility to generate false positives is very high. To prevent false positives there are 2 ways of dealing with it: separate rooms for assay setup and running post-PCR tests also Uracil-N glycosylase: replace dTTP with hydrolysable dUTP substrate. *
False negatives (inhibitors)
Agents that bind DNA or inhibit Taq (template/inhibitor dilutions)
Template DNA/RNA extraction method (purity)
Concentration of isolated template
Primers must be specific for the target gene/organism.
How can primer set design influence PCR?
Sequence: 100-1000 fold amplification difference for different primer sets amplifying the same gene.
Length 18-30 nucleotides
GC content 40-60%
Melting temperature (Tm): similar for both primers
No dimerization between primers: **primer dimers **or **hairpin **structures~ primers shouldn’t be able to bind to themselves.
Check primer specificity for target gene (BLAST homology against GenBank)
How can choice of target genes influence PCR?
Single copy gene~specifically to the X or Y chromosome so that you’re able to differentiate which is male and which is female. (e.g. gender typing prior to in vitro fertilization)
Multiple copy number gene= single amplification (identification of infectious agents)
RT-PCR (using mRNA; mRNA has multiple transcript copies but mRNA has rapid turnover.)~ using mRNA has a template, the problem with this is that it’s labile and subject to very rapid breakdown and turnover.
Describe product detection with PCR.
Classic agarose gel electrophoresis (end point PCR)
Run amplicon out on agarose gel, the size of amplicon with molecular weight standard will tell you whether you’ve ID’d the organism from the nucleic acids isolated.
Describe End Point PCR in terms of this being a consideration for even running PCR.
You get product detection at the end of running the assay.
Prescence or absence only.
Yes or no result~ doesn’t tell you anything about the # of organisms that you have in a sample. It is non-quantitative.
What are some features of RT-PCR (Real Time quantitative PCR)?
- Detection of product amplification during each cycle of the PCR reaction.
- Detection using measurements of fluorescent dyes.
- Fluorescent probes.
- Taqman Chemistry
- Absolute quantification
- Relative quantification
- Using different fluorescent dyes allows multiplexing
- Minimal information for quantitative Real Time PCR Experiments. (MIQE)
- Quantification allows disease progression to be monitored.
Describe in more detail detection of product amplification during each cycle.
Exponential amplification plot dependent on starting concentration of the template.
Describe in more detail detection using florescent dyes.
SybrGreen (dsDNA intercalating agent)
Forward and reverse primers only: to be able to detect the product.
Melting curve analysis (temperature at which amplicon becomes single stranded.) **Dependent on base composition of amplicon: Increased %GC leads to an increase in melting point. Single peak=single product.
Describe in more detail fluorescent probes.
Third oligonucleotide in addition to the 2 primers (greater specificity).
Different chemistries (e.g. Taqman, FRET, Scorpion, Molecular Beacons.)
Has a fluorescent group attached to it and binds to amplified DNA between 2 primers that you’re using. Generally at one end of nucleotide you will have a fluorescent group, etc…
Describe in more detail Taqman chemisry.
Probe has fluorescent reporter at one end and florescent quencher at the other end (F & Q are close together= signal quenched.)
Taq polymerase has 5’–>3’ exonuclease activity
Digests probe releasing fluorescent reporter and quencher (probe hydrolysis) (F & Q are far apart, no quenching= fluorescent signal)
Reporter (F) release proportional to product concentration.
Describe in more detail Absolute Quantification.
Standard curve from quantified template.
Describe in more detail relative quantification.
Target compared to endogenous gene or to an exogenous internal control added to the reaction.
Describe in more detail using different fluorescent dyes allowing multiplexing.
Can detect multiple targets in a single reaction.
Caution: reagent depletion
Describe in more detail minimal information for quantitative real time PCR experiments (MIQE).
Considers 42 variables in experimental design.
Assay validation is essential.
PCR **cannot discriminate **between ______ and ______ organisms.
*Living *and Dead
Because it’s simply nucleic acid.
What is Digital PCR?
Separation of a single reaction in thousands or partitions or nanodrops.
Amplicon in each nanodrop reaction is detected individually.
The number of nanodrops with detected amplicon versus those without give a quantitative result.
What are some examples of veterinary diagnosis using PCR assays?
Ehrlichia canis
Toxoplasmosis
Babesia
Leptospirosis
and many more….
