Bacteriology Final Lab Exam Flashcards
(192 cards)
1
Q
What are the features of Microsporum spp.?
A
Macroconidia: Spindle shaped
Microconidia: Tear shaped & few or absent
2
Q
What are the features of Trichophyton spp.?
A
Macroconidia: Cigar shaped & few or absent.
Microconidia: Grape like clusters & Numerous
3
Q
What are the features of Microsporum canis?
A
Infects dogs, cats, humans.
6-15 cells within MACROconidia.
Spindle shaped.
4
Q
What is this?
A
Microsporum canis
~remember spindle shaped & 6-15 cells for MACROconidia.
5
Q
What are the features of Microsporum gypseum?
A
Infects: rodents, horses and dogs.
4-6 cells within MACROconidia.
Boat shaped.
6
Q
What is this?
A
Microsporum gypseum
~remember 4-6 cells within MACROconidia, this is fewer than M. canis.
~Gypseum=gypsy, frizzy gypsy hair fringed around edges.
7
Q
What are the features of Microsporum audouinii?
A
Most plates are sterile.
Think houdini=audouinii, they are never around.
Macro- and Microconidia are rare.
Most closely resembles M. canis.
8
Q
What are the features of Trichophyton mentagrophytes?
A
Infects: rodents, dogs and horses.
3-7 celled MACROconidia.
Cigar shaped.
2 colony types: granular (animal pathogen) and downy.
9
Q
What is this?
A
Trichophyton mentagrophytes
10
Q
What are the features of Trichophyton equinum?
A
Infects: horses.
3-5 celled MACROconidia.
MACROs are rare.
Can see chlamydospores with old cultures.
11
Q
What is this?
A
Trichophyton equinum
~since MACRO’s are rare should be looking more at the hyphae.
12
Q
What are the features of Trichophyton rubrum?
A
- **Abundant **clavate to pyriform MICROconidia.
- Moderate to abundant cigar-shaped MACROconidia.
13
Q
What is this?
A
Trichophyton rubrum
~resembles a very skinny cigar
14
Q
What are the features of Trichophyton tonsurans?
A
- 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.
15
Q
What is this?
A
Trichophyton tonsurans
~remember broad hyphae and cigar shaped MACROcondidia
~ MICROconidia forming right angles.
16
Q
What are the features of Epidermophyton floccosum?
A
- On Sab’s agar is slow growing.
- Smooth thin walled MACROconidia
- Clusters of MACRO’s growing directly from the hyphae.
17
Q
What is this?
A
Epidermophyton floccosum
18
Q
What are the features of Geotrichum candidum?
A
-Fragmented hyphae, **rectangular one-celled arthrospores **in chains
19
Q
What is this?
A
Geotrichum candidum
~Geo: think geometric shapes as in rectangular shaped arthrospores.
20
Q
What are the defining features of Sprothrix schenckii?
A
-Flowerettes of conidia
21
Q
What is this?
A
Sporothrix schenckii
~shank you very much for the “flowers”>>I know, corny as hell.
22
Q
What is the fungus on the left?
What is the fungus on the right?
A
Left: Penicillium ~ Paint brush-illium
Right: Aspergillus~ Afro-gillus
23
Q
What are the features of Aspergillus fumigatus?
A
- Afro shaped
- Chains of bluish green conidia
- Stem= conidiophore
- Have FOOT at base.
24
Q
What is this?
A
Aspergillus fumigatus
~remember: Afrogillus fumigatus
25
What are the features of Aspergillus flavus?
- **Dandelion shaped**
- Round vesicles
- Round conidia
26
What is this?
Aspergillus flavus
~**Flava flava dandy dandelion shaped**
27
What are the features of Aspergillus niger?
-Small black balls that are rough
28
What is this?
Aspergillus niger
~the word niger in Latin means **black,** these are your little black balls.
29
What are the features of Penicillium spp.?
- Paint brushed, **Paint-brushillium**
- Blue green
30
What is this?
Penicllium spp.
~remember: **Paint-brushillium**
31
What are the features of Bipolaris spp.?
- Pale brown
- Condia rounded at ends
- Acorn shaped
32
What is this?
Bipolaris spp.
~**The bipolar squirrels eat their acorns,** I get it...I'm crazy.
33
What are the features of Curvularis?
- Brown
- **3 or more transverse septa**
- Central ball is larger
- *Curvy*
34
What is this?
Curvularis
-**The *curvier* acorn, hence it has 3 or more transverse septa.**
35
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.**
36
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.***
37
What are the features of Candida albicans?
-Budding yeast cells in Sab's or blood agar
38
What is this?
Candida albicans
~Remember:**My old "buds" from "Canada", yeast buds are Candida.**
39
What are the features of Fusarium?
-Banana shaped MACROconidia
40
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.*
41
What are the steps in performing a Gram Stain?
1. With plastic loop, add a small amount of bacteria to slide mixed with water.
2. Heat and dry slide
3. Primary stain: with **Crystal violet~ 1 min**
4. Mordant: **Iodine~ 2 min**
5. Decolorizer: **Alcohol or Acetone~ seconds**
6. Counter stain: **Safranin~ 1 min**
(Make sure to rinse in between each step)
42
What is TSA?
Tryptic soy agar
- General purpose medium
- Useful for : culture storage, enumeration, isolation of pure culture and general cultures.
43
What are the components of Tryptic Soy Agar?
- 15 g Tryptone
- 5 g Soytone
- 5 g NaCl
- 15 g agar
44
What is blood agar?
- General purpose enriched medium
- Grows fastidious organisms
- **Differentiates bacteria based on their hemolytic properties.**
-
45
What type of hemolysis is this?
What type of toxin produces this?
**Beta hemolysis** or (complete hemolysis)
*Alpha toxin* produces this.
46
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.***
47
What type of hemolysis is this?
**Gamma hemolysis** or (no lysis)
48
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
49
What is PEA?
Phenylethyl Alchohol Agar *with 5% sheep's blood*
-Selective medium used to **isolate most Gram (+) bacteria**
50
How does PEA inhibit growth of most Gram (-) bacteria?
It disrupts the lipid structure of Gram (-) resulting in stunted or no growth.
51
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.***
52
What is in MacConkey Agar that prevents Gram (+) bacteria from growing?
Bile salts (most Gram +)
Crystal Violet dye (some Gram +)
53
What is present in MacConkey Agar that stains microbes that ferment lactose?
Neutral red dye
54
What other 2 components are included in MacConkey Agar?
Lactose and Peptone
55
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.***
56
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.***
57
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**
58
What is Hektoen enteric agar?
A differential selective medium for *isolation of Salmonella and Shigella.*
59
On Hektoen Enteric Agar, Shigella will give what type of apperance?
**Green** colonies
60
On Hektoen Enteric Agar, Salmonella will give what type of apperance?
**Blue/green** colonies, with or without **black centers.**
61
Hektoen Enteric Agar includes indicators for what?
Lactose fermentation and H2S production
62
What are the 2 things used to indicate H2S production on Hektoen Enteric Agar?
**Thiosulfate** and **ferric ammonium citrate**
63
Hektoen Enteric Agar contains inhibitors for what?
**Gram (+) bacteria**
64
What are the 2 things used to prevent growth of Gram (+) bacteria on Hektoen Enteric Agar?
Bile salts and Bromothymol Blue
65
What other things does Hektoen Enteric Agar contain?
Lactose, Sucrose and Salicin
66
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.***
67
What does Brilliant Green (BG) Agar look for?
If certain bacteria are able to ferment sugars: **lactose and sucrose.**
68
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.*
69
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.***
70
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)
71
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.***
72
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.
73
What is the pH indicator used for XLD agar?
Phenol red
~**Red **at pH 8.2 (alkaline)
**~Yellow** at pH 6.4 (acidic)
74
What are the inhibitors present in XLD agar?
What are they inhibiting?
Bile salts
Inhibiting Gram (+) bacteria
75
What substrates are used to detect H2S with XLD agar?
Lysine
76
Salmonella will appear to have what color colonies on XLD agar?
Red with a black center (**checkers**)
77
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.***
78
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._***
79
What is Eosin Methylene Blue Agar (EMBA)?
A selective stain for **Gram (-) organisms.**
80
What is both the pH indicator and selective agents of Eosin Methylene Blue Agar?
**Eosin Y** and **Methylene Blue**
81
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.)
82
What is the fermentable sugar in Eosin Methylene Blue Agar?
Lactose 1%
83
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*
84
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*
85
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.**
86
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.**
87
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!!!**
88
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.
89
T/F: A Gram (-) cell will lose it's outer membrane and the peptidoglycan layer is left exposed
**True!!!**
90
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.**
91
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!!!**
92
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!!!**
93
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.
94
T/F: At the end of the process, Gram (+) cells appear pink.
**False: Gram (+) cells will be purple!!**
95
T/F: At the end of the Gram Stain process, Gram (-) bacteria will appear pink.
**True!!!**
96
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!!!**
97
What is this?
Gram (+) bacteria: Streptococcus
~Gram (+) we know because it's **purple**
**~Strep**=**chains**
~**Coccus/cocci**= circles
98
What is this?
**Gram (-) bacteria:** **Staphylobacilli**
~Gram (-) because of the **pink color.**
~**Staph**=**groups/clusters**
~**Bacillus/bacilli**=**rods**
99
How does the catalase test work?
Detects the enzyme catalase which converts **H2O2 (hydrogen peroxide) into H2O and O2.**
100
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*
101
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*
102
The Oxidase Test is detecting the presence of what?
**Cytochrome C Oxidase **in the bacterial cell wall.
103
What type of Oxidase test result is this?
What type of bacteria would produce this result?
**Oxidase negative**
Anaerobic bacteria
*Staphylococcus aureus*
104
What type of Oxidase test result is this?
What type of bacteria would produce this result?
**Oxidase positive**
Aerobic bacteria
*Pseudomonas aeruginosa*
105
What is added to the medium of motility test to detect the presence of motility in a bacteria?
Tetrazolium salts
106
Tetrazolium salts are colored/colorless?
Colorless
107
As the bacterium grows the dye is incorporated into the bacterial _____ \_\_\_\_\_\_where it is reduced to an insoluble red pigment.
Cell wall
108
What is the name of the insoluble red pigment in the motility test?
**Formazan**
109
What type of motility test result is this?
What type of bacteria produce this result?
**Positive motility test**
*Proteus mirabilis*
110
What type of motility test result is this?
What type of bacteria would give this result?
**Negative motility test**
*Klebsiella pneumoniae*
111
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.***
112
What is the medium present with the Urease Test?
Urea broth (contains ~2% urea)
113
What is the indicator in the Urease Test?
**Phenol red**
114
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.*
115
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
116
What does IMViC stand for?
I-Indole
M-Methyl
V- Voges Proskauer
C- Citrate
117
What is the IMViC test, testing for?
It is a group of **individual tests** use to **differentiate the Family Enterobacteriaceae.**
118
What is the Indole Test, testing for?
To see if the bacteria can convert **Tryptophan to indole. **
119
What is the indicator used for the Indole Test?
Kovac's Reagant
120
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
121
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
122
What is the Methyl Red (MR) test used for?
Testing for **low molecular weight acids: such as* lactic, formic and acetic acid.***
123
What is the medium used with Methyl Red (MR) test?
Peptone-glucose broth
124
What is the indicator used for the Methyl Red (MR) test?
**Methyl Red....***duhhhh......*
125
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, ++++++++***
126
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
127
What is the Voges-Proskauer Test, testing for?
To see if bacteria can **convert glucose to acetoin.**
128
What is the medium used for the Voges-Proskauer test?
Peptone-Glucose broth
129
What is the indicator used for the Voges-Proskauer test?
40% KOH (Potassium hydroxide) and α-naphthol
130
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_**
131
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_**
132
What is the Citrate Test, testing for?
The ability of the bacteria to **use citrate as it's sole carbon source.**
133
What is the medium present in the Citrate test?
Simmons citrate agar
134
What is the indicator present with the Citrate Test?
Bromothymol Blue
135
What type of Citrate test result is this?
What type of bacteria would produce this result?
**Negative test result**
**Green** color
*E. coli*
136
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.*
137
What are the 3 methods of *antimicrobial susceptibility* in the lab?
1. Kirby Bauer (disc diffusion)
2. E-test (MIC)
3. Sensitire broth microdiliution (MIC)
138
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.
139
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.*
140
For the Kirby Bauer Disc Diffusion what are the 3 possible results you can get?
~Susceptible
~Intermediate
~Resistant
141
What are the discs that are used in the Kirby Bauer test?
Can be: **Antibiotic impregnated paper discs (several suppliers) **or **tablets (Rosco)~ neosensitabs**
142
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.**
143
What is the *zone of inhibition*?
The area around an antibiotic disc that contains no bacterial growth.
Measured in mm's
144
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._
145
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**
146
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*)
147
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.
148
What is the MIC?
Minimum inhibitory concentration
## Footnote
*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.**
149
What is the MBC?
Minimum bactericidal concentration
## Footnote
*The concentration of the antibiotic by which the strain does not grown anymore after subculture.*
150
The E-test is a combination of _______ and \_\_\_\_\_\_\_.
**Dilution and Diffusion**
151
When reading an E-test what does \<1 correspond with?
Susceptible
152
When reading an E-test what does 1-4 correspond with?
Intermediate
153
When reading an E-test what does \>4 correspond with?
Resistant
154
What is the upside to using an E-test?
It's a good and fairly accurate test.
Few methodological problems.
155
What does the E in E-test stand for?
Ellipsoid
156
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.
157
What are 2 dilution tests used?
Agar dilution
Broth dilution (*Macro-broth dilution **and **Micro-broth dilution.)*
158
Between the 2 dilution tests which one is the **best** one?
Agar dilution
159
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.
160
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.**
161
Which of these tubes would be your MIC?
162
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.
163
Micro broth dilution is most suitable for what?
Surveillances
~microbiological criterion
164
Who is the founder of modern microbiology?
Robert Koch
165
What are Koch's Postulates?
**Cause and effect relationship in infectious disease.**
* 1. The organism **or parts thereof (e.g. PAMPs) **must always be present in disease.*
* 2. 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.***
* 3. The cultured microbe **should** cause disease when transferred to a healthy animal. **Emphasis on should and not will--there is also asymptomatic carriers.***
* 4. The same type of microorganism **or parts thereof that cause disease **should be isolated from the newly infected animal.*
166
What are 2 types of PCR?
RT-PCR (Real Time *quantitativ*e PCR)
End-point PCR ***(convential)***
167
What is considered the Gold Standard diagnostic test?
Culture and biochemical identification
168
What is gene cloning?
It is where DNA can be isolated and manipulated (cut and joined) by enzymes
169
What is nucleic acid hybridization?
Single-stranded nucleic acids will form a double-stranded complex if the two strands are complementary to one another.
170
Single stranded DNA can be used as a template to make complementary DNA using ________ \_\_\_\_\_\_\_\_\_.
Polymerase enzymes
171
Polymerases require a ______ that create regions of double-stranded DNA to initiate DNA synthesis.
Primer
172
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
173
What are the steps of PCR?
1. **Heating to denature** the original DNA (~95 C)
2. **Annealing** [glueing] **of the primers** to the template DNA. (~50-60 C)
3. **Taq product synthesis** and subsequent primer extension (72 C)
* Repeat process: thermocycling*
174
What does PCR stand for and what is it?
Polymerase chain reaction
Exponential amplification of a segment of DNA
*Doubling with each cycle.*
175
What are some things to take into consideration when performing PCR?
1. Contamination
2. Primer set design
3. Choice of target genes
4. Product detection
5. End point PCR
176
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.
177
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)
178
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.*
179
Describe product detection with PCR.
Classic agarose gel electrophoresis (**end point PCR**)
## Footnote
*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.*
180
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.
## Footnote
***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.*
181
What are some features of RT-PCR (Real Time *quantitative* PCR)?
1. Detection of product amplification during each cycle of the PCR reaction.
2. Detection using measurements of fluorescent dyes.
3. Fluorescent probes.
4. Taqman Chemistry
5. Absolute quantification
6. Relative quantification
7. Using different fluorescent dyes allows multiplexing
8. Minimal information for quantitative Real Time PCR Experiments. (MIQE)
9. Quantification allows disease progression to be monitored.
182
Describe in more detail detection of product amplification during each cycle.
Exponential amplification plot dependent on starting concentration of the template.
183
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.*
184
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...*
185
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.
186
Describe in more detail *Absolute Quantification.*
**Standard curve from quantified template.**
187
Describe in more detail *relative quantification.*
**Target compared to endogenous gene or to an exogenous internal control added to the reaction.**
188
Describe in more detail using different fluorescent dyes allowing multiplexing.
Can detect multiple targets in a single reaction.
## Footnote
**Caution: reagent depletion**
189
Describe in more detail minimal information for quantitative real time PCR experiments (MIQE).
Considers 42 variables in experimental design.
Assay validation is essential.
190
PCR **cannot discriminate **between ______ and ______ organisms.
*Living *and *Dead*
## Footnote
***Because it's simply nucleic acid.***
191
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.*
192
What are some examples of veterinary diagnosis using PCR assays?
Ehrlichia canis
Toxoplasmosis
Babesia
Leptospirosis
*and many more....*
