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Bac-T Lab Exam > Bacteriology Final Lab Exam > Flashcards

Bacteriology Final Lab Exam Flashcards

1
Q

What are the features of Microsporum spp.?

A

Macroconidia: Spindle shaped

Microconidia: Tear shaped & few or absent

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2
Q

What are the features of Trichophyton spp.?

A

Macroconidia: Cigar shaped & few or absent.

Microconidia: Grape like clusters & Numerous

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3
Q

What are the features of Microsporum canis?

A

Infects dogs, cats, humans.

6-15 cells within MACROconidia.

Spindle shaped.

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4
Q

What is this?

A

Microsporum canis

~remember spindle shaped & 6-15 cells for MACROconidia.

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5
Q

What are the features of Microsporum gypseum?

A

Infects: rodents, horses and dogs.

4-6 cells within MACROconidia.

Boat shaped.

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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.

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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.

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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.

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9
Q

What is this?

A

Trichophyton mentagrophytes

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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.

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11
Q

What is this?

A

Trichophyton equinum

~since MACRO’s are rare should be looking more at the hyphae.

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12
Q

What are the features of Trichophyton rubrum?

A
  • **Abundant **clavate to pyriform MICROconidia.
  • Moderate to abundant cigar-shaped MACROconidia.
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13
Q

What is this?

A

Trichophyton rubrum

~resembles a very skinny cigar

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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.
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15
Q

What is this?

A

Trichophyton tonsurans

~remember broad hyphae and cigar shaped MACROcondidia

~ MICROconidia forming right angles.

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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.
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17
Q

What is this?

A

Epidermophyton floccosum

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18
Q

What are the features of Geotrichum candidum?

A

-Fragmented hyphae, **rectangular one-celled arthrospores **in chains

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19
Q

What is this?

A

Geotrichum candidum

~Geo: think geometric shapes as in rectangular shaped arthrospores.

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20
Q

What are the defining features of Sprothrix schenckii?

A

-Flowerettes of conidia

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21
Q

What is this?

A

Sporothrix schenckii

~shank you very much for the “flowers”>>I know, corny as hell.

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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

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23
Q

What are the features of Aspergillus fumigatus?

A
  • Afro shaped
  • Chains of bluish green conidia
  • Stem= conidiophore
  • Have FOOT at base.
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24
Q

What is this?

A

Aspergillus fumigatus

~remember: Afrogillus fumigatus

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25
Q

What are the features of Aspergillus flavus?

A
  • Dandelion shaped
  • Round vesicles
  • Round conidia
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26
Q

What is this?

A

Aspergillus flavus

~Flava flava dandy dandelion shaped

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27
Q

What are the features of Aspergillus niger?

A

-Small black balls that are rough

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28
Q

What is this?

A

Aspergillus niger

~the word niger in Latin means black, these are your little black balls.

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29
Q

What are the features of Penicillium spp.?

A
  • Paint brushed, Paint-brushillium
  • Blue green
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30
Q

What is this?

A

Penicllium spp.

~remember: Paint-brushillium

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31
Q

What are the features of Bipolaris spp.?

A
  • Pale brown
  • Condia rounded at ends
  • Acorn shaped
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32
Q

What is this?

A

Bipolaris spp.

~The bipolar squirrels eat their acorns, I get it…I’m crazy.

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33
Q

What are the features of Curvularis?

A
  • Brown
  • 3 or more transverse septa
  • Central ball is larger
  • Curvy
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34
Q

What is this?

A

Curvularis

-The curvier acorn, hence it has 3 or more transverse septa.

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35
Q

What are the features of Cryptococcus neoformans?

A
  • 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.
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36
Q

What is this?

A

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.

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37
Q

What are the features of Candida albicans?

A

-Budding yeast cells in Sab’s or blood agar

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38
Q

What is this?

A

Candida albicans

~Remember:My old “buds” from “Canada”, yeast buds are Candida.

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39
Q

What are the features of Fusarium?

A

-Banana shaped MACROconidia

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40
Q

What is this?

A

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.

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41
Q

What are the steps in performing a Gram Stain?

A
  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)

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42
Q

What is TSA?

A

Tryptic soy agar

  • General purpose medium
  • Useful for : culture storage, enumeration, isolation of pure culture and general cultures.
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43
Q

What are the components of Tryptic Soy Agar?

A
  • 15 g Tryptone
  • 5 g Soytone
  • 5 g NaCl
  • 15 g agar
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44
Q

What is blood agar?

A
  • General purpose enriched medium
  • Grows fastidious organisms
  • Differentiates bacteria based on their hemolytic properties.

-

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45
Q

What type of hemolysis is this?

What type of toxin produces this?

A

Beta hemolysis or (complete hemolysis)

Alpha toxin produces this.

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46
Q

What type of hemolysis is this?

What type of toxin produces this?

A

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.

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47
Q

What type of hemolysis is this?

A

Gamma hemolysis or (no lysis)

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48
Q

What is an example of the type of bacteria that would produce this type of hemolysis?

What type of hemolysis is this?

A

Staphylococcus aureus

Beta-hemolysis

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49
Q

What is PEA?

A

Phenylethyl Alchohol Agar with 5% sheep’s blood

-Selective medium used to isolate most Gram (+) bacteria

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50
Q

How does PEA inhibit growth of most Gram (-) bacteria?

A

It disrupts the lipid structure of Gram (-) resulting in stunted or no growth.

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51
Q

What is MacConkey Agar?

A
  • Agar designed to grow Gram (-) bacteria.
  • Used to: differentiate lactose fermenters from non-fermenters.

Remember: Mac sounds like Lac, MacConkey used for Lactose differentiation.

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52
Q

What is in MacConkey Agar that prevents Gram (+) bacteria from growing?

A

Bile salts (most Gram +)

Crystal Violet dye (some Gram +)

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53
Q

What is present in MacConkey Agar that stains microbes that ferment lactose?

A

Neutral red dye

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54
Q

What other 2 components are included in MacConkey Agar?

A

Lactose and Peptone

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55
Q

What are the features of a lactose fermenter?

A

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.

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56
Q

What are the features of a non-lactose fermenter?

A

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.

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57
Q

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?

A

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

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58
Q

What is Hektoen enteric agar?

A

A differential selective medium for isolation of Salmonella and Shigella.

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59
Q

On Hektoen Enteric Agar, Shigella will give what type of apperance?

A

Green colonies

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60
Q

On Hektoen Enteric Agar, Salmonella will give what type of apperance?

A

Blue/green colonies, with or without black centers.

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61
Q

Hektoen Enteric Agar includes indicators for what?

A

Lactose fermentation and H2S production

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62
Q

What are the 2 things used to indicate H2S production on Hektoen Enteric Agar?

A

Thiosulfate and ferric ammonium citrate

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63
Q

Hektoen Enteric Agar contains inhibitors for what?

A

Gram (+) bacteria

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64
Q

What are the 2 things used to prevent growth of Gram (+) bacteria on Hektoen Enteric Agar?

A

Bile salts and Bromothymol Blue

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65
Q

What other things does Hektoen Enteric Agar contain?

A

Lactose, Sucrose and Salicin

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66
Q

Since Hektoen Enteric Agar contains indicators for lactose fermentation, how would something that does ferment lactose appear on HEA?

A

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.

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67
Q

What does Brilliant Green (BG) Agar look for?

A

If certain bacteria are able to ferment sugars: lactose and sucrose.

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68
Q

On Brilliant Green Agar how would non lactose/sucrose fermenting organisms appear?

What are some examples of bacteria that do this?

A

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.

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69
Q

On Brilliant Green how would lactose/sucrose fermenters appear?

What are examples of bacteria that do this?

A

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.

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70
Q

What is the pH indicator for Brilliant Green Agar?

A

Phenol Red

~**Red **at pH of 8.2 (alkaline)

~Yellow at pH of 6.4 (acidic)

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71
Q

What is the inhibitor in Brilliant Green (BG) Agar?

What is it inhibiting?

A

The inhibitor is brilliant green dye.

Inhibits the growth of most enterobacteria (all Gram (-) organisms) **except: Salmonella.

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72
Q

What is XLD agar?

A

Stands for: Xylose Lysine Deoxycholate agar.

It is used to see if certain bacteria can ferment these different sugars: lactose, sucrose, and xylose.

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73
Q

What is the pH indicator used for XLD agar?

A

Phenol red

~**Red **at pH 8.2 (alkaline)

~Yellow at pH 6.4 (acidic)

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74
Q

What are the inhibitors present in XLD agar?

What are they inhibiting?

A

Bile salts

Inhibiting Gram (+) bacteria

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75
Q

What substrates are used to detect H2S with XLD agar?

A

Lysine

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76
Q

Salmonella will appear to have what color colonies on XLD agar?

A

Red with a black center (checkers)

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77
Q

Why do Salmonella have this appearance (red with black center) on XLD agar?

A

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
Q

The alternative to Salmonella having (red with black centered) colonies on XLD is what?

A

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
Q

What is Eosin Methylene Blue Agar (EMBA)?

A

A selective stain for Gram (-) organisms.

80
Q

What is both the pH indicator and selective agents of Eosin Methylene Blue Agar?

A

Eosin Y and Methylene Blue

81
Q

What is the amino acid contained within Eosin Methylene Blue Agar?

What is the purpose of this amino acid?

A

Peptone

It may be deaminated (forming an alkaline reaction.)

82
Q

What is the fermentable sugar in Eosin Methylene Blue Agar?

A

Lactose 1%

83
Q

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?

A

Metallic green/with dark centers

Net producers of acid.

ex: E. coli

84
Q

On Eosin Methylene Blue Agar, what would something look like that does not ferment lactose?

What is an example of bacteria that display this?

A

Lightly colored

Net alkaline

ex: Salmonella and Shigella

85
Q

T/F: With the Gram Stain, Crystal violet only penetrates Gram (+) cell walls.

A

**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
Q

T/F: CV+ ions interact with positively charged components of bacterial cells.

A

**False: **CV+ ions interact with negatively charged components of bacterial cells and stain them purple.

87
Q

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.

A

True!!!

88
Q

T/F: When a decolorizer such as alcohol or acetone is added, it interacts with the protein of the cell membrane.

A

**False: **It interacts with the lipid of the cell membrane.

89
Q

T/F: A Gram (-) cell will lose it’s outer membrane and the peptidoglycan layer is left exposed

A

True!!!

90
Q

T/F: The CV-1 complexes stay intact with the Gram (-) cell wall while the rest is washed away.

A

**False: **The CV-1 complexes are washed away as well.

91
Q

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.

A

True!!!

92
Q

T/F: The counterstaining is the critical step of the Gram Stain process.

A

**False: **the decolorizing step is the critical step and must be timed correctly!!!

93
Q

Why is it important that the decolorization be timed precisely?

A

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
Q

T/F: At the end of the process, Gram (+) cells appear pink.

A

False: Gram (+) cells will be purple!!

95
Q

T/F: At the end of the Gram Stain process, Gram (-) bacteria will appear pink.

A

True!!!

96
Q

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.

A

True!!!

97
Q

What is this?

A

Gram (+) bacteria: Streptococcus

~Gram (+) we know because it’s purple

~Strep=chains

~Coccus/cocci= circles

98
Q

What is this?

A

Gram (-) bacteria: Staphylobacilli

~Gram (-) because of the pink color.

~Staph=groups/clusters

~Bacillus/bacilli=rods

99
Q

How does the catalase test work?

A

Detects the enzyme catalase which converts H2O2 (hydrogen peroxide) into H2O and O2.

100
Q

What type of catalase result is this?

What type of bacteria produce this type of result?

A

Positive result

Most aerobic bacteria

“Staph species” like Staphylococcus aureus

101
Q

What type of catalase test result is this?

What type of bacteria would produce this result?

A

Negative result

Obligate anaerobes, mainly produce this

“Strep species” like Streptococcus equi

102
Q

The Oxidase Test is detecting the presence of what?

A

**Cytochrome C Oxidase **in the bacterial cell wall.

103
Q

What type of Oxidase test result is this?

What type of bacteria would produce this result?

A

Oxidase negative

Anaerobic bacteria

Staphylococcus aureus

104
Q

What type of Oxidase test result is this?

What type of bacteria would produce this result?

A

Oxidase positive

Aerobic bacteria

Pseudomonas aeruginosa

105
Q

What is added to the medium of motility test to detect the presence of motility in a bacteria?

A

Tetrazolium salts

106
Q

Tetrazolium salts are colored/colorless?

A

Colorless

107
Q

As the bacterium grows the dye is incorporated into the bacterial _____ ______where it is reduced to an insoluble red pigment.

A

Cell wall

108
Q

What is the name of the insoluble red pigment in the motility test?

A

Formazan

109
Q

What type of motility test result is this?

What type of bacteria produce this result?

A

Positive motility test

Proteus mirabilis

110
Q

What type of motility test result is this?

What type of bacteria would give this result?

A

Negative motility test

Klebsiella pneumoniae

111
Q

What is the Urease Test looking for?

A

If the bacteria contains urease and is able to split urea with water to form ammonia and CO2.

112
Q

What is the medium present with the Urease Test?

A

Urea broth (contains ~2% urea)

113
Q

What is the indicator in the Urease Test?

A

Phenol red

114
Q

What type of Urease Test result is this?

What type of bacteria would produce this result?

A

Negative result

Produces a yellow-orange color

Salmonella spp.

115
Q

What type of Urease Test result is this?

What type of bacteria would result in this?

A

Positive test result

Hot pink color

Proteus spp.

Alkaline

116
Q

What does IMViC stand for?

A

I-Indole

M-Methyl

V- Voges Proskauer

C- Citrate

117
Q

What is the IMViC test, testing for?

A

It is a group of individual tests use to differentiate the Family Enterobacteriaceae.

118
Q

What is the Indole Test, testing for?

A

To see if the bacteria can convert **Tryptophan to indole. **

119
Q

What is the indicator used for the Indole Test?

A

Kovac’s Reagant

120
Q

What type of Indole Test result is this?

What type of bacteria would produce this type of result?

A

Negative indole test

Enterobacter spp.

Yellow color

121
Q

What type of Indole test result is this?

What type of bacteria would produce this type of result?

A

Positive indole test

E. coli

Red color

122
Q

What is the Methyl Red (MR) test used for?

A

Testing for low molecular weight acids: such as* lactic, formic and acetic acid.*

123
Q

What is the medium used with Methyl Red (MR) test?

A

Peptone-glucose broth

124
Q

What is the indicator used for the Methyl Red (MR) test?

A

Methyl Red….duhhhh……

125
Q

What type of Methyl Red test result is this?

What type of bacteria would produce this type of result?

A

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
Q

What type of test result is this for the Methyl Red test?

What type of bacteria would produce this result?

A

Negative methyl red test

Enterobacter spp.

Yellow color

127
Q

What is the Voges-Proskauer Test, testing for?

A

To see if bacteria can convert glucose to acetoin.

128
Q

What is the medium used for the Voges-Proskauer test?

A

Peptone-Glucose broth

129
Q

What is the indicator used for the Voges-Proskauer test?

A

40% KOH (Potassium hydroxide) and α-naphthol

130
Q

What type of Voges-Proskauer test result is this?

What type of bacteria would produce this result?

A

Negative test result

E. coli

Reagant layer yellow

131
Q

What type of Voges-Proskauer test result is this?

What type of bacteria would produce this type of result?

A

Positive test result

Enterobacter spp.

Reagant layer red

132
Q

What is the Citrate Test, testing for?

A

The ability of the bacteria to use citrate as it’s sole carbon source.

133
Q

What is the medium present in the Citrate test?

A

Simmons citrate agar

134
Q

What is the indicator present with the Citrate Test?

A

Bromothymol Blue

135
Q

What type of Citrate test result is this?

What type of bacteria would produce this result?

A

Negative test result

Green color

E. coli

136
Q

What type of Citrate test result is this?

What type of bacteria would produce this type of result?

A

Positive test result

Blue color

Salmonella spp.

137
Q

What are the 3 methods of antimicrobial susceptibility in the lab?

A
  1. Kirby Bauer (disc diffusion)
  2. E-test (MIC)
  3. Sensitire broth microdiliution (MIC)
138
Q

When you test for susceptibility in the lab, how many ways possible are there and what are they called?

A

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
Q

The Kirby Bauer Disc Diffusion method is a qualitative or quantitative interpretation?

A

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
Q

For the Kirby Bauer Disc Diffusion what are the 3 possible results you can get?

A

~Susceptible

~Intermediate

~Resistant

141
Q

What are the discs that are used in the Kirby Bauer test?

A

Can be: **Antibiotic impregnated paper discs (several suppliers) **or tablets (Rosco)~ neosensitabs

142
Q

What are the steps you’d use in performing the Kirby Bauer Disc Diffusion test?

A

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
Q

What is the zone of inhibition?

A

The area around an antibiotic disc that contains no bacterial growth.

Measured in mm’s

144
Q

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?

A

If it has a fuzzy edge: Report as susceptible.

If it has a sharp edge: Report as resistant.

145
Q

What is an example of a bacteria and an antibiotic where you may need to decipher fuzzy from sharp zone of inhibition borders?

A

*Staphylococcus aureaus *with Benzylpenicillin

146
Q

What are factors that influence the zone of inhibition?

A
  • 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
Q

What is the breakpoint?

A

A level of minimum inhibitory concentration (MIC) at which a bacterium is deemed either susceptible or resistant to an antibiotic being used.

148
Q

What is the MIC?

A

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.

149
Q

What is the MBC?

A

Minimum bactericidal concentration

The concentration of the antibiotic by which the strain does not grown anymore after subculture.

150
Q

The E-test is a combination of _______ and _______.

A

Dilution and Diffusion

151
Q

When reading an E-test what does <1 correspond with?

A

Susceptible

152
Q

When reading an E-test what does 1-4 correspond with?

A

Intermediate

153
Q

When reading an E-test what does >4 correspond with?

A

Resistant

154
Q

What is the upside to using an E-test?

A

It’s a good and fairly accurate test.

Few methodological problems.

155
Q

What does the E in E-test stand for?

A

Ellipsoid

156
Q

What does the E-test give you?

A

Your MIC which is quantitative data.

Can also be used in a specific research setting when a single antibiotic is involved.

157
Q

What are 2 dilution tests used?

A

Agar dilution

Broth dilution (Macro-broth dilution **and **Micro-broth dilution.)

158
Q

Between the 2 dilution tests which one is the best one?

A

Agar dilution

159
Q

How is the agar dilution test performed?

A

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
Q

If you’re doing a broth macro-dilution which of these tubes would be considered your MIC?

A

It would be the next most concentrated tube but with no growth.

161
Q

Which of these tubes would be your MIC?

A
162
Q

How is micro broth dilution performed?

A

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
Q

Micro broth dilution is most suitable for what?

A

Surveillances

~microbiological criterion

164
Q

Who is the founder of modern microbiology?

A

Robert Koch

165
Q

What are Koch’s Postulates?

A

Cause and effect relationship in infectious disease.

    1. The organism **or parts thereof (e.g. PAMPs) **must always be present in disease.*
    1. 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.*
    1. The cultured microbe should cause disease when transferred to a healthy animal. Emphasis on should and not will–there is also asymptomatic carriers.*
    1. The same type of microorganism **or parts thereof that cause disease **should be isolated from the newly infected animal.*
166
Q

What are 2 types of PCR?

A

RT-PCR (Real Time quantitative PCR)

End-point PCR (convential)

167
Q

What is considered the Gold Standard diagnostic test?

A

Culture and biochemical identification

168
Q

What is gene cloning?

A

It is where DNA can be isolated and manipulated (cut and joined) by enzymes

169
Q

What is nucleic acid hybridization?

A

Single-stranded nucleic acids will form a double-stranded complex if the two strands are complementary to one another.

170
Q

Single stranded DNA can be used as a template to make complementary DNA using ________ _________.

A

Polymerase enzymes

171
Q

Polymerases require a ______ that create regions of double-stranded DNA to initiate DNA synthesis.

A

Primer

172
Q

What are the basic reagants you need for PCR?

A

Template DNA (must be an isolated template)

Target gene specific primers

Taq polymerase (thermostable)

Nucleotides

Divalent cation (Mg2+)

Buffer (pH)

Thermocycler

173
Q

What are the steps of PCR?

A
  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
Q

What does PCR stand for and what is it?

A

Polymerase chain reaction

Exponential amplification of a segment of DNA

Doubling with each cycle.

175
Q

What are some things to take into consideration when performing PCR?

A
  1. Contamination
  2. Primer set design
  3. Choice of target genes
  4. Product detection
  5. End point PCR
176
Q

Describe what can happen with contamination when performing PCR?

A

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
Q

How can primer set design influence PCR?

A

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
Q

How can choice of target genes influence PCR?

A

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
Q

Describe product detection with PCR.

A

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.

180
Q

Describe End Point PCR in terms of this being a consideration for even running PCR.

A

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.

181
Q

What are some features of RT-PCR (Real Time quantitative PCR)?

A
  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
Q

Describe in more detail detection of product amplification during each cycle.

A

Exponential amplification plot dependent on starting concentration of the template.

183
Q

Describe in more detail detection using florescent dyes.

A

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
Q

Describe in more detail fluorescent probes.

A

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
Q

Describe in more detail Taqman chemisry.

A

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
Q

Describe in more detail Absolute Quantification.

A

Standard curve from quantified template.

187
Q

Describe in more detail relative quantification.

A

Target compared to endogenous gene or to an exogenous internal control added to the reaction.

188
Q

Describe in more detail using different fluorescent dyes allowing multiplexing.

A

Can detect multiple targets in a single reaction.

Caution: reagent depletion

189
Q

Describe in more detail minimal information for quantitative real time PCR experiments (MIQE).

A

Considers 42 variables in experimental design.

Assay validation is essential.

190
Q

PCR **cannot discriminate **between ______ and ______ organisms.

A

*Living *and Dead

Because it’s simply nucleic acid.

191
Q

What is Digital PCR?

A

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
Q

What are some examples of veterinary diagnosis using PCR assays?

A

Ehrlichia canis

Toxoplasmosis

Babesia

Leptospirosis

and many more….

Bac-T Lab Exam (1 decks)

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