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Micro Lab > Experiments 5-8 > Flashcards

Experiments 5-8 Flashcards

1
Q

Listeria blackening of medium

A

Hydrolysis of esculin
Product (esculetin) reacts with ferric iron
Creates black/brown complex

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2
Q

Listeria acidification

A

Fermentation of mannitol changes color from red to yellow

PALCAM

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3
Q

Selectivity PALCAM

A

LiCl
Polymyxin B Sulfate
Acriflavine
Ceftazidime

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4
Q

Differentiation PALCAM

Listeria

A

Esculin
Mannitol
Phenol Red

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5
Q

EB selective (Listeria)

A

Nalidixic acid
Acriflavin
Cycloheximide

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6
Q

Catalase reaction

A

2 H2O2 –> 2 H2O + O2

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7
Q

Alpha hemolysis

A

Cloudy zone under colony

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8
Q

Beta hemolysis

A

Clear zone around colony

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9
Q

Gamma hemolysis

A

No hemolysis

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10
Q

TASYE purpose

A

Further purification

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11
Q

Listeria Characteristics

A

Small
Catalase +
Gram +
Non spore forming
Rods

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12
Q

Issue with antibiotic residues

A

Inhibition of growth of lactic acid bacteria

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13
Q

Mastitis

A

Bacterial infection of mammary glands

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14
Q

Beta lactam antibiotics

A

Bind to peptidoglycan and inhibit cell wall synthesis

Penicillin
Ampicillin
Amoxicillin
Cephalosporin

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15
Q

Antibiotic regulations

A

Cow cannot be supplemented with antibiotics for 48 hours prior to milking

All milk that is shipped from farm to factory to be tested for antibiotics

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16
Q

Antibiotic tests

A

Disc assay
Delvo P

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17
Q

Delvo P test

A

If antibiotic is not present: media changes from purple to yellow (shows growth via acid production)

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18
Q

BSDA:

Negative for inhibitory substances

A

No zone around disk containing untreated milk

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19
Q

BSDA:

Positive for β-lactam residue

A

A zone around disk containing untreated milk but no zone around disk containing penicillinase-treated milk

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20
Q

BSDA:

Presence of inhibitors other than β-lactam residues

A

Clear zone of equal size around both disks

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21
Q

BSDA:

Presence of β-lactam residues as well as another inhibitor(s)

A

Clear zone of 4 mm around penicillinase-treated milk smaller than that around untreated milk disk

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22
Q

Penicillin positive control

A

Clear, well defined zones
16-20 mm

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23
Q

Antibiotic assay controls

A

PM

PM + A

PM + A + B

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24
Q

Vehicle of transmissions for listeriosis

A

Consumption of contaminated food products

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25
Q

Listeria plating order

A

Enrichment broth (selective)
PALCAM (black halo)
TASYE (purify)

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26
Q

Listeria Blood Agar results (if pathogenic)

A

Narrow zone of clearing around colonies
Hemolytic

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27
Q

Antibiotics select for

A

Bacteria that have acquired mutations/genes that confer antibiotic resistance

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28
Q

Beta lactamase is

A

An enzyme that some bacteria produce that renders them resistant to beta lactam antibiotics

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29
Q

Sulfonamides

A

A group of antibiotics

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30
Q

Lactic ring has

A

1 x N and 3 x C

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31
Q

Organisms used as standard test for antibiotic residues

A

Bacillus stearothermophilus
Lactococcus lactis

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32
Q

Delvo P
Result: purple medium

A

An antibiotic is present, no growth of B. stearothermophilus will occur

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33
Q

LST MUG Assay

A

Based on presence of GUD
GUD cleave MUG
Creates MU (fluorescent)

Identify coliforms by gas production from lactose

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34
Q

Methyl Red

A

1-2 drops of methyl red
MR VP broth

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35
Q

Methyl Red: Red color

A

Mixed acid fermentation
pH < 4.4

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36
Q

Methyl Red: Yellow color

A

Butanediol fermentation
pH > 6.2

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37
Q

Methyl Red: Orange color

A

equivocal reaction
pH 4.4 - 6.2

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38
Q

VP Test

A

Detect presence of acetoin

α-naphthol reagent
KOH

MR VP broth

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39
Q

VP: red color

A

Positive reaction
Presence of acetoin (neutral end product)

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40
Q

Indole test

A

Kovac’s reagent
Tryptone broth

Determine the ability of an organism to split tryptophan to form indole

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41
Q

Indole test: Red ring

A

Positive result for indole

42
Q

Simmon citrate

A

Citrate agar

Determine if citrate is used as a carbon and energy source

43
Q

Simmons citrate: blue color

A

Positive for citrate utilization

Alkaline reaction turns agar blue due to removal of citrate

44
Q

What does IMVic Stand for

A

Methyl Red
Indole
VP
Simmons citrate

45
Q

E.coli IMViC results
Biotype 1

A

Indole +
MR +
VP -
Simmons -

46
Q

E.coli IMViC results
Biotype 2

A

Biotype 2:
Indole -
MR +
VP -
Simmons -

47
Q

What does GUD stand for

A

β-glucouronidase

48
Q

What does MU stand for

A

4-methylumbelliferone

49
Q

E.coli characteristics

A

Gram -
Rod
Facultative
Ferments lactose
Non fastidious

50
Q

E.coli on EMB agar

A

E. coli colonies are typically dark purple or blue and convex shaped due to fermentation of lactose in the EMB agar (agar color changes from green to purple)

Acid complex w/ EMB agents

51
Q

EMB agents

A

Eosin
Methylene Blue

Inhibit growth of gram +

52
Q

API strip test

A

Rapid test
Fast way to inoculate

Easy
Expensive

53
Q

E.coli fermentation products

A

Ethanol
CO2
H2

Lactic acid
Acetic acid
Succinic acid
Formic acid

54
Q

Acids produced from E.coli glucose fermentation

A

Lactic
Succinic
Acetic
Formic

55
Q

E.coli produces per mol of glucose

A

4 mol acidic products
1 mol EtOH
1 mol CO2
1 mol H2

56
Q

ETEC

A

Enterotoxigenic E. coli

Traveler’s diarrhea
High infectious dose
Outbreaks rare

57
Q

EPEC

A

Enteropathogenic E. Coli

Infant diarrhea
Poor sanitation practices

58
Q

EIEC

A

Enteroinvasive E. coli

Dysentery
Low infectious dose
Fecally contaminated foods
Cramps, diarrhea
Self limiting

59
Q

EHEC

A

Enterohemorrhagic E. coli

Low infectious dose
Shiga toxins
Intestinal disease
HUS
Renal failure

60
Q

PCR Steps

A

Microwave (1 min)

Denaturation (5 min) (95 C)

Denaturation (30 sec) (95 C)
Annealing (30 sec) (55 C)
Elongation (30 sec) (72 C)
Repeat 30 x total

Final elongation (2 min) (72 C)
Cooling (4 C) (infinitive)

61
Q

PCR Product purification

A

Column Purification

62
Q

Column purification

A

DNA charged
Bind to filter

dNTP too small to bind
Very large fragments don’t bind either

Add water or buffer releases purified DNA

63
Q

PCR
What fragment is copied

A

16 s rRNA gene
V3 to V4
550 base pairs

64
Q

Phred score

A

Chance that a base in the sequence is incorrect

65
Q

Eliminate listeria

A

Pasteurization
Irradiation

66
Q

Why not use LMP + esculin/Fe3+
instead of PALCAM

A

Expensive
Hard to read

67
Q

MRS plate

A

Favor lactobacilli

Ammonium citrate
Sodium acetate

68
Q

BHI Plate

A

Brain heart infusion

Non selective

Yeast, mold, bacteria

69
Q

Nucleic acid sequencing

A

Allows the researcher to determine the exact order of nucleotides that are present in a DNA or RNA molecule

70
Q

Sanger ideal for

A

Small-scale identification of microbes that can be isolated from any source

71
Q

Purpose of microwaving: PCR

A

Break open the cell and release the DNA template

72
Q

PCR Mix

A

25 uL 2x mastermix
10 uL forward primer
10 uL reverse primer
5 uL water

73
Q

Purpose of gel electrophoresis

A

Verify whether your reaction yielded a single amplicon, a clean amplicon (no smear), and an amplicon of the desired size

74
Q

Gel Loading Buffer

A

Contains a mixture of dye and glycerol

75
Q

Purpose of dye in gel electrophoresis

A

Visualize the sample when loading the well
Track the DNA while running the gel

76
Q

Purpose of glycerol/sucrose in gel electrophoresis

A

makes DNA sample heavy so it sinks to the bottom of the well

77
Q

DNA ladder

A

Contains fragments of known sizes

78
Q

Gel speed

A

Smaller segments move faster than larger ones

79
Q

DNA running buffer

A

TAE
Tris Acetate ADTA

80
Q

Listeria
Blood Agar + Catalase

A

Catalase +
narrow zone of clearing (hemolytic)

81
Q

Identification of E coli (FDA)

A

Homogenate in PBS
Enrichment step
Plate onto MAC agar
Hybridization with specific gene probes for virulence genes

82
Q

E.coli EMB agar (agar color)

A

Acid –> metachromatic effect
Turns agar from green to purple

83
Q

Methyl Red control

A

Turns the broth yellow

84
Q

Primer

A

Short piece of DNA that is complementary to known DNA sequence

85
Q

Separation of fragments on a gel

A

DNA = negatively charged

Migrate towards positive end

Small fragments are faster than large ones

86
Q

Why do the first 30 bases have poor phred scores

A

Flourescently labelled fragments that are very small can’t be separated at high resolution in the gel matrix

The laser can’t determine the base call with high accuracy

87
Q

Purpose of microwaving DNA

A

Weaken the cell wall

Break open the cells

Denaturation

Release the DNA

88
Q

Loading Dye Ex

A

Orange G

Xylene Cyanol

Bromophenol Blue

Tartazine

Cresol Red

89
Q

Adjust the migration pattern by

A

Adjusting the concentration of agarose

90
Q

Small DNA fragment

Concentration of agarose should be

A

Relatively higher

91
Q

Piece of agarose gel prepared with

A

Agarose powder
EtBr
TAE buffer

92
Q

EtBr

A

Visable under UV

Intercalates btwn bases of ds DNA

Used to visualize DNA bands

Carcinogen

93
Q

Why is LST MUG presumptive for E. coli?

A

Other microbes can grow and cause fluorescence

94
Q

EtOH in purification

A

Maintains DNA
Dehydrates

95
Q

Working stock calculations

A

C1 V1 = C2 V2

(C = concentration)

96
Q

Opportunistic Pathogen

A

Only cause disease when the opportunity arises

97
Q

Ingredients that yield black halo

A

Esculin
Ferric ammonium citrate

98
Q

Internalin A

A

Protein on cell surface of Listeria

Play a key role in invasion of LM

99
Q

Virulence of LM

A

Invade intestinal cells
Hemolytic activity
Spread from cell to cell

100
Q

Listeria on TASYE

A

Smooth, convex, white colonies

101
Q

BSDA Microbe

A

Bacillus stearothermophilus

Lactococcus lactis used in class

Micro Lab (4 decks)

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