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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
Listeria plating order
Enrichment broth (selective) PALCAM (black halo) TASYE (purify)
26
Listeria Blood Agar results (if pathogenic)
Narrow zone of clearing around colonies Hemolytic
27
Antibiotics select for
Bacteria that have acquired mutations/genes that confer antibiotic resistance
28
Beta lactamase is
An enzyme that some bacteria produce that renders them resistant to beta lactam antibiotics
29
Sulfonamides
A group of antibiotics
30
Lactic ring has
1 x N and 3 x C
31
Organisms used as standard test for antibiotic residues
Bacillus stearothermophilus Lactococcus lactis
32
Delvo P Result: purple medium
An antibiotic is present, no growth of B. stearothermophilus will occur
33
LST MUG Assay
Based on presence of GUD GUD cleave MUG Creates MU (fluorescent) Identify coliforms by gas production from lactose
34
Methyl Red
1-2 drops of methyl red MR VP broth
35
Methyl Red: Red color
Mixed acid fermentation pH < 4.4
36
Methyl Red: Yellow color
Butanediol fermentation pH > 6.2
37
Methyl Red: Orange color
equivocal reaction pH 4.4 - 6.2
38
VP Test
Detect presence of acetoin α-naphthol reagent KOH MR VP broth
39
VP: red color
Positive reaction Presence of acetoin (neutral end product)
40
Indole test
Kovac's reagent Tryptone broth Determine the ability of an organism to split tryptophan to form indole
41
Indole test: Red ring
Positive result for indole
42
Simmon citrate
Citrate agar Determine if citrate is used as a carbon and energy source
43
Simmons citrate: blue color
Positive for citrate utilization Alkaline reaction turns agar blue due to removal of citrate
44
What does IMVic Stand for
Methyl Red Indole VP Simmons citrate
45
E.coli IMViC results Biotype 1
Indole + MR + VP - Simmons -
46
E.coli IMViC results Biotype 2
Biotype 2: Indole - MR + VP - Simmons -
47
What does GUD stand for
β-glucouronidase
48
What does MU stand for
4-methylumbelliferone
49
E.coli characteristics
Gram - Rod Facultative Ferments lactose Non fastidious
50
E.coli on EMB agar
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
EMB agents
Eosin Methylene Blue Inhibit growth of gram +
52
API strip test
Rapid test Fast way to inoculate Easy Expensive
53
E.coli fermentation products
Ethanol CO2 H2 Lactic acid Acetic acid Succinic acid Formic acid
54
Acids produced from E.coli glucose fermentation
Lactic Succinic Acetic Formic
55
E.coli produces per mol of glucose
4 mol acidic products 1 mol EtOH 1 mol CO2 1 mol H2
56
ETEC
Enterotoxigenic E. coli Traveler’s diarrhea High infectious dose Outbreaks rare
57
EPEC
Enteropathogenic E. Coli Infant diarrhea Poor sanitation practices
58
EIEC
Enteroinvasive E. coli Dysentery Low infectious dose Fecally contaminated foods Cramps, diarrhea Self limiting
59
EHEC
Enterohemorrhagic E. coli Low infectious dose Shiga toxins Intestinal disease HUS Renal failure
60
PCR Steps
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
PCR Product purification
Column Purification
62
Column purification
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
PCR What fragment is copied
16 s rRNA gene V3 to V4 550 base pairs
64
Phred score
Chance that a base in the sequence is incorrect
65
Eliminate listeria
Pasteurization Irradiation
66
Why not use LMP + esculin/Fe3+ instead of PALCAM
Expensive Hard to read
67
MRS plate
Favor lactobacilli Ammonium citrate Sodium acetate
68
BHI Plate
Brain heart infusion Non selective Yeast, mold, bacteria
69
Nucleic acid sequencing
Allows the researcher to determine the exact order of nucleotides that are present in a DNA or RNA molecule
70
Sanger ideal for
Small-scale identification of microbes that can be isolated from any source
71
Purpose of microwaving: PCR
Break open the cell and release the DNA template
72
PCR Mix
25 uL 2x mastermix 10 uL forward primer 10 uL reverse primer 5 uL water
73
Purpose of gel electrophoresis
Verify whether your reaction yielded a single amplicon, a clean amplicon (no smear), and an amplicon of the desired size
74
Gel Loading Buffer
Contains a mixture of dye and glycerol
75
Purpose of dye in gel electrophoresis
Visualize the sample when loading the well Track the DNA while running the gel
76
Purpose of glycerol/sucrose in gel electrophoresis
makes DNA sample heavy so it sinks to the bottom of the well
77
DNA ladder
Contains fragments of known sizes
78
Gel speed
Smaller segments move faster than larger ones
79
DNA running buffer
TAE Tris Acetate ADTA
80
Listeria Blood Agar + Catalase
Catalase + narrow zone of clearing (hemolytic)
81
Identification of E coli (FDA)
Homogenate in PBS Enrichment step Plate onto MAC agar Hybridization with specific gene probes for virulence genes
82
E.coli EMB agar (agar color)
Acid --> metachromatic effect Turns agar from green to purple
83
Methyl Red control
Turns the broth yellow
84
Primer
Short piece of DNA that is complementary to known DNA sequence
85
Separation of fragments on a gel
DNA = negatively charged Migrate towards positive end Small fragments are faster than large ones
86
Why do the first 30 bases have poor phred scores
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
Purpose of microwaving DNA
Weaken the cell wall Break open the cells Denaturation Release the DNA
88
Loading Dye Ex
Orange G Xylene Cyanol Bromophenol Blue Tartazine Cresol Red
89
Adjust the migration pattern by
Adjusting the concentration of agarose
90
Small DNA fragment Concentration of agarose should be
Relatively higher
91
Piece of agarose gel prepared with
Agarose powder EtBr TAE buffer
92
EtBr
Visable under UV Intercalates btwn bases of ds DNA Used to visualize DNA bands Carcinogen
93
Why is LST MUG presumptive for E. coli?
Other microbes can grow and cause fluorescence
94
EtOH in purification
Maintains DNA Dehydrates
95
Working stock calculations
C1 V1 = C2 V2 (C = concentration)
96
Opportunistic Pathogen
Only cause disease when the opportunity arises
97
Ingredients that yield black halo
Esculin Ferric ammonium citrate
98
Internalin A
Protein on cell surface of Listeria Play a key role in invasion of LM
99
Virulence of LM
Invade intestinal cells Hemolytic activity Spread from cell to cell
100
Listeria on TASYE
Smooth, convex, white colonies
101
BSDA Microbe
Bacillus stearothermophilus Lactococcus lactis used in class

Micro Lab (4 decks)

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