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MBIO345 MB > Module 8 > Flashcards

Module 8 Flashcards

(53 cards)

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1
Q

Catalase test

A

Staphylococcus and streptococcus Genus test
detects the enzyme catalase which causes breakdown of H2O2 into water and oxygen
Bubbles of oxygen = positive test
Nichrome wire or wooden sticks used

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2
Q

3 groups of bacteria that Catalase test is useful for

A

Differentiating Staphylococci from Streptococci
Species identification of Mycobacterium
Identification of anaerobes

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3
Q

Reagent used in catalase test

A

3% hydrogen peroxide

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4
Q

What causes H2O2 to break down

A

exposure to light and warm temps

Do QC daily or per shift

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5
Q

What happens if decomposed H2O2 is used for catalase test

A

Tests would be falsely negative as the reagent is actually just water

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6
Q

3 methods of the Catalase test

A

Rapid tube method
Rapid slide test
Overnight culture method

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7
Q

Catalase test: Rapid tube method

A

Control: Dip wire/stick into H2O2 and observe for bubbles
Should show no bubbles = used as negative control

Pick up portion of colony, immerse in small test tube with H2O2
Positive test = bubbles form
Negative test = no bubbles

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8
Q

Catalase test: Rapid Slide Test method

A

Control: sterile stick for negative control

Place drop of H2O2 on glass slide
Pick up small portion of colony on stick and immerse in drop
Pos test = bubbles
neg test = no bubbles

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9
Q

Catalase test: Overnight culture method

A 
Pour 1mL of H2O2 reagent over 24hr growth on a nutrient agar slant or plate
Observe for bubbles
Bubbles = pos test
no bubbles = neg test
May also be done with 24hr broth culture
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10
Q

Use of platinum wires in catalase test

A

DO NOT USE
PLAT = BAD
platinum may cause breakdown of H2O2 with formation of bubbles (false positive result)

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11
Q

Why to be careful when taking colonies off blood agar plates for catalase test

A

blood contains the enzymes catalase
If you accidentally scrape the plate when picking up a colony, the H2O2 may react with the agar to produce a FALSE POSITIVE
This can be avoided by using a chocolate agar plate

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12
Q

How old should cultures be when doing a catalase test

A

incubated for 18-24hrs

Older cultures may lose catalase activity resulting in FALSE NEGATIVE

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13
Q

Controls for Catalase test

A

Positive: Any staphylococcus species
Negative: Any streptococcus species from blood agar

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14
Q

Nitrate Reduction test

A

determines ability of an organism to reduce nitrate
Many different forms of reduced nitrogen, therefore the medium is tested for UNREDUCED nitrate
If unreduced nitrate is found in the medium, nitrate reduction has NOT taken place and the test result is NEGATIVE

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15
Q

4 groups that the Nitrate Reduction test is useful for

A

Enterobacteriaceae
non fermentative gram negative bacilli
Separation of streptococci and staphylococci
anaerobes

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16
Q

Durham tube

A

“upside-down barbie test tube”

Nitrogen gas collects in tube in Nitrate reduction test

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17
Q

2 reagents required for Nitrate Reduction test

A

Reagent A: sulfanilic acid in acetic acid

Reagent B: alpha naphthylamine in acetic acid (stored in brown bottle/fridge)

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18
Q

End products for Nitrate reduction test

A

test medium inoculated/incubated for 18-24hrs
Checked for growth (cloudy) and for gas in tube
Gas in tube is assumed to be nitrogen gas and test is POS
No gas in tube? equal amounts of reagent A and B are added
Red color = presence of nitrite in the medium (POS RESULT FOR STAPH)
No red color after adding A and B?
Add zinc dust
Red color with zinc dust indicated unreduced nitrate remains in the medium = NEG RESULT (zinc reduces the unreduced nitrate present to nitrite which then reacts with reagents A and B that were previously added)

No color after zinc? Nitrate has been reduced beyond nitrite to one of the other end products = POS RESULTS

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19
Q

Quality control for Nitrate reduction test

A 
Pos = Staphylococcus aureus (nitrite endproduct)
Neg= Streptococcus species
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20
Q

Coagulase Test

A

Tests for bacterial enzyme, coagulase

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21
Q

2 forms of coagulase

A

Bound coagulase or clumping factor

Free coagulase

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22
Q

Bound coagulase

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A 
AKA clumping factor
Attached to bacterial cell wall
Not found in extracellular fluid
Acts of fibrinogen in plasma to form fibrin
Detected in the SLIDE coagulase test

clumping = pos test

23
Q

Free coagulase

Study These Flashcards
A

extracellular enzyme found in fluids and culture filtrates
Produces a thrombin-like substance from plasma which then converts into a fibrin clot
Detected in the TUBE coagulase test

24
Q

Reagents used in coagulase test

Study These Flashcards
A 
Human or rabbit plasma
Rabbit does not need to be screened for hep/HIV
Lyophilized form
Refrigerated
NOT BROKEN DOWN BY LIGHT
25
Anticoagulant used in coagulase test
heparin EDTA Not sodium citrate (contaminating bacteria may use it as a nutrient causing false clotting in tube = false positive)
26
Slide coagulase test procedure
for bound coagulase Place drop of saline on slide Emulsify several colonies of Staphylococci to give THICK emulsion Stir in loop of plasma and examine for macroscopic clumping or agglutination
27
Autoagglutination in coagulase test
when bacteria clump with sterile saline before plasma is added If this happens, slide coagulase cannot be performed and a tube coagulase is required
28
Most common error in performing slide coagulase
making too weak of a bacterial suspension | Will give false negative result
29
Tube coagulase test procedure
0.5ml plasma in small tube (may be diluted 1:4 for test) Emulsify a portion of a colony in the plasma Incubate at 35C for 4 hours If negative, incubate at RT for 24hrs and examine Formation of a partial or complete clot = positive
30
Incubation temp for tube coagulase
incubate at 35C | Temps over 35C may prevent clot formation in some strains of S. aureus = false negative tube coagulase
31
Excess agitation in coagulase incubation =
prevent clot formation | False negative
32
Over incubation in coagulase test
Full 24hrs required for clot to form | BUT Incubation should not exceed this time
33
Staphylokinase
produced by S. aureus that may cause clot dissociation on prolonged incubating Causes false negative
34
Use of citrated plasma in coagulase test
DO NOT use bacteria use citrate was nutrient which may use up the anticoagulant causing the plasma to clot and a false positive test result * staphylococci do not use citrate but other bacteria may
35
Use of serum in coagulase test
DO NOT use serum is plasma minus fibrinogen use will cause false neg/no clot formation
36
QC for coagulase test
``` Positive = Staphylococcus aureus Negative = Staphylococcus epidermidis ```
37
DNase test
Determines if bacteria produce enzymes that will depolymerize (unwind) DNA *virulence factor for bacteria
38
DNase test clinical applications
Differentiate S. aureus from other Staphylococci Differentiate Moraxella catarrhalis from Neisseria species Differentiate Serrate species from other Enterbacteriaceae Differentiate Xanthomonas maltophilia from other non-fermentative gram neg rods
39
Oligonucleotides
final product after DNase from bacteria has unwound DNA in the host cell
40
3 ways to test for DNase
Hydrochloric acid Methyl Green Toluidine Blue
41
Hydrochloric acid for DNase test
Inoculate a plate containing DNA with he bacterium in question Incubate overnight, then flood plate with HCl Whole/intact DNA is precipitated by HCl = cloudy appearance = Negative DNase Oligonucleotides are insoluble in HCl = clearing around bacterial growth = positive DNase
42
Methyl Green for DNase test
Inoculate Methyl Green Dnase agar with bacterium Incubate and observe for clear areas around colonies ``` Green = whole/intact DNA = Negative DNase Colorless = oligonucleotides present = positive DNase ``` Store plates in dark
43
Toluidine Blue for DNase test
Inoculate DNase agar with bacterium and incubate overnight Flood plate with 0.1% Toluidine blue dye after growth Blue= whole/intact DNA = Negative DNase Pink = oligonucleotides = Positive DNase
44
QC for DNase test
``` Pos = S. aureus, Serratia marcescens Neg = S. epidermidis, E. coli ```
45
Why is toluidine blue NOT incorporated into medium
it may inhibit growth of some bacteria
46
Beta-Lactamase Test
detect presence of beta-lactamase enzymes that inactivate certain beta-lactam antibiotics *considered virulence factor
47
2 groups of antibiotics with beta-lactam rings
penicillins and cephalosporins *Only those with EXPOSED B-lactam rings are susceptible to b-lactamase These include: Pen G (benzylpenicillin), Ampicillin, Carbenicillin and a few cephalosporins
48
Induced B-bactamase
Enzymes in bacteria can be induced by growing up organism in the presence of b-lactam antibiotics (eg. oxacillin disc) Bacterial growth taken from edge of disc and tested with B-lactamase reagent
49
3 methods of testing for B-lactamase
Acidimetric method Idometric method Chromogenic Cephalosporin test
50
Acidimetric Method for B-lactamase
Rub bacterial growth on a moistened strip (combined strip with benzylpenicillin and a pH indicator) and observe for color change indicating acid Can take up to 30 min Hydrolysis of b-lactam ring by b-lactamase yields an acid pH
51
Iodometric Method for B-lactamase
Uses combo of penicillin, starch and iodine When penicillin is hydrolyzed to penicilloic acid by B-bactamase, the acid converts iodine to iodide with a subsequent loss of purple color Purple + Bacterical B-lactamase enzyme = no purple (pos result) Performed by preparing filter paper strips soaked in benzylpenicillin and starch Iodine added to give deep purple color Test colonies rubbed on strip and clear area indicated B-lactamase Up to 30 min for reaction
52
Chromogenic Cephalosporin Test for B-lactamase
NITROCEFIN - cephalosporin with b-lactam ring that is hydrolyzed by b-lactamse Yellow in color but RED when hydrolyzed ``` Commercial strip is moistened and colonies are rubbed on the surface RED = POS YELLOW = NEG Up to 15 min Nitrocefin kept away from light Store in drawer while waiting the 15 min ```
53
Most sensitive b-lactamase test method
Chromogenic cephalosporin test | uses Nitrocefin

MBIO345 MB (13 decks)

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