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MBIO345 MB > Module 8 > Flashcards

Module 8 Flashcards

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

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

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

A 
Human or rabbit plasma
Rabbit does not need to be screened for hep/HIV
Lyophilized form
Refrigerated
NOT BROKEN DOWN BY LIGHT
25
Q

Anticoagulant used in coagulase test

A

heparin
EDTA
Not sodium citrate (contaminating bacteria may use it as a nutrient causing false clotting in tube = false positive)

26
Q

Slide coagulase test procedure

A

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
Q

Autoagglutination in coagulase test

A

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
Q

Most common error in performing slide coagulase

A

making too weak of a bacterial suspension

Will give false negative result

29
Q

Tube coagulase test procedure

A

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
Q

Incubation temp for tube coagulase

A

incubate at 35C

Temps over 35C may prevent clot formation in some strains of S. aureus = false negative tube coagulase

31
Q

Excess agitation in coagulase incubation =

A

prevent clot formation

False negative

32
Q

Over incubation in coagulase test

A

Full 24hrs required for clot to form

BUT Incubation should not exceed this time

33
Q

Staphylokinase

A

produced by S. aureus that may cause clot dissociation on prolonged incubating
Causes false negative

34
Q

Use of citrated plasma in coagulase test

A

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
Q

Use of serum in coagulase test

A

DO NOT use
serum is plasma minus fibrinogen
use will cause false neg/no clot formation

36
Q

QC for coagulase test

A 
Positive = Staphylococcus aureus 
Negative = Staphylococcus epidermidis
37
Q

DNase test

A

Determines if bacteria produce enzymes that will depolymerize (unwind) DNA
*virulence factor for bacteria

38
Q

DNase test clinical applications

A

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
Q

Oligonucleotides

A

final product after DNase from bacteria has unwound DNA in the host cell

40
Q

3 ways to test for DNase

A

Hydrochloric acid
Methyl Green
Toluidine Blue

41
Q

Hydrochloric acid for DNase test

A

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
Q

Methyl Green for DNase test

A

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
Q

Toluidine Blue for DNase test

A

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
Q

QC for DNase test

A 
Pos = S. aureus, Serratia marcescens 
Neg = S. epidermidis, E. coli
45
Q

Why is toluidine blue NOT incorporated into medium

A

it may inhibit growth of some bacteria

46
Q

Beta-Lactamase Test

A

detect presence of beta-lactamase enzymes that inactivate certain beta-lactam antibiotics
*considered virulence factor

47
Q

2 groups of antibiotics with beta-lactam rings

A

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
Q

Induced B-bactamase

A

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
Q

3 methods of testing for B-lactamase

A

Acidimetric method
Idometric method
Chromogenic Cephalosporin test

50
Q

Acidimetric Method for B-lactamase

A

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
Q

Iodometric Method for B-lactamase

A

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
Q

Chromogenic Cephalosporin Test for B-lactamase

A

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
Q

Most sensitive b-lactamase test method

A

Chromogenic cephalosporin test

uses Nitrocefin

MBIO345 MB (13 decks)

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