Module 8

Question 1

What is the catalase test useful in?

Answer 1

Differentiates Staph from Strep

Myobacterium species ID

Anaerobe ID

Question 2

What is a positive test for catalase?

Answer 2

Bubble formation

Question 3

What percentage of H2O2 is used?

Answer 3

3% from 30%

Question 4

How is H2O2 stored and why?

Answer 4

Brown/amber bottle to prevent breakdown by light.

Question 5

What is the rapid tube method catalase test?

Answer 5

Negative control- stick in H2O2

Pick colonies and immerse in tube with 0.5mL

Observe for bubbles

Question 6

What is the rapid slide catalase test?

Answer 6

Negative control

Place drop of reagent on slide

Pick up colonies and immerse

Observe for bubbles

Question 7

What is the overnight culture catalase method?

Answer 7

Pour 1mL reagent over 24hr growth on plate

Observe for bubbles

Question 8

Why should platinum wire not be used for catalase tests?

Answer 8

May cause H2O2 breakdown, false positive reaction.

Question 9

Why can blood alter catalase results?

Answer 9

It contains catalase and can therefore cause false positives.

If blood agar is picked up the results can be false.

Question 10

Why should old cultures not be used for catalase testing?

Answer 10

Can lose their catalase catalase activity, gives false negatives.

Question 11

What are the controls for the catalase test?

Answer 11

Positive- any Staph specie

Negative- any Strep species or stick

Question 12

What does the nitrate reduction test determine?

Answer 12

Whether an organism to reduce the nitrate.

Previously used as a Staph genus test.

Question 13

What can the nitrate reduction test ID?

Answer 13

Enteriobacteriaceae

Nonfermentative gram negative bacilli

Separation of Staph and Strep

Anaerobes

Question 14

How is the nitrate reduction test complicated?

Answer 14

A variety of end products can be produced, detection can be difficult.

Question 15

What are the end products of the nitrate reduction test? How are they detected?

Answer 15

Nitrogen gas- accumulates in Durham tube (not Staph)

Add reagents if no gas formed

Nitrite- red colour, Staph species

No red, add Zn

Unreduced nitrate to nitrite- red colour

Other endproducts- no red colour, not Staph

Question 16

What medium is used for the nitrate reduction test?

Answer 16

Nitrate medium- beef extract, peptone, potassium nitrate

Question 17

What reagents are used in nitrate reduction tests?

Answer 17

A- sulfanic acid in acetic acid

B- alpha naphthylamine in acetic acid

Zn

Question 18

What quality control is performed in the nitrate reduction test?

Answer 18

Medium tested for the presence of nitrate- add A, B and Zn, if red colour nitrate is in medium

Positive control- S. aureus

Negative control- Strep species

Question 19

What does the coagulase test detect?

Answer 19

The presence of catalase enzyme.

Question 20

What are the different types of coagulase enzyme?

Answer 20

Bound/clumping factor- part of cell wall, acts on fibrinogen to form fibrin, detected with slide test

Free- extra cellular enzyme, produces thrombin-like substance, converted to fibrin clot, detected with tube test

Question 21

What is the reagent for coagulase tests?

Answer 21

Lyophylized human or rabbit plasma.

Question 22

What anticoagulant is used for coagulase tests?

Answer 22

Heparin or EDTA

Question 23

Why is citrate not used for coagulase tests?

Answer 23

Some bacteria use citrate.

Can give false positive results.

Question 24

How is the slide coagulase test performed?

Answer 24

Drop of saline on slide

Emulsify several colonies

Stir in loop full of plasma

Observe for clumping or agglutination

Question 25

Why should a weak bacterial suspension not be used for coagulation tests?

Answer 25

Can give false negatives.

Question 26

How is the tube coagulase test performed?

Answer 26

0.5mL of plasma in tube Emulsify colony portion Incubate at 35°C for 4hrs If negative incubate at room temp for 24hrs Observe for full or partial clot

Question 27

What can alter the results of a coagulase test?

Answer 27

Temperature >35°C can prevent formation (some S. aureus)- false negative Frequent and excess agitation prevents formation- false negative >24hr incubation- S. aureus produces staphylokinase that can dissolve clots, false negative Serum- false positive

Question 28

What are the controls used for coagulase testing?

Answer 28

Positive- S. aureus Negative- S. epidermidis

Question 29

What does the DNase test detect?

Answer 29

Whether the bacteria produces enzymes that will depolymerize or unwind DNA.

Question 30

What are the applications of DNase testing?

Answer 30

S. aureus from other Staph Moraxella catarrhais from Neisseria species Serratia species from other Enterobacteriaceae Xanthomonas maltophilia from other nonfermentative gram negative rods

Question 31

What are the three indicator systems for DNase testing?

Answer 31

HCl acid Methyl green Toluidine blue

Question 32

How is HCl acid used to determine DNase presence?

Answer 32

Plate with DNA is inoculated and incubated overnight Flooded with HCl Positive- clear zones, oligonucleotides are soluble in HCl Negative- precipitate around growth, while DNA precipitated by the acid

Question 33

How is methyl green used to determine DNase presence?

Answer 33

Inoculate methyl green DNase agar Incubate Positive- clear zones, oligonucleotides make the solution colourless Negative- green up to growth, while DNA bound is green

Question 34

How is toluidine blue used to determine DNase presence?

Answer 34

Inoculate DNase agar Incubate overnight Flood with 0.1% toluidine blue dye Positive- pink colour in the presence of oligonucleotides Negative- blue colour in the presence of whole DNA

Question 35

What are the controls used for DNase tests?

Answer 35

Positive- S. aureus, Serratia marcescens Negative- S. epidermidis, E. coli

Question 36

What does the B-lactamase test detect?

Answer 36

The presence of B-lactamase enzymes which can inactivate penicillins and cephalosporins.

Question 37

Where is bacteria scraped from for B-lactamase testing?

Answer 37

The zone of inhibition.

Question 38

How can the production of B-lactamase be induced?

Answer 38

Growing the organism in the presence of a B-lactam antibiotic.

Question 39

What are the three methods for detection of B-lactamase?

Answer 39

Acidimetric method Iodometric method Chromogenic cephalosporin test

Question 40

How does the acidimetric method detect B-lactamase?

Answer 40

Hydrolysis of the B-lactam ring yields an acidic pH that can be detected. Growth is rubbed on a moistened strip of benzylpenicillin with indicator. Colour change indicates bacteria. 30 min.

Question 41

How does the iodometric method detect B-lactamase?

Answer 41

When penicillin is hydrolyzed to penicilloic acid iodine is converted to iodide, purple colour is lost. Filter paper soaked in antibiotic, starch and iodine has colonies rubbed on. Clear area indicates presence of the enzyme. 30 min.

Question 42

How does the chromogenic cephalosporin test detect B-lactamase?

Answer 42

Uses nitrocefin which changes from red to yellow when hydrolyzed. Strip/disc is moistened and bacterial colonies are added. Yellow to red indicates a positive test. 15 min. Discs are light sensitive.

Question 43

What does the catalase test detect?

Answer 43

Detects the catalase enzyme that causes the breakdown of H2O2. Genus test (Staph vs Strep).