Module 8

Question 1

What is the catalase test used to identify?

Answer 1

Differentiates Staph from Strep
ID species of Mycobacterium
ID anaerobes

Question 2

What does catalase do?

Answer 2

Causes the breakdown of hydrogen peroxide (H2O2) to water and oxygen, forming bubbles

Question 3

What concentration of hydrogen peroxide do we use in clinical testing?

Answer 3

3%

Question 4

3 methods of catalase testing

Answer 4

Rapid tube method
Rapid slide test
Overnight culture method

Question 5

Procedure for overnight culture method to test for catalase

Answer 5

1. Pour approx. 1mL of H2O2 over 24hr growth on a nutrient agar plate
2. Set the plate in an inclined position and observe for bubbles

Question 6

Presence of bubbles in a catalase test indicates:

Answer 6

A positive test (catalase enzyme is present)

Question 7

What can cause false positive catalase test results?

Answer 7

Using a wire that contains platinum
Scraping the BA plate when picking up colonies
Reagent spontaneously breaking down

Question 8

What can cause false negative catalase test results?

Answer 8

Reagent that is already broken down

Using a culture older than 24hrs

Question 9

Positive control for catalase test

Answer 9

Any Staphylococcus species

Question 10

Negative control for catalase test

Answer 10

Any Streptococcus species from blood agar

Question 11

What can the nitrate reduction test be used to identify?

Answer 11

Enterobacteriaceae
Non-fermentative Gram negative bacilli
Anaerobes
Differentiates between Strep and Staph

Question 12

Durham tube

Answer 12

Gas collection device
Used in nitrate reduction test
Small inverted tube

Question 13

Nitrate reagent A

Answer 13

Sulfanilic acid in acetic acid

Question 14

Nitrate reagent B

Answer 14

Alpha naphthylamine in acetic acid

Question 15

Bound coagulase / clumping factor

Answer 15

Attached to bacterial cell wall and not found in extracellular fluid
Acts on fibrinogen in plasma to form fibrin
Detected in the slide coagulase test

Question 16

Free coagulase

Answer 16

Extracellular enzyme found in fluids and culture filtrates
Produces a thrombin-like substance from plasma which is then converted to a fibrin clot
Detected in the rube coagulase test

Question 17

Reagent in coagulase test

Answer 17

Human or rabbit plasma (usually rabbit plasma)

Question 18

Positive control for coagulase test

Answer 18

Staphylococcus aureus

Question 19

Negative control for coagulase test

Answer 19

Staphylococcus epidermidis

Question 20

DNase test

Answer 20

Determines if bacteria produce enzymes that unwind DNA

Question 21

Applications of DNase test

Answer 21

Differentiate S. aureus from other Staph
Differentiate Moraxella catarrhalis from Neisseria species
Differentiate Serratia species from other Enterobacteriaceae
Differentiate Xanthomonas maltophilia from other non-fermentative GN rods

Question 22

Positive controls for DNase test

Answer 22

Staph aureus, Serratia marcescens

Question 23

Negative controls for DNase test

Answer 23

Staph epidermidis, E. coli

Question 24

3 indicator systems for DNase

Answer 24

Hydrochloric acid
Methyl green
Toluidine blue

Question 25

DNase test: hydrochloric acid system

Answer 25

Inoculate a plate containing DNA (deoxyribonuclease agar) with bacterium in question Incubate overnight, then flood plate with HCl. Whole/intact DNA is precipitated by HCl = cloudy appearance = negative DNase Oligonucleotides are soluble in HCl = clearing around bacterial growth = positive DNase

Question 26

DNase test: Methyl green system

Answer 26

Inoculate Methyl Green Dnase agar with bacterium. Incubate and observe for clear areas around colonies. Methyl green is incorporated into the media and a clearing around bacterial growth indicates positive DNase reaction Methyl green is green when bound to whole/intact DNA = negative DNase Methyl green is colorless when oligonucleotides are present = positive DNase

Question 27

DNase test: Toluidine blue system

Answer 27

Inoculate DNase agar with bacterium and incubate overnight. Flood plate with 0.1% Toluidine blue dye after growth blue color indicates whole/intact DNA = negative result pink color indicates oligonucleotides present = positive

Question 28

Induced beta-lactamase test

Answer 28

Grow organism up in the presence of beta-lactam antibiotics (Oxacillin or Methicillin) Scrape growth from edge of zone of inhibition Test with beta lactamase reagent

Question 29

Beta-lactamase test: acidimetric method

Answer 29

Presence of beta lactamase enzyme hydrolyzes beta lactam ring causing acid production pH change is detected by indicator Rub bacterial growth on moistened strip and observe color change Observe for color for up to 30 minutes

Question 30

Beta-lactamase test: iodometric method

Answer 30

Penicillin, starch and iodine are used Pen G-Starch strip is purple Iodine is added, result is a dark purple color When Pen is hydrolyzed to Penicilloic acid by beta lactamase, iodine is also converted to iodide = loss of purple color = positive beta lactamase result Procedure: Rub colonies on iodine soaked filter paper Purple = negative Colorless = positive Read for 30 minutes

Question 31

Beta-lactamase test: chromogenic cephalosporin method

Answer 31

**Nitrocefin is a cephalosporin that is hydrolyzed by beta lactamase Rub bacteria on moistened strip or disc and observe for color change – up to 15 minutes Intact Nitrocefin is yellow = negative test result Hydrolyzed form is red = positive test result

Question 32

Nitrate reduction test medium

Answer 32

Contains beef extract, peptone, and potassium nitrate dissolved in water

Question 33

Result of nitrate reduction test if Staphylococcus

Answer 33

Nitrate broth showing growth and no gas in Durham tube, and a red color after addition of reagents A and B Nitrate has been reduced to nitrite