Testing Methods

Question 1

Catalase Test

Answer 1

Utilizes 3% hydrogen peroxide to determine whether an organism contains the enzyme catalase (convert H2O2 to water and oxygen)
If present there is vigorous bubbling (Ex. Staph species)
If not present there is no bubbling (Ex. Strep species)

Bubbles after 20-30 seconds aren’t considered a positive reaction
Catalase from RBCs in blood agar may produce false positive

Question 2

Hippurate Hydrolysis

Answer 2

Detects hippuricase and is to differentiate groups of strep (Group B) as well as Gardnerella vaginalis and Campylobacter jejuni
Hippuric acid is hydrolyzed by hippuricase to form glycine and sodium benzoate
Utilizes a heavy suspension of organism and 1% aqueous Hippurate solution and is incubated for 2 hours
Ninhydrin is then added and complexes with the glycine and produces a purple color (positive)

Question 3

Indole

Answer 3

Utilizes an organisms ability to produce tryptophanase which converts to tryptophan and then to indole
Detected by the addition of Ehlich or Kovac’s reagent to a broth solution
If a red ring forms it is positive (Ex. E coli)
If no color change occurs it is negative (Ex. Klebsiella)

Question 4

Oxidase Test

Answer 4

Tetramethyl-para-phenylene-diamine dihydrochloride reacts with cytochrome C to produce a blue/purple color
Positive = purple (Pseudomonas)
Negative = no color change (E. coli)

Question 5

PYR Test

Question 6

ONPG Test

Answer 6

Tests for slow lactose fermentation
Detects the change from orthonitrophenol by beta-galactosidase
Positive = yellow (Citrobacter)
Negative = no color change (Salmonella)

Question 7

Methyl Red (MR) Test

Answer 7

Detects acid products formed when glucose is metabolized by mixed acid fermentation pathways
Color change caused by change in pH
Positive = red (duh) (E. coli)
Negative = no color change (K. pneumo)

Question 8

Voges-Proskauer (VP) Test

Answer 8

Detects acetoin production from alternate pathway for glucose metabolism with the addition of alpha-naphthol and KOH
Little acid is produced by the pathway so organisms that are VP positive are usually MR neg and vice versa
Positive = red (Klebsiella)
Negative = no color change (Citrobacter)

Question 9

LAP Test

Question 10

PAD Test

Question 11

Coagulase Test

Answer 11

Enzyme coagulase causes coagulation (tube test) or agglutination (slide test)
S. aureus pos, CONS neg
Slide test is a screening test that detects bound coagulase
If slide test is negative, tube test should be performed which tests for free coagulase
Largely replaced by latex agglutination tests

Question 12

Optochin disk

Answer 12

Zone of inhibition > or equal to 14 mm with a 6 mm disk or > or equal to 16 mm with a 10 mm disk
Contains ethylhydrocupreine hydrochloride
If zone is < 14 mm, sensitive to optochin and can be given presumptive identification of S. pneumoniae

Question 13

Bile solubility test

Answer 13

Bile salts cause lysis of some organisms

Question 14

Urease test

Answer 14

Detects the breakdown of urea, releasing ammonia and increasing the pH, causing a color change
Positive = pink (Proteus)
Negative = yellow (Salmonella)

Question 15

Citrate test

Answer 15

Detects an organisms ability to use citrate as a sole source of carbon
Increases the pH, causing a color change
Positive = blue (Citrobacter)
Negative = green (E. coli)

False negative can occur if cap is on too tight

Question 16

Bacitracin susceptibility

Answer 16

For the presumptive identification and differentiation of beta-hemolytic Group A streptococcus (S. pyogenes) from other beta-hemolytic streptococci
Also used to distinguish staph species (resistant) from micrococcus species (susceptible)

Bacitracin inhibits the synthesis of bacterial cell walls
Disk impregnated with a small amount of bacitracin is placed on agar allows for the antibiotic to diffuse throughout the plate
After incubation, the inoculated plates are examined for zone of inhibition surrounding the disks

Positive: any zone of inhibition greater than 10 mm (susceptible)
Negative: no zone of inhibition