Methods

Question 1

ALA Disk Test
(Identification of Pathogenic Haemophilus)

Answer 1

Disk contains delta-amino levulenic acid
Converted to porphobilinogen and porphyrins if the organism contains the enzyme to make hemin

Question 2

Bacitracin Susceptibility Disk Test
(Identification of Streptococcus pyogenes)

Answer 2

Susceptibility to low concentrations of polypeptide antibiotic

Question 3

Beta-Lactamase Disk Test
(Cefinase Disk Method)

Answer 3

Detection of Penicillin Resistance
Bacterial enzymes that cleave the Beta-lactam ring of Penicllins and Cephalosporins to inactivate the antibiotic

Cefinase disk contains chromogenic cephalosporin, nitrocefin

Question 4

Bile Solubility
(Identification of Streptococcus pneumoniae)

Answer 4

10% sodium desoxycholate reagent
Colonies lyse in presence of sodium desoxycholate
Autolytic enzyme that cleaves bonds and cell wall peptidoglycan

Question 5

Bile Esculin Hydrolysis
(Identificaion of Enterococci and Group D Streptococci)

Answer 5

Medium contains Esculin, ferric Citrate
Esculin is hydrolyzed by Enterococci and Group D Streptococci to form destrose and esculetin
Esculetin reacts with ferric ions contained in medium

Question 6

CAMP Test
(Identification of S. agalactiae, L. moncytogenes)

Answer 6

Beta-hemolytic activity of Staphylococcus (ATCC 25293) is enhanced by extracellular protein B

Question 7

Catalase Test

Answer 7

Enzyme that decomposes Hydrogen Peroxide into O2 and H2O
Hemoprotein with similar structure similar to Heme but with Iron in the Fe3+

Question 8

Catarrhalis Test
(Identification of Moraxella catarrhalis)

Answer 8

Enzyme butyrate esterase acts on disk substrate bromo-chloro-indolyl butyrate
Forms indigo in presence of O2

Question 9

Citrate Agar
(Identification of Enteric GNRs)

Answer 9

Organism utilizes ammonium dihydrogen phosphate and sodium citrate as sole sources of nitrogen and carbon

Question 10

Coagulase Test

Answer 10

Uses lyophilized rabbit plasma

Question 11

CTA Sugars
(Cystine Trypticase Agar)

Answer 11

Organism metabolizes carbohydrate = organic acids are produced and medium becomes acidified
Peptone in medium also degraded by bacteria

No fermentable carbohydrate is present = most cultures show alkaline shift

Phenol red indicator changes from reddish-orange to yellow when amount of acid produced by carbohydrate fermentation is greater than alkaline products of peptone degradation

Question 12

DNAse Test Agar

Answer 12

Agar with toluidine blue, dye forms a complex with intact DNA
DNAse activity depolymerizes the DNA, breaking down the dye-DNA complex

Question 13

Germ Tube Test

Answer 13

Represents initiation of hypha directly from yeast cell

Question 14

LIM or Carrot Broth

Answer 14

Enhances Beta hemolysis on Group B Streptococcus

Question 15

Hippurate Hydrolysis Disk Method

Answer 15

Ninhydrin reagent
Hippuric acid is hydrolyzed by enzyme hippuricase to glycine and benzoic acid
Ninhydrin evokes a five step reaction beginning with deamination of glycine to form hydrindantin, CO2, and Ammonia
Ammonia and residual ninhydrin reacts with hydrindantin to produce purple color

Question 16

Hippurate Hydrolysis Tube Method

Answer 16

Enzyme Hippuricase hydrolyzes sodium hippurate present in broth culture medium to form bezoic acid and glycine
Addition of ferric chloride results in ferric ion combining with benzoate to form insoluble precipitate, ferric benzoate

Question 17

H-Quad

Answer 17

Growth Factor Detection
Growth Factor from Blood = X factor or hemin
Factor supplied by S. aureus = V factor or Nicotinamide adenine dinucleotide (NAD)

Quad 1: Hemin (X Factor)
Quad 2: NAD (V Factor)
Quad 3: Both Hemin and NAD factors
Quad 4: NAD + Horse Blood to demonstrate hemolysis

Question 18

Indole Test

Answer 18

Benzyl pyrrole is one of the metabolic degradtion products of amino acid tryptophan
Enzyme tryptophanase hydrolyses and deaminating tryptophan and production of indole, pyruvic acid, and ammonia

Tryptophan + Water ==== (Tryptophanase) ====> Indole + Pyruvate + Ammonium

Question 19

Leucine Amino Peptidase (LAP) Test
(Identification of Streptococcus and Related organisms)

Answer 19

Disk is infused with substrate leucine-B-naphthylamide hydrochloride

Enzyme leucine aminopeptidase which hydrolyzed to release leucine and B-naphthylamide
B-naphthylamide combines with cinnamaldehyde to turn pink

Question 20

Methyl Red

Answer 20

Organisms produce high levels of acid during fermentation of dextrose, overcome phosphate buffer system and produce red color

Question 21

Voges-Proskauer Test

Answer 21

Addition of Potassium hydroxide to cultures
Organisms produce a neutral product, acetoin (acetylmethylcarbinol) from fermentation of dextrose
Acetoin is oxidized in presence of O2 and alkali to produce a red color

Question 22

Microdase Disk Test

Answer 22

Detects Oxidase enzyme in Micrococcus
DMSO in Microdase disk provides solubility and stability against auto-oxidation and enhances permeability of tetramethyl-p-phenylenediamine (TMDP)

Presence of O2, oxidase enzyme reacts with TMPD and cytochrome c in organism to form a color compound (Indophenol)

Question 23

Nitrate Broth

Answer 23

Capability of organism to reduce nitrates to nitrites
Nitrate (NO3-) + 2e- + 2H ===> Nitrite (NO2) + H2O
Reagents:
Nitrate A (0.8% sulfanilic acid in 5N acetic acid)
Nitrate B (0.6% N,N-dimethy;-alpha-naphthylamine in 5N acetic acid)

Detect the presence of a catabolic end product

Addition of Nitrate C (Zinc dust) Detects the absence of remaining nitrate in medium

Question 24

Optochin Susceptibility (P Disk)

Answer 24

Eythihydrocupreine hydropchloride - quinine derivative
Selectively inhibits S. pneumoniae

Question 25

Oxidase Test

Answer 25

Enzyme indophenol oxidase oxidizes a redox dye which results in color change to dark purple or blue

Question 26

PDA

Answer 26

Penylalanine is an amino acid that upon deamination forms a keto acid, penylpyruvic acid
Test is positive with addition of 12% ferric chloride acidified with HCl

Question 27

PYR

Answer 27

Substrate is L-pyrrolindonyl-B-nephuthylamide
Compound is hydrolyzed by enzyme aminopeptidase and releases free B-naphthylamide and detected by addition of N,N-dimethylaminocinnamaldehyde which couples with naphthylamide

Question 28

Staphaurex

Answer 28

Protein A has specific affinity for F c moiety of IgG
Latex particles coated with fibrinogen and IgG

Question 29

Streptex

Answer 29

Latex particles coated with group specific antibodies
Group specific antigens - A, B, C, D, F, and G

Question 30

Superoxol Test

Answer 30

Uses 30% H2O2 to detect Neisseria gonorrhoea

Question 31

Urea

Answer 31

Organisms utilize urea, ammonia is produced. The media becomes alkaline, producing red-pink color