Methods Flashcards
ALA Disk Test
(Identification of Pathogenic Haemophilus)
Disk contains delta-amino levulenic acid
Converted to porphobilinogen and porphyrins if the organism contains the enzyme to make hemin
Bacitracin Susceptibility Disk Test
(Identification of Streptococcus pyogenes)
Susceptibility to low concentrations of polypeptide antibiotic
Beta-Lactamase Disk Test
(Cefinase Disk Method)
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
Bile Solubility
(Identification of Streptococcus pneumoniae)
10% sodium desoxycholate reagent
Colonies lyse in presence of sodium desoxycholate
Autolytic enzyme that cleaves bonds and cell wall peptidoglycan
Bile Esculin Hydrolysis
(Identificaion of Enterococci and Group D Streptococci)
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
CAMP Test
(Identification of S. agalactiae, L. moncytogenes)
Beta-hemolytic activity of Staphylococcus (ATCC 25293) is enhanced by extracellular protein B
Catalase Test
Enzyme that decomposes Hydrogen Peroxide into O2 and H2O
Hemoprotein with similar structure similar to Heme but with Iron in the Fe3+
Catarrhalis Test
(Identification of Moraxella catarrhalis)
Enzyme butyrate esterase acts on disk substrate bromo-chloro-indolyl butyrate
Forms indigo in presence of O2
Citrate Agar
(Identification of Enteric GNRs)
Organism utilizes ammonium dihydrogen phosphate and sodium citrate as sole sources of nitrogen and carbon
Coagulase Test
Uses lyophilized rabbit plasma
CTA Sugars
(Cystine Trypticase Agar)
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
DNAse Test Agar
Agar with toluidine blue, dye forms a complex with intact DNA
DNAse activity depolymerizes the DNA, breaking down the dye-DNA complex
Germ Tube Test
Represents initiation of hypha directly from yeast cell
LIM or Carrot Broth
Enhances Beta hemolysis on Group B Streptococcus
Hippurate Hydrolysis Disk Method
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
Hippurate Hydrolysis Tube Method
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
H-Quad
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
Indole Test
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
Leucine Amino Peptidase (LAP) Test
(Identification of Streptococcus and Related organisms)
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
Methyl Red
Organisms produce high levels of acid during fermentation of dextrose, overcome phosphate buffer system and produce red color
Voges-Proskauer Test
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
Microdase Disk Test
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)
Nitrate Broth
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
Optochin Susceptibility (P Disk)
Eythihydrocupreine hydropchloride - quinine derivative
Selectively inhibits S. pneumoniae
