Indicator System: Utilizes a double-indicator system involving two color-changing indicators:
Methyl Red: - Changes color from red to yellow within the pH range of 4 to 6.
Bromothymol Blue: - Changes color from yellow to blue within the pH range of 6 to 9.
pH Alteration:
No substances are added to alter the pH directly.
Bacterial growth can make urine alkaline by the conversion of urea to ammonia.
Protein Testing
Indicators Used: Different manufacturers may use:
Tetrabromophenol Blue
3',3",5',5"-Tetrachlorophenol
3,4,5,6-Tetrabromosulfonphthalein
Buffer: An acid buffer is utilized to maintain a constant pH level.
Color Change Reaction:
At a pH of 3, no protein exhibits yellow color.
As protein levels increase, the color transitions from yellow through shades of green to blue.
This reaction occurs as protein molecules accept hydrogen ions from the indicator.
Glucose Testing
Testing Pad Composition: Contains:
Glucose Oxidase
Peroxidase
Chromogen
Buffer
Enzyme Reaction:
A double sequential enzyme reaction is produced leading to color change.
Different chromogens such as:
Potassium Iodide: Changes from green to brown.
Tetramethylbenzidine: Changes from yellow to green.
Reaction Pathway:
Glucose oxidase catalyzes the reaction between glucose and oxygen yielding gluconic acid and peroxide.
Subsequently, peroxidase catalyzes the reaction between peroxide and chromogen, resulting in an oxidized colored compound.
Ketone Testing
Chemical Reaction Used: Utilizes sodium nitroprusside reaction for ketone detection.
Reaction Mechanism:
Acetoacetic acid reacts in an alkaline medium with sodium nitroprusside producing a purple color.
Composition of Ketones in Urine:
70% is beta-hydroxybutyric acid.
20% is acetoacetic acid.
2% is acetone.
Blood Testing
Detection Method: Employs pseudo-peroxidase activity of hemoglobin.
Reaction Details:
Catalyzes a reaction between the heme of hemoglobin/myoglobin and the chromogen tetramethylbenzidine.
Outcomes include:
Free hemoglobin/myoglobin: Color transitions from yellow to green and then to positive green-blue.
Intact red blood cells (RBCs) lysed on contact with the pad produce a speckled pattern.
Bilirubin Testing
Diazo Reaction: Utilizes a diazo reaction for bilirubin detection.
Chemical Reaction:
Bilirubin reacts with either:
2,4-Dichloroanaline diazonium salt
2,6-Dichlorobenzene-diazonium-tetrafluoroborate
This reaction occurs in an acidic medium, yielding an azo dye.
Color Outcomes: Colors typically range from tan or pink to violet depending on concentrations.
Urobilinogen Testing
Chemical Reaction Used: Ehrlich reaction or azo-coupling reaction is employed.
Ehrlich Reaction:
Urobilinogen reacts with p-dimethylaminobenzaldehyde producing colors from light to dark pink.
Azo-Coupling Reaction:
Involves 4-methoxybenzene-diazonium-tetrafluoroborate reacting with urobilinogen to yield colors from white to pink.
Limitations: Reagent strips are unable to detect the complete absence of urobilinogen, which may indicate biliary obstruction.
Nitrite Testing
Chemical Basis: Detects nitrite, a byproduct formed by certain gram-negative bacteria reducing nitrate to nitrite.
Detection Method: Greiss reaction.
At acidic pH, nitrites react with an aromatic amine producing a diazonium compound, which subsequently reacts with tetrahydrobenzoquinolin compounds to yield pink-colored azo dyes.
Leukocyte Testing
Testing Mechanism: Utilizes leukocyte esterase.
Reaction Summary:
Catalyzes the hydrolysis of an acid ester embedded in the pad to yield an aromatic compound and acid.
The resulting aromatic compound reacts with a diazonium salt on the pad creating a purple azo dye.
Specific Gravity (SPGR) Testing
Basis of Testing: Determined by changes in the pKa of a polyelectrolyte in alkaline media.
Mechanism of Action:
Polyelectrolyte ionizes, releasing hydrogen ions correlating to the ion concentration in solution.
Indicator Used: Bromothymol blue on the pad measures pH changes.
Color Transition: As SPGR increases, color shifts from blue (alkaline) through various shades of green to yellow (acidic).