URINE ANALYSIS

1.Formation of Urine
2.Normal constituents and composition 3.Collection of urine specimens 4.Preservation of urine 5.Physical examination of urine 6.Chemical Examination of Urine 7. Microscopic Examination Of Urine Deposits

 Urine --- most easily obtained Examination of urine Information--- about the functioning of
the kidney diagnosis ---of urinary tract diseases diagnosis -----of certain metabolic and systemic diseases.

 Urine is formed in the kidney. The functional unit of kidney is called nephrons, where in the ultrafilteration of plasma takes

place, followed by absorption of most of the water and some of the solutes. The kidneys through the nephrons ---excrete many of the waste products of the body

3 NORMAL CONSTITUENTS AND COMPOSITION OF URINE

Constituents A.Inorganic Chloride Phosphorous Sulfur Sodium Potassium Calcium Magnesium Iron B.Organic Urea Uric acid Creatinine Ammonia Sugar not detected by benedicts test Ketone bodies Carbonates, bicarbonates & free carbonic acid Mucin & mucin like substances Diastase grams/litre 9.0 2.0 1.5 4.0 2.0 0.2 0.2 003 25 0.6 1.5 0.6 trace trace trace

COLLECTION OF URINE SPECIMENS In. Improper collection---- may invalidate the results Containers for collection of urine should be wide mouthed, clean and dry First morning sample concentrated urine --chemical constituents casts and crystals Random specimen chemical screening microscopic examinations 24 hours urine sample quantitative estimation of proteins, sugars, electrolytes, and hormones. Urine specimen is collected in a clean 2 liters bottle with a stopper. The first morning sample is not collected. All the urine passed during the rest of the day and night and next day I st sample is collected. Volume is measured and immediately sent to the laboratory.

Midstream urine specimen

Urine should be collected in a clean, dry and,
preferably, sterilized container. urethral opening is cleaned with a moist cotton swab. first 10 25 ml of urine is not collected (discarded) since it contains urethral and prostatic secretions which may be required if investigating urethra and prostrate. First morning samples ---- these are the concentrated samples and casts, crystals etc .if present

 Terminal urine sample patient voids the last portion of

urine in an open container. Urine specimen collection using a catheter. This procedure is used for certain bacteriological tests Urine specimens from infants Urine can be collected into a plastic bag with an adhesive mouth. The bag is fixed around the genitalia and left in place for 1- 3 hours, depending on the examination requested. Colostomy bags can be used

PRESERVATION OF URINE
Urine sample < within 2 hours. If delay ----preservation. If more than 2 hours
Keep the sample in the refrigerator without any preservative. Toluene add a few drops till it forms a thin layer on the surface of urine. Conc.HCL 1 ml of conc.HCL for 125-150mlof urine. Formaldehyde -1 drop for 15 ml of urine. Cells and casts are well preserved.

PHYSICAL EXAMINATION
Volume
normal -- 1.2-2 L /day the day is 3-4 times > night. night is < 400 ml. polyuria >2000ml / day. Oliguria <500ml / day. Anuria is total suppression of urine <100 ml per day. Appearance color & turbidity 1. Color - normal ---- amber yellow ( to the presence of urobilin,uroerythrin,and urochromes)

1.Colorless---Very dilute urine ---Polyuria,diabetes 2.Yellow orange(high colored)--Concentrated urine -----------Excess urobilin ------------Bile pigments ------------Intake of carrots (Yellow foam ++) 3.Red/ smoky------Hemoglobin/ ---- RBC ----MyoglobinBeetroot / ---------- anilinedyes -------Menstrual contamination -------porphyrins 4.Cloudy--------Phosphates & carbonates, Urates & uric acid Pus cells bacteria,yeast,spermatozoa 5.Milky-------Pyuria,Fat ,chyluria 6. Brown black---Methemoglobin,,Homogenesticacid(alkaptonuria),Melanin 7. orange--------Bile pigments,Drugs pyridium,rifampicin

 2. Turbidity normal urine----clear. Turbidity in urine may be due to:

a. amorphous phosphate and carbonates alkaline or neutral urine disappears on addition of dilute acetic acid b. crystals, cellular exudates, bacteria and fungus c. chyle and fat d. pus

e. amorphous urates in acidic urine

 b. Odour: Normal-- aromatic odour

On standing--- ammonical there is decomposition of urea forming ammonia which gives a strong ammonical smell.. Abnormal odour of urine Fruity---- Ketonuria Mousy---- Phenylketonuria Rancid------ Tyrosinaemia

pH

ability to maintain normal hydrogen ion concentration varies from 4.5 to slightly higher than 8.0.

Normal kidneys are capable of producing urine the pH of which A pH below 7 indicates acid urine and a pH above7 alkaline urine PROCEDURE Dip the litumus paper strips in the urine, remove and read the color
change immediately. Blue litmus turns red - acid Red litmus turns blue - alkaline Blue and red litmus turns purple - neutral nitrazine paper method :The Ph---- nitrazine paper which are sensitive and specific in the pH range of 4.5 8.0 range.

SPECIFIC GRAVITY

degree of concentration or dilution of the

specimen. specific gravity measures the concentrating and diluting abilities of the kidney. The normal --------------1.015 and 1.025 (in a 24 hours specimen). ESTIMATION OF SPECIFIC GRAVITY BY URINOMETER urinometer: This is a weighted bulbshaped a scale calibrated----- from 1.000 to 1.060

PROCEDURE
Urine is poured into a cylindrical or conical glass so that the vessel is nearly full. Froth-- removed with a filter The instrument is floated in the urine and care should be taken to see that it does not touch the slides.

The depth to which it sinks in urine indicates the specific gravity of

urine air.

read on the urinometer scale at the junction of the urine with the
The reading is taken at eye level, the lowest part of the meniscus
being taken.

In case the urine is insufficient, it may be diluted with an equal

volume of distilled water and the last two figures of the reading are then multiplied by 2.

 Correction for temperature The urinometer is calibrated to read 1.000 in distilled

water at a specific temperature, indicated on each instrument e.g. 15 C or 20 C. There is a change in the specific gravity of 0.001 for each 3 C above and below this temperature. Therefore add 0.001 to the reading for each 3 C above the temperature for which the urinometer is calibrated, substract 0.001 for each 3 C, the temperature below the standard temperature. For example - urinometer calibrated for 20 C Specific gravity of urine at 32 C is 1.001 Corrected specific gravity {(32 20)/ 3 X 0.001} + 1.001 = 1.005 Correction is also recommended when glucose or protein are present. It is recommended that .003 be subtracted from the urinometer reading for each 1000 mg / dl of glucose or protein.

 1. SUGARS IN URINE: This is a non-specific test useful for semiquantitation of marked

glucosuria

Benedicts qualitative test: This test is not specific for sugars and is affected by most of the
reducing substances.

Composition of Benedicts reagent:

Copper sulphate - 17.3 gms Sodium carbonate (anhydrous) 100 gms Or Crystalline sodium carbonate - 200 Gms Sodium citrate - 175 gms Or Potassium citrate

 Dissolve crystalline copper sulphate in 100 ml of

distilled water. Dissolve sodium carbonate and sodium citrate in 700 cc. of distilled water. Slowly add the latter to the former solution with constant stirring. When complete, make up the volume to 1000ml with distilled water. Procedure Take 5ml of benedicts reagent Boil for 3 5 minutes add to it 0.5ml (8 drops)of protein free urine.

Cool and note the color.

Recording results The color varies from blue through green yellow- orange- brick red. Negative no change in color. Trace - solution appears pale green to slightly cloudy. 1+ - Definite cloudy green (0.5% sugar) 2+ - Yellow to orange precipitate, supernatant fluid pale blue (1% sugar) 3+ - Orange to red precipitate, supernatant fluid pale blue (1.5% sugars) 4+ - Brick red precipitate, supernatant fluid decolorizes (2 % sugar)

 COLORIMETRIC REAGENT STRIP TEST

Principle: this test is based on a double sequential enzyme reaction. One enzyme, glucose oxidase, catalyzes the formation of gluconic acid and hydrogen peroxide from the oxidation of glucose. A second enzyme, peroxides catalyzes the reaction of hydrogen peroxide with potassium iodide chromogen to oxidize the chromogen to colors ranging from green to brown.

KETONES IN URINE (ketonuria):

fats--------carbon dioxide and water. inadequate carbohydrate in the diet or a defect in carbohydrate metabolism or absorption, fatty acids------------metabolized. When the fatty acid utilization is incomplete------- the intermediary products of fat metabolism appear in the blood and the urine. These products -------- acetone, diacetic acid (acetoacetic acid) betahydroxybutyric acid. Diabetes mellitus, Other Causes of Ketonuria:
Fever Anorexia Gastrointestinal disturbances Fasting Starvation Severe vomiting

 Rotheras Test for Acetone and

Acetoacetic Acid: Procedure:
Take 5ml of urine in a test tube and saturate it with ammonium sulphate. Add 1 crystal of sodium nitroprusside.Run liquor ammonia carefully at the side of the tube so as to form a layer on top of the saturated urine.

Result: a permanganate calomel red (pink purple) ring forms at the junction of the two layers------positive. Negative result shows no ring or a brown ring. Ferric Chloride Test for Diacetic Acid Gerhardts Test Harts Test for Beta-Hydroxybutyric Acid Colorimetric Reagent Strip Test

 3. Proteins in urine:
Normal <30 mgms / 100 ml (30 50 mgms/24 hours) Tests for Detection of Proteins Semiquantitative precipitation tests: 1.The heat and acetic acid method, 2.sulfosalicyclic acid method and

3.the concentrated nitric acid protein precipitation method precipitation.

 Negative No turbidity or cloudiness.

Trace - A faint precipitate visible against a
black background, equivalent to about 5 mg / dl protein. 1+ - Definite cloud without flocculation equivalent to 10 30 mg / dl. 2+ - Heavy and granular cloud without flocculation equivalent to 40 100 mg / dl. 3+ - dense cloud with marked flocculation equivalent to 200 500 mg / dl . 4+ - cloudiness with precipitation equivalent to 500 mg / dl or more.

 Heat and Acetic Acid Method

Procedure : Take a long test tube and fill the tube with clear urine. Boil the upper portion over a flame .the lower portion serves as the control. If proteins, phosphates or carbonates are present in the urine a white cloud develops. Add 1-3 drops of glacial acetic acid. Any turbidity due to phosphate precipitation will clear or if it is due to carbonates they disappear with effervescence. If it persists, it is due to albumin . precipitates due to mucin or nucleoproteins will disappear on addition of 2 drops of nitric acid.

Sulphosalicylic acid method Procedure Take 2 ml of clear urine in a test tube. Add an equal volume of 30 % sulphosalicylic acid. Mix thoroughly, allow it to stand for 10 minutes and estimate the amount of turbidity.

 Nitric acid method Procedure Take 2 3 ml of concentrated nitric acid in a

test tube. Carefully pour 5ml of clear urine down the inner side of the inclined test tube so that the urine forms a layer over the nitric acid. A ring of white precipitated protein will form at the interface. Estimates the amount of precipitate.

 Colorimetric reagent strip test

PRINCIPLE: the colorimetric reagent strip test is based upon the ability of proteins to alter the color of some acid-base indicators without altering the pH. When an indicator such as tetrabromphenol blue is buffered at pH 3.it is yellow in solutions without protein. But in the presence of protein the color will change to green and then to blue with increasing protein concentration. PROCEDURE: protein is determined by dipping the strip into well mixed un centrifuged urine and immediately comparing the resultant color with the chart provided on the reagent strip bottle. RESULTS: the results are reported as negative (yellow color), trace, 1+ to 4+.trace readings may detect 5 to 20 mg of protein/dl. Albumin reacts more strongly than the other proteins. Highly buffered alkaline urine and contamination of the urine specimen with quaternary ammonium compounds or skin cleansers containing chlorohexidine may produce false results.

Bence Jones Proteinuria:

Bence Jones protein is soluble at room

and body temperatures. It precipitates upon heating between 45 C and 60 C redissolves when the urine is further heated to the boiling point.

Reappear on cooling

OCCULT BLOOD IN URINE:

red blood cells or haemoglobin

When hemolysis occurs in circulation urine Normally an occasional red cell may be found

on microscopic examination of the urine sediment. In women during menstruation, the urine may get contaminated with menstrual blood and hence examination of urine should not be done during that period.

 Haematuria: Denotes the presence of red

blood cells in urine. It is seen in various renal disorders, infectious or neoplastic or trauma related to any part of urinary tract.

Hemoglobinuria: is the presence of blood

pigments in the urine without the presence of red blood cells. It is associated with certain hemolytic anemias that cause hemolytic anemia, transfusion reactions, malaria, and paroxysmal nocturnal hemoglobinuria

 Tests for Detection of Blood: 1. Microscopic Examination:

sediment------------RBCS/HPF

2. Benzidine Test
PRINCIPLE Heme acts as a catalyst when hydrogen peroxide is mixed with benzidine. REAGENTS A: Saturated solution of benzidine in glacial acetic acid B: Hydrogen peroxide PROCEDURE Mix equal parts of A and B in a test tube and an equal amount of the mixed reagent. RESULTS A blue color indicates the presence of hemoglobin.

Colorimetric reagent strip method

Principle: the reagent area is impregnated with tetramethylbenzidine and buffered organic peroxide. This forms a green to dark blue compound when hemoglobin catalyzes the oxidation reaction of tetramethylbenzidine with a peroxide.

BILE IN THE URINE:

The constituents 1. bilirubin (bile pigments), 2. bile salts, 3. urobilin and urobilinogen. Bilirubin in appears IN JAUNDICE
Bilirubin in Urine: bilirubin in the urine indicates the presence of hepatocellular disease or intra or extrahepatic biliary obstruction in the reticuloendothelial cells breakdown of hemoglobin. linked to albumin---------- liver. This albumin-bound form, which is also known as unconjugated bilirubin( indirect bilirubin) insoluble in water and does not appear in the urine. In the liver cells,--conjugated with glucuronic and sulfuric acids to form water soluble conjugated bilirubin (direct bilirubin). It is secreted into the bile and then excreted into the intestinal tract through the bile duct. This conjugated bilirubin -------------to urobilinogen.

 certain liver diseases --unable to

conjugate all the bilirubin-----an increase in both conjugated and unconjugated bilirubin --bilirubinuria.

obstructive biliary tract disease-bilirubinuria.

hemolytic anemias unconjugated bilirubin------- urine free from bilirubin(Acholuric)

Tests for detection of bile salts:

Hay Test Procedure: flowers of sulphur --sprinkled on the surface of the urine, Results:if bile salts are present they sink to the bottom. Otherwise they float on the surface. This is due to the property of bile salts to lower surface tension

Tests for detection of bile pigments

1. Foam Test: Shake urine in a test tube. If the foam on top is yellow, bile pigments are present. 2. Gmelins test: PROCEDURE: 1. Place inch column of yellow nitric acid in a test tube. 2. Overlay with equal amounts of urine RESULT: A play of colored rings, the most distinct being green indicates the presence of bile pigments.

FOUCHETS REAGENT Trichloroacetic acid 25 gms Distilled water - 100 ml 10% Ferric chloride solution 10 ml. PROCEDURE 1. Place 10 ml of acidified urine in a test tube. 2. Add 2.5ml of 10 % barium chloride. 3. Mix and filter. 4. Unfold the filter paper and spread it on a dry filter. 5. Add 1 drop of Fouchets reagent to the residual precipitate. RESULT: A green or blue color indicates the presence of bilirubin.

3. Fouchets Test:

Colorimetric Strip Reagent Test

Principle: this test is base on the coupling of

bilirubin with diazotized 2, 4dichloroaniline in a strong acid medium to form a brown purple azobilirubin Compound. The color ranges through various shades of tan. Procedure: the reagent strip is dipped into fresh, uncentrifuged urine tapped to remove excess urine and after 20 seconds, compared to the color chart on the reagent strip bottle Result: the results are interpreted as negative, small (+), moderate (++), and large (+++) amounts of bilirubin.

Tests for detection of urobilinogen:

1. Qualitative Ehrlichs Test EHRLICHS REAGENT Paradimethylaminobenzaldehyde 2 gms 20%5 HCL - 100 ml PROCEDURE a. Place 10 ml of urine in a test tube. b. Add 2.5 ml of barium chloride (to remove bilirubin). c. Mix well and filter. d. Add 0.5 ml of Ehrlichs reagent e. Allow it stand for 3 5 minutes.

Results:
pink color observable when viewed from the top of the test tube. Against a white background placed beneath the bottom of the test tube. cherry red color Abnormally high amounts of This test must be done with fresh urine or else urobilinogen is oxidized on exposure to air urobilin. Excessively cold water should not be used in diluting the urine.

MICROSCOPIC EXAMINATION
Qualitative technique: the urine must be freshly voided examined without excessive delay in order to prevent cellular deterioration. Cellular debris from the urethral meatus and secretions from the vagina may contaminate the urine specimen.

10-15 ml of urine ----from freshly mixed urine specimen and centrifuged at a standard speed, usually 1500 to 2000 rpm for 5 minutes. This is sufficient to bring to the bottom casts, pus cells, blood and crystals. For bacteria however a higher speed of 3,000 rpm is required.

the sediment resuspended in 1 ml.of the same fluid.

A drop of resuspended sediment is placed directly on a microscope slide and covered with a cover slip.

low power- Casts tend to congregate at the edges of the cover slip. A minimum of 10 15 high power fields should be scanned for this examination.

Red blood cells, leucocytes,epithelial cells--- per high power field(/hpf); casts --- per low power fields(/lpf).

Other elements such as bacteria, parasites, crystals and spermatozoa are reported as well

 NORMAL SEDIMENT
Normal sediment contains a limited number of formed elements. it can be divided into two classes.

unorganized sediment Organized sediment

A. Unorganized sediment - these are the crystals of various substances present in the urine and they vary with the pH of the urine .crystals of normal urine is formed as the specimen cools. 1. Crystals in acidic urine: a. Uric acid and Urates; crystals are seen when the urine is allowed to stand for sometime and not seen in freshly passed urine. Amorphous urates appear as red granules and are dissolved by heat and sodium hydroxide but not acetic acid. Uric acid crystals vary in shape and are yellow brown in colour and are not dissolved by heat, acetic acid or HCL but are soluble when heated with sodium hydroxide. disturbances of uric acid metabolism fevers where the urine is concentrated.

Acid uric crystals

B.Calcium Oxalate: They are commonly found in diets rich in tomatoes, spinach etc. they are typically envelope shaped crystals but occasionally appear dumb- bell shaped .they are insoluble in strong Hcl. c. Cystine Crystals: highly refractile, hexagonal plates and are soluble in Hcl but insoluble in acetic acid. They are seen cystinosis which is an inborn error of metabolism in which cystine crystals are found in the urine, reticuloendothelial system and eyes. d.Leucine: slightly yellow, oily looking spheres with radial and concentric striations .they are not soluble in Hcl or ether .they are found in liver disorders.

Calcium Oxalate

Cystine Crystals

Leucine Crystals

 e.Tyrosine:

fine needles arranged in concentric sheaves, constriction at the middle. liver disorders. patients taking sulphonamides.

f.Sulpha crystals :-

2. Crystals in alkaline urine:

a. Ammonium Magnesium Phosphates:

( triple phosphate) coffin lid, feathery or leaf like forms. In freshly passed urine they indicate stones in the bladder or kidney. Phosphates may occur as amorphous deposits in alkaline urine and are dissolved in acetic acid. b. Dicalcium Phosphates: hey are also seen in slightly acid or neutral urine. They are colorless prisms arranged in stars and rosettes They are soluble in acetic acid. c. Calcium carbonate: amorphous granules or colorless spheres and dumb-bells which are soluble in acetic acid with gas formation.

Tyrosine crystals

Ammonium Magnesium Phosphates

Ammonium Biurate Crystals

Calcium Phosphate

Crystals in acid urine

Amorphous urates-Brick Red- Granules Uric Acid -Yellow Brown-Polymorphous Whetstones,Rosettes of prisms,Rhombohedral prisms, hexagonal plate Sodium urateColorless to YellowFan of slender prisms Cystine (rare)1. Colorless2.Highly refractileFlat hexagonal plates with well defined edges singly or in clusters

Cholestrol (rare) -ColorlessBroken window panes with notched corners ,Flat plates
Leucine (rare)-1. Yellow or Brown2.Highly refractile-Spheroids with striations pure form hexagonal Tyrosine (rare) Colorless or YellowFine silky needles in sheaves or rosettes Bilirubin-Reddish Brown--Cubes, Rhombic plates, Amorphous needles

Cholesterol Crystals

Acid, Neutral or Slightly Alkaline Urine

Calcium oxalate--Colorless Octahedral Dumbbells,Often small Hippuric acid Colorless- Rhombic plates Four sided prisms

Alkaline Urine
Calcium Carbonate-Colorless -Needles,Spheres,Dumbbells Ammonium biurate-Yellow Opaque BrownThorn apple,Spheres,Dumbbells,Sheaves of needles

Calcium Phosphate-Colorless- Prisms, Plates, Needles - - - -

A- represents the residue of normal human urine, as seen under the microscope. B is represented oxalate of urea. An excess of this element indicates indigestion

Nitrate of urea is represented in division C.

A and B--highly magnified urinary deposits, which indicate different degrees of impairment of the digestive functions are represented.

epithelial cells

In division A is represented pus and mucus, the presence of which indicates suppuration of the kidneys (Bright's disease). In B pus globules are alone represented. In the division marked C are shown blood corpuscles as they are arranged in blood drawn from a vein or artery. D represents the same separated, as they always are when present in the urine. In E highly magnified oil globules are represented. If present in the urine, they indicate disease of the kidneys. In the division marked F are represented epithelial cells, the presence of which in large numbers is indicative of disease of the mucous lining of the urinary organs.

Uric acid

In division A are represented the mixed urates as they appear

during idiopathic fevers, as intermittent, remittent, etc. When appearing as seen in division B, a less violent affection of the same character is indicated. Division C represents urate of ammonia, occasionally observed when there is a tendency towards albuminuria, In division D which is present in the urine of persons suffering from gout. The crystals shown in division E consist of the same salt.

varieties of cancer.

 Organized Sediment: the components of organized

sediment include casts, red blood cells, white blood cells, epithelial cells, bacteria, yeast, parasites, spermatozoa and artifacts. a. Casts: Casts are formed in the tubules and is composed of proteinaceous material. They are washed out by the glomerular secretion into the collecting tubules and the bladder. They are cylindrical in shape with round or broken ends with uniform diameter but varying in length. They require acidic conditions, high salt concentration, reduced urine flow and protein to be formed. Practically all casts have a hyaline matrix, which may or may not contain inclusions such as desquamated cells. The casts are named according to the matrix of the inclusions contained in them e.g. red blood cell casts.

1. Hyaline casts: are colorless, semi-transparent and occasionally refractile cylinders and are soluble in acetic acid. They are seen when there is damage to the glomerular capillary membrane, permitting leakage of proteins through the glomerular filtrate. They are seen in fever, orthostatic proteinuria, and emotional stress or strenuous exercise. 2. Granular casts: are casts containing large or fine granules embedded in coagulated protein. They are not found in normal urine and their presences indicate pyelonephritis. They are also seen in chronic lead poisoning. 3. Epithelial casts: are formed of fused desquamated tubular cells. They are coagulated protein in which are embedded desquamated epithelial cells from the renal tubules .they are seen in diseases where there is damage to the tubular epithelium as in nephrosis, eclampsia, amyloidosis and heavy metal poisoning.

Hyaline casts

Granular casts

4. Red Blood Cell Casts: are casts with red blood cells embedded in the coagulated protein in the tubule. Their presences indicate acute inflammation or vascular disorder in the glomerulus causing hematuria. They are seen in pathological conditions such as acute glomerulonephritis, renal infarction and collagen vascular disorder. 5. White Blood Cell Casts (Pus cell) 6. Fatty Casts 7. Waxy Casts

Red Blood Cell Casts

White Blood Cell Casts

Waxy Casts

 CELLS: a. Red blood cells: Normally 1-2 red blood cell

are found per high power field .they appear pale , light refractive, biconcave discs when viewed under high power magnification .they have no nuclei. Red blood cells in fresh, unstained sediment appear pale in color; in urine that is not fresh, they are pale or colorless shadow cells .in concentrated urine, they may be small and crenated; and in dilute urine, they are large and swollen and sometimes rupture to produce ghost cells. b. White cells c. Epithelial Cells: Normally a few epithelial cells occur in the urine .A marked increase is these cells in the urine is seen destruction of the tissues in the urinary tract.

Red blood cells

Red blood cells

White cells

White cells

Squamous Epithelial Cells

Transitional Epithelial Cells

Renal Tubular Epithelial Cells

 Quantitative Evaluation of the urine

sediment Addis count The Addis count is a quantitative measurement of the excretion of red cells, leucocytes and casts in the urine during a 12 hour period. e. Bacteria: Bacteruria is considered significant when there is the presence of 100,000 or more bacteria per ml of urine specimen.

DETECTION OF BACTERIA
Microscopic Examination:
sediment - >20 or more bacteria per high power field may indicate a urinary tract infection Reagent strips: PRINCIPLE: This test depends upon the conversion of nitrate to nitrite by the action of gram negative bacteria in urine. At the acid pH of the reagent area, nitrite in the urine reacts with p-arsanilic acid to form diazonium compound. This compound in turn couples with 1, 2, 3, 4-tetra hydrobenzoquinolin-3-ol to produce pink color. Procedure: The strip is dipped in the urine specimen for 5 seconds.

Results: uniform pink color --positive result the presence of 100,000 or more organisms per ml, A negative result should never be interpreted as indicating absence of bacteruria. There are several reasons for this. First morning urine or urine that has remained in the bladder for several hours is more likely to yield a positive nitrite test result in the presence of significant bacteruria than a random urine sample that may have been in the bladder for short time. in the latter type of specimen there may have been insufficient time(less than 4 hours) for conversion of nitrate to nitrite to have occurred. When dietary nitrate is absent, even if organisms containing reductase are present and bladder incubation is ample. Ascorbic acid concentrations of more than 25 mg / dl or greater may cause false negative results with specimens containing small amounts of nitrate.