2). URINE:
Bacteriologic examination of the urine is done mainly when signs or symptoms point to urinary tract infection,
renal insufficiency ,  or hypertension.  It should always be done in persons with suspected systemic infection or  fever of unknown origin.  It is desirable for women in the first trimester of pregnancy .
Urine secreted in the kidney is sterile unless the kidney is infected.  Uncontaminated bladder urine is also
normally sterile.  The urethra,  however ,  contains a normal flora,  so that normal voided urine contains small
numbers of bacteria.  Because it is necessary to distinguish contaminating organisms from etiologically
important organisms,  only quantitative urine examination can yield meaningful results.
The following steps are essential in proper urine examination.
Proper collection of the specimen is the single most important step in a urine culture and the most difficult.
Satisfactory specimens from females are problematic.
1.   Have at hand a sterile,  screw-cap specimen container and two to three gauze sponges soaked with
non-bacteriostatic saline (antibacterial soaps for cleansing are not recommended).
2.   Spread the labia with two fingers and keep them spread during the cleansing and collection process.  Wipe the urethra area once from front to back with each of the saline gauzes.
3.   Start the urine stream and,  using the urine cup,  collect a midstream specimen.  Properly label the cup.
The same method is used to collect specimens from males; the foreskin should be kept retracted in uncircumcised males. Catheterization carries a risk of introducing microorganisms into the bladder ,  but it is sometimes unavoidable. Separate specimens from the right and left kidneys and ureters can be obtained by the urologist using a catheter at cystoscopy .  When an indwelling catheter and closed collection system are in place,  urine should be obtained by sterile aspiration of the catheter with needle and syringe,  not from the collection bag.  
T o resolve diagnostic problems,  urine can be aspirated aseptically directly from the full bladder by means of suprapubic puncture of the abdominal wall. For most examinations,  0.5 mL of ureteral urine or 5 mL of voided urine is sufficient.  Because many types of microorganisms multiply rapidly in urine at room or body temperature,  urine specimens must be delivered to the laboratory rapidly or refrigerated not longer than overnight.
Much can be learned from simple microscopic examination of urine.  A drop of fresh uncentrifuged urine placed on a slide,  covered with a coverglass,  and examined with restricted light intensity under the high-dry objective of an ordinary clinical microscope can reveal leukocytes,  epithelial cells,  and bacteria if more than 10^5 /mL are present.Finding 10^5 organisms per milliliter in a properly collected and examined urine specimen is strong evidence of active urinary tract infection.  A Gram-stained smear of uncentrifuged midstream urine that shows gram-negative rods is diagnostic of a urinary tract infection.
          Brief centrifugation of urine readily sediments pus cells,  which may carry along bacteria and thus may help in microscopic diagnosis of infection.  The presence of other formed elements in the sediments—or the presence of proteinuria—is of little direct aid in the specific identification of active urinary tract infection.   Pus cells may be present without bacteria,  and,  conversely ,  bacteriuria may be present without pyuria.  The presence of many squamous epithelial cells,  lactobacilli,  or mixed flora on culture suggests improper urine collection.Some urine dipsticks contain leukocyte esterase and nitrite,  measurements of polymorphonuclear cells and bacteria,  respectively , in the urine.   Positive reactions are strongly suggestive of bacterial urinary tract infection.
Although not readily embraced by clinical microbiology laboratories,  many chemistry laboratories have
implemented automated or semi-automated instruments for routine performance of urinalysis.  A variety of
techniques are used by these instruments to detect leukocytes and bacteria.  The performance of these systems varies,  but they bring a level of standardization for high volume testing that may not be accomplished using dipstick methods.

Culture of the urine,  to be meaningful,  must be performed quantitatively .  Properly collected urine is cultured in measured amounts on solid media,  and the colonies that appear after incubation are counted to indicate the number of bacteria per milliliter .  The usual procedure is to spread 0.001–0.05 mL of undiluted urine on blood agar plates and other solid media for quantitative culture.  All media are incubated overnight at 37°C; growth density is then compared with photographs of different densities of growth for similar bacteria,  yielding semi quantitative data.
In active pyelonephritis,  the number of bacteria in urine collected by ureteral catheter is relatively low.  While

accumulating in the bladder ,  bacteria multiply rapidly and soon reach numbers in excess of 10^5/mL —far more than could occur as a result of contamination by urethral or skin flora or from the air .  Therefore,  it is generally agreed that if more than 10^5 colonies/mL are cultivated from a properly collected and properly cultured urine specimen,  this constitutes strong evidence of active urinary tract infection.The presence of more than 10^5 bacteria of the same type per milliliter in two consecutive specimens establishes a diagnosis of active infection of the urinary tract with 95% certainty .  If fewer bacteria are cultivated,  repeated examination of urine is indicated to establish the presence of infection.
The presence of fewer than 10^4 bacteria per milliliter ,  including several different types of bacteria,suggests that organisms come from the normal flora and are contaminants,  usually from an improperly collected specimen.The presence of 10^4/mL of a single type of enteric gram-negative rod is strongly suggestive of urinary tract infection,  especially in men. Occasionally ,  young women with acute dysuria and urinary tract infection will have 10^2 to 10^3/mL.  If cultures are negative but clinical signs of urinary tract infection are present,  "urethral syndrome," ureteral obstruction,  tuberculosis of the bladder ,  gonococcal infection,  or other disease must be considered. 

Bsc Microbiology, PBPC,Nayabazar-9, Pokhara.


 Forbes BA,  Sahm DF ,  Weissfeld AS (editors): Bailey and Scott's Diagnostic Microbiology,  12th ed.  ASM Press,Washington,  DC,  2007.
Winn W et al (editors): Koneman's Color Atlas and Textbook of Diagnostic Microbiology 6th ed.  Lippincott Williams and Wilkins,  2006.


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