Urine Culture And Its Microbiological Analysis

Laboratory analysis is the initial step of examination of urine specimen. The midstream urine is required and that should be collected in a sterile container following the cleaning of external genitalia. The first portion of the urine contains most of the contaminants. As the urine passes out from urethra, it can be contaminated with genital microflora. It is necessary to analyse the urine sample immediately after its collection otherwise normal organisms may overgrow on more slowly growing pathogens. If it is not possible, it can be stored for a short time in refrigerator.

Clinical analysis of urine requires quantitative determination of the number of microorganisms per ml of urine sample. If the count is more than 10−5 it indicates urinary tract infection (UTI). The count ranges from 0 to 1000 per ml are treated as normal.

The organisms commonly responsible for infections of the genito-urinary tract are given below.

Gram-positive bacteria : Staphylococcus aureus, Staphylococcus pyogenes, Streptococcus faecalis, Streptococcus faecium

Gram-negative bacteria : Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, Klebsiella pneumoniae

Besides, some organisms are responsible for veneral diseases. These are given below:

Treponema pallidium, Neisseria gonorrhoeae, Haemophilus ducreyi, Calymmatobacterium granulomatis and Herpes hominus.

Fungi: Candida albicans, Blastomyces dermatidis, Coccidioides immitis

Protozoa: Trichomonas vagilis, and Entamoeba histolytica

Helminths: Schistosoma haematobium, Wuchereria bancrofti

Requirements

• Urine sample
• Sterile container
• Blood agar plate
• Tubes of saline water
• Bunsen burner
• Pipette
• Incubator.

Procedure

Streaking a urine sample for isolation of microorganisms is the recommended method.

1. Prepare one plate of blood agar and other plate of MacConkey’s agar medium and streak urine collected in a special vial.
2. Incubate the plates at 37oC for 48 hours.

1. Examine the plates for the presence of bacterial colonies. If the blood agar plate has significant growth of bacteria, the causative organism is Gram-positive cocci. On the other hand, if the growth occurs on both the plates, the organism is a Gram-negative. Be careful while preparing a testing the cultures for Grams’ stain. Further identification is possible for specific genera and species.
2. If Gram-positive cocci are visible, examine the arrangement of the cells and other tests from a separate exercise for the identification of staphylococci and streptococci.
3. Similarly, for Gram-negative bacteria, perform carbohydrate fermentation using TSI slants.

For the detailed procedure consult the exercise for carbohydrate fermentation. This will

make you acquainted with the causal organism of UTI. After purification of the isolate perform test for sensitivity for antibiotics as given below. If you obtain the growth of the causative organism, following summary will help you to identify the UTI causing bacteria.

Staphylococci, Gram-positive; enterococci (Enterococcus faecalis), Gram-positive; Streptococcus pyogenes, Gram-positive; Escherichia coli, Gram-negative and perform differentiation test and lactose fermentation ; Proteus spp., Gram-negative and determine urease test; Pseudomonas spp., Gramnegative; Enterobacter-Klebsiella group, Gram-negative and lactose fermenting.