Urinary tract infections represent one of the most common bacterial infections encountered in clinical practice. Accurate and timely laboratory diagnosis is paramount for effective patient management, preventing complications, and guiding appropriate antimicrobial therapy.
Staining Methods and Their Principles for Urine Specimens
Microscopic examination of a urine specimen is a critical first step in UTI diagnostics. Staining allows for the visualization of bacteria, host cells (like white blood cells), and other formed elements that are otherwise transparent. The primary method used is the Gram stain.
The Gram Stain: The Cornerstone of Bacteriological Identification
The Gram stain is a differential staining technique that separates bacteria into two major groups—Gram-positive and Gram-negative—based on differences in their cell wall structure.
Principle: The key differentiator is the thickness of the peptidoglycan layer in the bacterial cell wall.
- Gram-positive bacteria possess a thick, multi-layered peptidoglycan wall that readily retains the primary stain (Crystal Violet) even after a decolorization step.
- Gram-negative bacteria have a much thinner peptidoglycan layer, which is surrounded by an outer lipid membrane. This structure cannot retain the Crystal Violet-Iodine complex during decolorization. It is then visualized by applying a counterstain (Safranin).
Step-by-Step Staining Procedure:
- Specimen Preparation:
- Centrifuge approximately 10-15 mL of urine at 2000 rpm for 5 minutes.
- Discard the supernatant, leaving a small amount of urine to resuspend the sediment.
- Using a sterile loop or pipette, place a small drop of the resuspended sediment onto a clean, dry glass slide and spread it into a thin, even smear.
- Fixation:
- Allow the smear to air dry completely.
- Heat-fix the smear by passing the slide quickly through a Bunsen burner flame 2-3 times. This adheres the bacteria to the slide and kills them.
- Staining:
- Step A: Primary Stain: Flood the slide with Crystal Violet and let it stand for 1 minute. This stains all cells purple. Rinse gently with water.
- Step B: Mordant: Flood the slide with Gram’s Iodine and let it stand for 1 minute. The iodine forms an insoluble Crystal Violet-Iodine (CV-I) complex within the cell wall. Rinse gently with water.
- Step C: Decolorization: This is the most critical step. Briefly rinse the slide with a decolorizer (95% Ethanol or an Acetone-Alcohol mixture) until the purple color ceases to run off (typically 5-15 seconds). This removes the CV-I complex from Gram-negative bacteria but not from Gram-positive bacteria. Immediately rinse with water to stop the decolorization process.
- Step D: Counterstain: Flood the slide with Safranin and let it stand for 1 minute. This stains the now-colorless Gram-negative bacteria pink or red. Gram-positive bacteria remain purple. Rinse with water, blot dry gently, and examine under a microscope using the oil immersion lens (100x).
Microscopic Findings in Acute vs. Chronic UTIs
- Acute UTI: The microscopic view is often straightforward. Key findings include:
- Significant Bacteriuria: A high number of a single morphological type of bacteria (e.g., thousands of Gram-negative rods per oil immersion field).
- Pyuria: A large number of neutrophils (pus cells), typically >10 per high-power field, indicating an active inflammatory response.
- Chronic or Complicated UTI: The findings can be more complex. They may include:
- Lower Bacterial Counts: The number of organisms may be lower than in an acute infection.
- Polymicrobial Flora: The presence of two or more different types of bacteria may be observed, suggesting a complicated infection, catheter association, or contamination.
- Presence of Yeast: Organisms like Candida albicans may be seen, especially in immunocompromised or catheterized patients.
- Epithelial Cells: An increased number of transitional or squamous epithelial cells might be present.
Identifying Common Uropathogens on a Stained Slide
Based on the Gram stain reaction and morphology, a presumptive identification of the causative agent can be made.
- Gram-Negative Rods (Bacilli): These are the most frequent cause of UTIs. They appear as pink or red rod-shaped organisms.
- Escherichia coli (E. coli): The leading uropathogen (>80% of cases). Appears as standard, medium-sized, pink/red rods.
- Klebsiella pneumoniae: Often appears as shorter, plumper, non-motile pink/red rods. They frequently produce a polysaccharide capsule, which may appear as a clear halo around the cells.
- Proteus mirabilis: A highly motile, pleomorphic Gram-negative rod. Its appearance can be variable.
- Gram-Positive Cocci: These appear as purple, spherical organisms.
- Enterococcus faecalis: Appears as purple cocci, typically arranged in pairs (diplococci) or short chains.
- Staphylococcus saprophyticus: A common cause of UTIs in young, sexually active females. Appears as purple cocci arranged in grape-like clusters.
- Yeast:
- Candida albicans: Appears as large, oval, Gram-positive (purple) budding cells. They are significantly larger than bacteria. Pseudohyphae may also be present.
Sterilized Methods for Collection of Specimens for Culture
The accuracy of a urine culture is entirely dependent on the quality of the specimen. Contamination during collection is the most common source of error.
Principle: The goal is to collect a sample of bladder urine with minimal contamination from the distal urethra, vagina, or skin.
The Midstream Clean-Catch (MSU) Method
This is the standard, non-invasive method for routine urine culture.
Step-by-Step Instructions for the Patient:
- Preparation: Wash hands thoroughly with soap and water.
- Container Handling: Open the sterile container provided, being careful not to touch the inside of the cup or the lid. Place the lid with the inside facing up.
- Cleansing:
- Females: Use the provided antiseptic towelettes to cleanse the labia. Separate the labial folds with one hand and wipe from front to back, using a new towelette for each side and a final one for the urethral opening itself.
- Males: If uncircumcised, retract the foreskin. Use an antiseptic towelette to cleanse the glans penis.
- Collection:
- Begin urinating into the toilet. This initial stream flushes out contaminants from the urethra.
- After a few seconds, without stopping the flow of urine, position the sterile cup to collect the “midstream” portion of the urine (approximately 30-50 mL).
- Finish urinating into the toilet.
- Sealing: Securely screw the lid onto the cup. Ensure the outside of the container is clean.
- Submission: Label the container with the patient’s name and date/time of collection. The specimen should be transported to the laboratory immediately or refrigerated if a delay is unavoidable.
Other Collection Methods:
- Catheter Specimen: For patients with an indwelling catheter, the sample should be collected aseptically from the collection port of the catheter tubing, not from the drainage bag.
- Suprapubic Aspiration (SPA): An invasive procedure where a needle is inserted directly into the bladder through the abdominal wall. This is the gold standard for obtaining an uncontaminated specimen, typically reserved for infants or when other methods are inconclusive.
Practical for Culture and Sensitivity by Disc Diffusion Method
Once a pathogen is isolated in culture, antimicrobial susceptibility testing (AST) is performed to determine which antibiotics will be effective for treatment. The Kirby-Bauer disc diffusion method is a widely used standard.
Principle: The Kirby-Bauer test is based on the diffusion of a pre-set concentration of an antibiotic from a paper disc into an agar plate that has been seeded with a uniform “lawn” of the test bacterium. If the bacterium is susceptible to the antibiotic, a clear circular area of no growth, called the zone of inhibition, will form around the disc. The diameter of this zone is proportional to the bacterium’s sensitivity to that antibiotic.
Practical Step-by-Step Procedure (using an isolated uropathogen, e.g., E. coli):
- Preparation of Inoculum:
- Using a sterile loop, select 3-5 well-isolated colonies of the uropathogen from a primary culture plate.
- Suspend these colonies in a tube of sterile saline or broth.
- Vortex the tube to create a smooth, homogenous suspension.
- Adjust the turbidity (cloudiness) of the suspension to match a 0.5 McFarland standard. This ensures a standardized bacterial concentration for reproducible results.
- Inoculation of the Agar Plate:
- Use a sterile cotton swab to inoculate a Mueller-Hinton agar (MHA) plate. MHA is the standard medium for AST.
- Dip the swab into the standardized bacterial suspension and remove excess fluid by pressing it against the inside of the tube.
- Streak the swab evenly over the entire surface of the MHA plate to create a confluent “lawn” of growth. Rotate the plate 60 degrees and repeat the streaking two more times to ensure complete coverage.
- Application of Antibiotic Discs:
- Using a sterile forceps or an antibiotic disc dispenser, place the appropriate antibiotic discs onto the surface of the inoculated agar.
- Discs should be spaced at least 24 mm apart from center to center to prevent the zones of inhibition from overlapping.
- Gently press each disc down to ensure full contact with the agar surface.
- Incubation:
- Invert the plates and place them in an incubator at 35-37°C for 18-24 hours.
- Reading and Interpretation of Results:
- After incubation, examine the plate for zones of inhibition around each disc.
- Using a ruler or calipers, measure the diameter of the zone of inhibition (including the disc) for each antibiotic in millimeters (mm).
- Compare these measurements to a standardized interpretation chart, such as those provided by the Clinical and Laboratory Standards Institute (CLSI).
- Based on the zone diameter, classify the organism’s susceptibility to each antibiotic as:
- S (Susceptible): The infection is likely to respond to treatment with this drug at the recommended dosage.
- I (Intermediate): The drug may be effective in body sites where it is concentrated (like the urinary tract) or if a higher dosage can be used.
- R (Resistant): The organism is not inhibited by the achievable concentrations of the drug, and clinical efficacy is unlikely.
This final report, combining the pathogen identification with its susceptibility profile, provides the clinician with the critical information needed to prescribe targeted, effective antibiotic therapy for the patient’s UTI.
