Cystitis Research Center
Dr. Paul Fugazzotto used a very effective culturing technique to isolate what he considered to be the real pathogens in chronic cystitis. It was used many years ago but fell out of use. He simple adapted it to use for culturing urine specimens of symptomatic patients and after many years of use he found it very accurate and helped many patients in their struggle to find answers for their symptoms. This has been verified by other microbiolgists as a reliable method and thus it is reprinted here for perusal and possible use by laboratory professionals. After he retired Dr. Fugazzotto continued to work and to research in his own lab. He called it the Cystitis Research Center and for many years there was a website describing his views and work to further the understanding of chronic cystitis.
Below is his method:
Laboratory Test Technology For Diagnosis And Monitoring Of Urinary Tract Disorder
Update and revision of the “Method for Revealing Hidden Pathogens”; in Laboratory Management: 24-29, Oct. 1987; A clinical laboratory diagnostic procedure.
Test #1: Test for Presence of Antibiotic in the Urine
- This is done on all specimens, initial to the program, in order to detect evidence of false reactions due to extraneous components antagonistic to recovery of offending organisms.
- This is also done periodically on participant urines, to monitor the antibiotic treatment program on previously diagnosed study participants.
- Utilizing a Trypticase Soy Agar (TSA) Plate, with a crayon line drawn on the plate bottom, to divide the plate into halves: Apply a suspension of E.coli (Gram-negative) to one-half of the agar and a suspension of Enterococci (Gram-positive) to the other half. Spread each out for confluent growth.
- Utilizing two _” blank disks immersed in each urine specimen, place one disk onto the E.coli side and the other disk onto the enterococci side of the plate. Incubate plate at 35 degrees Celsius.
- Determine if antibiotics are present by observing inhibitory zones around the discs. Measure the zones in mm and document. As many as 6 specimens can be tested per plate.
- In a chart such as the following, record the size of the inhibitory zone in mm observed for each specimen on its tests, indicating the amount of antibiotic excreted into the bladder.
|outer diameter of inhibitory zone||mm found||amount of antibiotic excreted into bladder|
|mm Found G+ G-||0 to 12 mm||small|
|Amount of Antibiotic Excreted into Bladder||14 to 20 mm||medium|
|level 3||22 to 28 mm||therapeutic|
|level 4||29 and above||high|
Evaluation of Results
The value recorded in the G+ test is used in the evaluation of patient response.
The result in the G- is used as a negative control.
Wet Prep Procedure (optional)
This test is done in anticipation of viewing the organisms to be expected as emerging from transfer of the broth culture; or to confirm the result of the broth transfer.
Transfer one loop of each broth to a microscope slide. Cover with cover slip and examine at 430X magnification to record the nature and morphology of the organisms present, i.e., strep, other cocci, motile and non-motile bacilli, other strains. Although the sub-culturing can be made to the respective differential media, prior to examinations of wet mounts, cocci in the presence of other organisms may be missed if one is not aware of their presence in the broth, and fails to make special efforts to isolate and identify them.
I. Purging of the raw specimen.
- All raw urines voided by the patients can be expected to contain body waste products from foods, ingestibles, medications (antibiotics), toxins; antagonistic to the emergence of offending pathogens. Aliquots of all urines must first be purged of these aqueous soluble components.
- Using a cotton tipped applicator, stir the urine well, and with the saturated swab transfer one or two portions of the urine to a 13 x 100 ml screw cap tube containing 5 or 6 ml of sterile saline solution. The saline solution should consist of 0.85 g NaC1, plus 0.1 gram proteose-peptone as a buffer per 100 ml distilled water. Securely replace the cap. Hold tube in an upright position, and pass it with smooth inversion excursions by twist of the wrist, completely inverting two times per second, for 10 seconds (20 complete inversions).
- Centrifuge the tube at 2,000 rpm for 20 minutes. Pour the supernate off; add 3.0 ml of Trypticase Soy Broth (TSB) to the residue and replace the tube cap. Shake the tube briefly and incubate at 35 degrees Celsius until growth appears (usually in 3 days), or for as long as 6 days if negative in 3 days. It is convenient to have a supply of TSB in 13 x 100 mm screw cap tubes already prepared for the transfer.
II. Isolation of the Offending Organisms
- To isolate the offending organisms, it is necessary to employ agar plates of differential media: Phenyl Ethanol blood agar (PEA) and Eosin Methylene Blue (EMB) agar. The plate bottoms are marked with wax crayon, in as many as 8 segments with lines crossing at the plate center, depending on the numbers of cultures to be processed.
- With a 0.1mm wire loop, transfer an aliquot of each culture to respective segments of both plates; but carefully spread it on the PEA for distinct colony isolation.
- After inoculation of the PEA segments, puncture the agar for possible hemolysis. Place a NA disc (Negram) on the PEA surface, where the marks cross at the center. Incubate both plates at 35 degrees Celsius for about 24 hours or until significant bacteria emerge on the plate.
- Determine which prominent discrete organisms are significant on the PEA agar plate in the area close to the NA disk. Find one or more totally isolated, completely round colony on the plate, making sure that it has no imperfections such as mixed colony growth.
- Utilizing a straight wire, transfer and spread the chosen colony from the PEA plate to a separate routine blood agar plate. Puncture this blood plate a few times to show its hemolytic characteristic. Incubate at 35 degrees Celsius overnight and observe growth to ensure a pure culture confluent growth (with no breakthrough of submerged colonies).
- The EMB (Gram-negative) plate culture growth is identified merely for the record, as these organisms are not considered significant of infection. (UTI/IC are Gram-positive cocci infections.) The Gram-negative produces discomfort by fermentation.
III. ABST (Antibiotic Sensitivity Test) of infecting Gram-positive pathogens.
- After the infecting organism is identified, perform its ABST (Antibiotic Sensitivity Test) on the pure culture. Utilize only the agar diffusion technique. (Do not use the multiple plastic well plate method.)
- From the confluent growth in step 5 above, prepare a light saline suspension of the etiologic agent with a sterile cotton tip applicator.
- Transfer this suspension sparingly over the surface of a TSA plate, streaking at two right angles for confluent growth.
- Space the antibiotic discs equally on the agar surface.
- Invert the plate and incubate at 35 degrees Celsius for overnight (16-20 hrs.).
- Read and record the diameters of the inhibitory zones around each antibiotic.
Antibiotics Used, and Their Respective Manufacturers Minimal Inhibitor Concentration For Gram-Positive Pathogens
- In our reporting, for purpose of patient treatment, we do not consider an antibiotic effective for treatment, unless the antibiotic reading in the test, amounts to 5mm and higher than the manufacturer MIC value.
- The organisms emerging on the EMB are not pathogenic, but do cause some discomfort by reason of fermentation. They also require treatment.
- Gram-negative (intestinal) bacilli respond to customary Cipro (and other similar antibiotics).
- Yeasts respond to Nystatin, Diflucan, and other anti-yeast treatment.