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4 five conventional media, supplemented with either Helicobacter pylori or Campylobacter selective supplement (Table 2). Positive controls were the same as described above. All plates were incubated in microaerophillic atmosphere at 37oC for up to 7 days to provide the optimal environment for culturing Helicobacter. Formulation of enhanced selectivity (HP) media HP media preparation requires a sequential addition of components. The mixture of special peptone, beef extract, yeast extract, NaCl, phenol red, agar, and water was autoclaved for 20 minutes at 121oC and then tempered to 50oC. Then calf serum with iron, antibiotics (vancomycin trimethoprim, cefsulodin, Amphotericin B, and Polymixin B), and urea were aseptically added with constant stirring. Finally 0.8 ml of 1N HCl was added drop-wise to the media as color changed from red to yellow/orange (final pH 5.7 @ 45oC, pH 6.0 @ 22oC). The media was then poured into petri plates. Table 3. Media components evaluated over a range of concentrations to develop the final HP formulation. Component Range tested per liter a optimum level Porcine mucin 0 - 4 g O Ferrous sulfate 0 - 500 mg O Na pyruvate 0 - 500 mg O Polymixin B 0 - 4000 units 3500 units Amphotericin B 0 - 7.5 mg 7.5 mg Vancomycin 0 - 10 mg 10 mg Trimethoprim 0 - 5 mg 5 mg Cefsulodin 0 - 5 mg 5 mg Urea 0 - 1 mg 600 mg Phenol red 0 - 200 mg 100 mg 1 N HCl 0 - 2 ml 0.8 ml a optimum level for recovering H. pylori while inhibiting background flora. Well sample testing About three hundred fifty private drinking water wells across Wisconsin were screened using HP agar, immunomagnetic separation (IMS), and/or fluorescent antibody staining (FA) for the presence of H. pylori. The samples were grouped into two types, designated random or requested. Random samples were collected by taking aliquots from samples sent to the WSLH Water Bacteriology Department for routine coliform testing. For this group, fifty to one hundred ml aliquots were centrifuged at 2500 x g for 15 minutes and then all but the bottom 5 mls of supernatant was carefully siphoned away. 0.5 ml aliquots of the resulting 5 mls (or dilutions thereof) were then spread plated onto HP agar plates and incubated in a microaerophilic environment at 37oC for up to 7 days. Colonies appearing after 3 days with reddish "halos" (urease +), 1-2 mm in diameter, catalase and urease positive were considered positive. Subsequent microscopic examination showing helical, horseshoe, and/or coccoid morphology was used for verification. About 275 wells were represented in the grouping of random samples.
5 The requested sample group consisted of wells where a specific request to test for H. pylori was received from the well owner. These requests most likely resulted from the fact that a resident in the home served by the well had suffered from an H. pylori infection. For this group, 0.5 liter was concentrated to 5 ml via centrifugation. Four mls of the final concentration was spread onto HP agar plates (0.5 ml per plate) and incubated as described. For the requested sample group, two additional methods were employed in an attempt to recover H. pylori. In addition to the culture another 1 ml aliquot was assayed using immunomagnetic separation (IMS). Briefly, the 1 ml aliquot is mixed with microscopic magnetic beads (Dynabeads M-450, Dynal ASA, Oslo, Norway) coated with antibodies specific to H. pylori. The quantity of beads and duration of contact mixing time was determined by manufacturer's recommendations to provide the optimal exposure of capture beads to target organism. During this process the bacteria immunologically attach to the magnetic particles. A magnetic tube rack is then used to retrieve the beads and attached H. pylori. The retrieved beads are then plated onto the HP agar. A third procedure involving direct fluorescent antibody staining of a filtered sample was also employed as a non culture method control on some samples. For the fluorescent antibody (FA) staining, 0.5 liter of sample water was passed through a 0.4 micron pore size filter to capture H. pylori. The filter was treated with fluorescent stain (IgG Fluor, Chemicon International, Temecula, CA) attached to H. pylori-specific antibodies (Biodesign International, Saco, ME) that attach to the target cell if present. Cells reflecting green under fluorescent light microscope and helical, horse-shoe, or folded shape were considered H. pylori (note: non-viable as well as viable cells will fluoresce). IMS was used when samples contained visibly high particulate matter and FA staining was used to assay clear water. RESULTS & DISCUSSION Media evaluation Growth of pure cultures on conventional media. Table 2 lists the five conventional media formulations evaluated. Each of these media was evaluated for its ability to recover H. pylori from among a population of 7 spiked strains of bacteria and various indigenous strains contained in a sample of well water (Table 5). As expected, media without the antibiotic supplement allowed the growth of all organisms tested. The rapid growth of the non H. pylori bacteria covered the plates precluding any chance of detecting the slow growing H. pylori organisms. The addition of selective supplements provided some measure of selective pressure, however, some of the organisms (Acinetobacter, E. coli, Flavobactrum, Pasteurella, Ochrobactrum) were not inhibited and unacceptable levels of overgrowth still occurred. The selectivity profiles were identical among the five conventional media formulations, although it was noted that H. pylori colonies formed most rapidly (84 hours) on HPSPA media. Since the selectivity of the five conventional media proved inadequate to isolate H. pylori from the complex flora water samples it was determined that an enhanced selective media would be required. To develop the formula for this new media, components were individually evaluated to determine their contribution to the selective and nutritive properties necessary to isolate H. pylori from a mixed population of microbial contaminants. Some nutritive components (yeast extract, beef extract, special peptone [Oxoid], NaCl) were incorporated at conventional concentrations without further evaluation. In order to develop a media with enhanced selectivity for Helicobacter, a list of selective, nutritional, and differential components were evaluated (Table 3). The properties of an improved formulation would include a broader inhibition