Helicobacter pylori ATCC 43504 BioVector NTCC中国质粒载体菌种细胞基因保藏中心

Helicobacter pylori ATCC 43504 BioVector NTCC中国质粒载体菌种细胞基因保藏中心 Designation: NCTC 11637 [JCM 7653, RPH 13487] Deposited Name: Campylobacter pyloridis Marshall et al. Product Description: Type strain. Quality control strain for API products. CLSI control strain for susceptibility testing. Medium ATCC® Medium 18: Trypticase Soy Agar/Broth ATCC® Medium 260: Trypticase soy agar/broth with defibrinated sheep blood Growth Conditions Temperature: 37°C Atmosphere: Microaerophilic, 35% O210% CO2 Propagation Procedure 1. This organism is shipped frozen in dry ice and should be stored in vapor phase liquid nitrogen until ready for use. Just prior to use, thaw vial in water bath at approximately 37°C with gentle agitation until no ice remains. When thawed, a drop of the suspension may be used to do an immediate wet mount to observe the unique morphology of this organism and verify its viability by checking for motility. 2. Aseptically transfer the entire thawed suspension into a fresh #18 broth (35 mL). Mix well. This suspension can now be used to inoculate agar slant(s), plate(s), or the preferred biphasic culture. Two #260 plates should be inoculated, one for microaerophilic growth and the second for aerobic growth. No growth should occur on the plate incubated aerobically. 3. To obtain a biphasic culture, add 0.6 mL of the suspension to a #260 slant. The resulting pool at the bottom of the slant is where the best, most rapid growth will occur. 4. Incubate at 37°C under microaerophilic conditions using an anaerobe jar with an active catalyst and a microaerophilic gas generator pack, or other acceptable method, to obtain microaerophilic conditions. Incubate tubes with caps loose. 5. Within 3 days, good growth should be obtained in the broth pool at the bottom of the slant. Additional incubation may be required for colonies to appear on the agar plate. Further subcultures can be made using the broth pool as the inoculum source. Subcultures to biphasic cultures will require only 24 to 48 hours of incubation for good growth. Colonies on #260 agar are small, entire, glistening, circular, smooth, and raised. This is a slowing organism that requires moist conditions for best growth. Growth at the broth/agar interface of the biphasic slant should occur within 3 days, but only light turbidity will be seen. To observe growth, examine a wet mount of the broth under phase microscopy. This organism displays motile corkscrew rods arranged singles. Growth on agar takes longer than in the biphasic culture. It is essential to use fresh, moist plates. The cells do not Gram stain well using traditional procedures. For best results, use a basic fuchsin counterstain in place of the safranin. Once good growth in obtained, transfer or freeze the culture. Adding an equal amount of 20% sterile glycerol to pooled broth from several biphasic slants, followed by freezing in liquid nitrogen or "ultralow temperature" freezer is recommended. Purified genomic DNA of this strain is available