Ohkusa Toshifumi, Miwa Hiroto, Endo Shu, Okayasu Isao, Sato Nobuhiro
Department of Gastroenterology, Juntendo University School of Medicine, Division of Animal Center, Tokyo Medical and Dental University, Tokyo, Japan.
J Gastroenterol Hepatol. 2004 Feb;19(2):200-4. doi: 10.1111/j.1440-1746.2004.03266.x.
Usually, bacteria are cryopreserved for short-term storage at low and ultra-low temperatures. There are no reports as to whether Helicobacter pylori is a fragile bacteria when stored at low and ultra-low temperatures as compared with other intestinal bacteria. A study was done on seven H. pylori strains and other intestinal bacteria to compare different temperatures for storage of organisms in saline solution.
Seven H. pylori strains, specifically American Type Culture Collection (ATCC) strains 43504 and TN2GF4, and five strains isolated from the present patients were grown on a modified Skirrow's agar for H. pylori. Escherichia coli and Bacteroides distasonis, both representing isolates from the present patients, were grown on trypticase soy blood agar for E. coli, and EG agar for B. distasonis. Culture was for 4-5 days under microaerobic, aerobic and anaerobic conditions at 37 degrees C. Cells were harvested by scraping growth from the solid medium and into sterile saline. The cells were adjusted to concentrations of 109 viable cells/mL in saline and preserved at 4 degrees C, -20 degrees C, or -80 degrees C for 3 weeks before reculture under microaerobic, aerobic and anaerobic conditions at 37 degrees C for 7 days. After incubation, morphologically distinct colonies were counted, isolated, and identified by standard bacteriologic techniques. The H. pylori were morphologically analyzed by electronic microscopy before and after preservation. Mongolian gerbils were inoculated with the cryopreserved H. pylori to evaluate the bacterial infectivity.
Six of the seven H. pylori strains failed to culture after being preserved at 4 degrees C, -20 degrees C, or -80 degrees C. Only ATCC 43504 could be cultured after freezing at -80 degrees C. The number of H. pylori ATCC 43504 before preservation was 9.0 +/- 0.5 (log10 no. organisms/mL) and decreased to 5.7 +/- 0.6 after preservation. Morphologically, all H. pylori except ATCC 43504 strains transformed from a bacillary to a coccoid form after preservation. In addition, none of the H. pylori strains could infect Mongolian gerbils after preservation. Escherichia coli and B. distasonis were recovered. Titers before and after 4 degrees C, -20 degrees C, and -80 degrees C, respectively, were 9.1 +/- 0.2, 8.9 +/- 0.5, 8.6 +/- 0.3, and 8.7 +/- 0.3 for E. coli and 9.1 +/- 0.4, 8.7 +/- 0.6, 8.6 +/- 0.5, and 8.8 +/- 0.3 for B. distasonis.
Helicobacter pylori is a fragile bacteria for storage at low and ultra-low temperatures in comparison with other intestinal bacteria.
通常，细菌在低温和超低温下进行短期保存。与其他肠道细菌相比，关于幽门螺杆菌在低温和超低温下保存时是否为脆弱细菌尚无相关报道。本研究针对7株幽门螺杆菌菌株和其他肠道细菌，比较了在盐溶液中保存生物体的不同温度。
7株幽门螺杆菌菌株，具体为美国典型培养物保藏中心（ATCC）菌株43504和TN2GF4，以及从当前患者中分离出的5株菌株，在改良的用于幽门螺杆菌的斯基罗琼脂上培养。代表当前患者分离株的大肠杆菌和狄氏拟杆菌，分别在用于大肠杆菌的胰蛋白酶大豆血琼脂和用于狄氏拟杆菌的EG琼脂上培养。在37℃的微需氧、需氧和厌氧条件下培养4 - 5天。通过从固体培养基上刮取生长物并放入无菌盐水中收获细胞。将细胞在盐水中调整至10⁹ 个活细胞/mL的浓度，并在4℃、-20℃或-80℃保存3周，然后在37℃的微需氧、需氧和厌氧条件下再培养7天。培养后，对形态不同的菌落进行计数、分离，并通过标准细菌学技术进行鉴定。在保存前后通过电子显微镜对幽门螺杆菌进行形态分析。用冷冻保存的幽门螺杆菌接种蒙古沙鼠以评估细菌的感染性。
7株幽门螺杆菌菌株中的6株在4℃、-20℃或-80℃保存后未能培养出。仅ATCC 43504在-80℃冷冻后能够培养。保存前幽门螺杆菌ATCC 43504的数量为9.0±0.5（log₁₀生物体数/mL），保存后降至5.7±0.6。形态上，除ATCC 43504菌株外，所有幽门螺杆菌菌株在保存后均从杆菌形态转变为球菌形态。此外，所有幽门螺杆菌菌株在保存后均不能感染蒙古沙鼠。大肠杆菌和狄氏拟杆菌得以复苏。大肠杆菌在4℃、-20℃和-80℃保存前后的滴度分别为9.1±0.2、8.9±0.5、8.6±0.3，狄氏拟杆菌分别为9.1±0.4、8.7±0.6、8.6±0.5、8.8±0.3。
与其他肠道细菌相比，幽门螺杆菌是一种在低温和超低温下保存时较为脆弱的细菌。
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