幽門螺桿菌在雙相培養(yǎng)基中的生長特性
發(fā)布時間：
2022-12-27
作者：
幽門螺桿菌[Helicobacter Pylori，Hp]的發(fā)現(xiàn)被認(rèn)為在人類胃炎病原學(xué)中具有重要 的作用，此菌1982年首次發(fā)現(xiàn)，由Marshall等所描述并命名為Campylobacter Pyloridis，后來根據(jù)國際細(xì)菌命名規(guī)則[ICNB]改正為C.Pylori。1989年Goodwin等建議把Hp從彎曲菌屬劃出來，而成立一個新的屬，稱為幽門螺桿菌[Helicobacter Pylori，Hp]，目前已在國際性刊物和國際會議上應(yīng)用此名稱。幽門螺桿菌為微需氧菌，地氧較敏感，此菌營養(yǎng)要求高，在普遍蛋白胨水或肉湯中不能生長，在固體培養(yǎng)基上Goodwin[1985]推薦以腦一心浸液瓊脂為基礎(chǔ)，加入7%馬血清，1%Isovitalix(BBL)，萬古霉素2mg/L，萘啶酮酸20mg/L，兩性霉素2mg/L。該菌最適宜生長溫度37°C，42°C條件下則不能生長，37°C下培養(yǎng)，細(xì)菌生長緩慢，培養(yǎng)3d—4d后在微氧條件下，固體培養(yǎng)基上可0.5mm—1mm直徑灰白色透明的菌落，在血平皿上有時可見菌落周圍有輕度溶血，該菌培養(yǎng)時要求溫度高，尤其初代分離時，相對濕度至少要在98%以上，為此我們用布氏瓊脂液體一固體雙相培養(yǎng)基，對幽門螺桿菌的生長曲線進(jìn)行了研究，現(xiàn)將結(jié)果報告如下。
1 材料和方法
1.1 菌株 幽門螺桿菌89404株自北京市積水潭醫(yī)院球潰瘍病患者的胃粘膜中分離；菌株鑒定結(jié)果：氧化酶試驗陽性，過氧化氫酶試驗陽性。1%甘氨酸生長，馬尿酸水解為陰性，37°C生長[+]，25°C生長為[－]。在3.5%NaCl中不生長，對慶大霉素、四環(huán)素、痢特靈等敏感，對萬古霉素抗藥，在布氏血瓊脂上生長良好，有輕微溶血。
1.2 液體—固體雙相培養(yǎng)基的制備 取25cm3組織培養(yǎng)瓶，高壓來菌后加入6%脫纖維羊血布氏瓊脂20ml，平放，待凝固后，將瓶子垂直，再加入新鮮配制的布氏肉湯9ml，置4°C冰箱內(nèi)保存?zhèn)溆谩?/span>
1.3 生長曲線的測定 將全羊備注中加89404的菌株自-70°C冷凍保存的幽門螺桿菌融化，取0.2ml菌液接種在6%布氏血瓊脂平思慮中，于5%O2，10%CO2，85%N2的混合氣體條件下于37°C培養(yǎng)3d后觀察，選取新培養(yǎng)的Hp89404用10%小牛血清布氏肉湯[pH7.2]洗下，作1:10稀釋，每個嘗試各換1支吸管，再經(jīng)充分混勻后，取10-4及10-5兩管進(jìn)行活菌計數(shù)，然后取10-4 菌液1ml(10.15×102cfu/ml)，直接接種在液體—固體雙相培養(yǎng)基中，此時按0h培養(yǎng)的菌數(shù)計算，即在雙相布氏血瓊脂培養(yǎng)中，每毫升含菌液濃度為1.02×102/cfu/ml，然后將雙相培養(yǎng)物置混合氣體培養(yǎng)箱中，經(jīng)不同時間[24h—144h]在布氏血瓊脂平皿上進(jìn)行活菌數(shù)測定。
1.4 pH值測定 將不同時間的菌株液取出，用精密pH試紙測定培養(yǎng)液的pH值，在測定前預(yù)備試驗用數(shù)字式電pH樣校正。
2 結(jié)果
2.1 幽門螺桿菌在固體培養(yǎng)基上生長特點
在固體布氏血瓊脂平皿上，經(jīng)37°C48h混合氣體培養(yǎng)后，用10%小牛血清布氏湯洗下，菌液濃度掃標(biāo)準(zhǔn)比濁管18億/ml菌配制，按10倍稀釋法，取10-4管接種子固體布氏血瓊脂上，經(jīng)37°C72h培養(yǎng)后，菌落計數(shù)為1.02×102cfu/ml，同樣以10-4接種在雙上培養(yǎng)基則為1.14×104cfu/ml，而且在雙相培養(yǎng)基上菌形彎曲較固體培養(yǎng)基上典型，球狀菌體數(shù)量也較少。
2.2 Hp89404在雙相培養(yǎng)基上的生長特點
為了解幽門螺桿菌生長特點，選擇自胃病患者胃粘膜標(biāo)本中分離的菌株[Hp89404]，采用布氏瓊脂雙相培養(yǎng)基進(jìn)行培養(yǎng)，于不同時間各取培養(yǎng)物進(jìn)行活菌計數(shù)，同時還進(jìn)行pH值測定。
生長時間菌落計數(shù)[cfu/ml] PH值 0 1.02×102 7.2 24 2.50×102 6.9 48 4.97×102 6.9 72 1.14×104 6.8 96 1.24×105 6.6 120 2.87×105 6.6 144 9.75×104 6.4
從附表可見，幽門螺桿菌經(jīng)培養(yǎng)0—24h，上潛伏期到緩慢生長期，在血雙相培養(yǎng)基中，于37°C培養(yǎng)12h—20h開始出現(xiàn)渾濁，24h—48h以后，即可出現(xiàn)迅速對數(shù)生長期，并持續(xù)到120h，達(dá)到生長高峰，此時菌落數(shù)最高達(dá)2.87×105cfu/ml，以后隨著培養(yǎng)時間的延長，菌落單位形成數(shù)則漸次下降，在144h時菌數(shù)為9.75104cfu/ml。從顯微鏡下觀察菌體形態(tài)，培養(yǎng)24h—48h,見菌形呈較為典型的彎曲形、螺桿狀或海鷗展翅樣，到72h—96h為多形態(tài)性，有球狀菌形出現(xiàn)。從液相培養(yǎng)液pH測定中24h—144h表明pH上原來7.2下降到6.4—6.9。
3 討論
幽門螺桿菌的生長營養(yǎng)條件要求較高，胃粘膜培養(yǎng)物在固體培養(yǎng)基布氏血瓊脂條件下，經(jīng)37°C24h—48h微氧培養(yǎng)，細(xì)菌生長緩慢，在固體培養(yǎng)基上可見成睛集落細(xì)小 的灰白色透明的菌苔，初代培養(yǎng)經(jīng)鏡檢形態(tài)為典型海鷗展翅樣或S形彎曲菌，但經(jīng)繼代培養(yǎng)，特別是72h以后的培養(yǎng)物菌體?？勺兂啥嘈螒B(tài)性，微球形則尤為多見，用雙相培養(yǎng)物菌液均勻混濁，菌生長較快，保存時間相對也較長，這是否由于液體—固體雙相培養(yǎng)基，其營養(yǎng)及溫度均有利幽門螺桿菌的存活，有待進(jìn)一步研究。
作者：陳晶晶，莊韜，蔣秀高，甘毓麟，劉強(qiáng)， 李文東
Growth characteristics of Helicobacter pylori in biphasic medium
The discovery of Helicobacter pylori (Hp) is believed to play an important role in the etiology of human gastritis. It was first discovered in 1982 and was described by Marshall et al. and named Campylobacter Pyloridis. It was later modified to C. Pylori according to the International Bacterial Nomenclature Rules (ICNB). In 1989, Goodwin and others suggested that Hp be classified from Campylobacter and a new genus, Helicobacter pylori (Hp), was established, which has been used in international journals and conferences. Helicobacter pylori is a micro-aerobic bacterium with high nutritional requirements. It can not grow in general peptone water or broth. Goodwin [1985] recommended to add 7% horse serum, 1% Isovitalix (BBL), 2 mg/L vancomycin, 20 mg/L naphthalene ketonate, 2 mg/L amphotericin on solid medium. . The bacteria can grow slowly at 37 C and 42 C. After 3-4 days of cultivation, the grey-white and transparent colony with 0.5mm-1 mm diameter can be seen on the solid medium. Mild hemolysis can be seen around the colony on the blood platter. The bacteria should be cultured at high temperature. Especially in the initial isolation, the relative humidity should be at least 98%. Therefore, we studied the growth curve of Helicobacter pylori in liquid-solid biphasic medium with Brinell agar. The results are reported as follows.
1 materials and methods
1.1 strain of Helicobacter pylori 89404 was isolated from gastric mucosa of patients with bulbar ulcer in Beijing Jishuitan Hospital. 1% growth of glycine, negative hydrolysis of horse acid, growth of + 37 degrees C, and growth of 25 degrees C. No growth was observed in 3.5% NaCl. It was sensitive to gentamicin, tetracycline and dysentery. It was resistant to vancomycin and grew well on Brinell blood agar with slight hemolysis.
1.2 Liquid-solid two-phase culture medium preparation of 25 cm 3 tissue culture flask, high-pressure bacteria after adding 6% de-fibrous sheep blood Brinell agar 20 ml, placing flat, after coagulation, the bottle will be vertical, and then add fresh preparation of Brinell broth 9 ml, stored in a 4 degree C refrigerator for reserve.
1.3 The growth curve was determined by melting H.pylori strain with 89404 in the whole sheep notes from - 70 degree C cryopreserved, inoculated with 0.2 ml of bacterial liquid in 6% Brucella blood agar, cultured at 37 degree C for 3 days under 5% O 2, 10% CO 2, 85% N 2 mixed gas conditions. The newly cultured Hp89404 was selected and cultured with 10% calf serum Brucella broth [pH7]. 2] Wash, dilute at 1:10, change one straw for each attempt, and then mix well, take 10-4 and 10-5 tubes to count the viable bacteria, then take 10-4 bacterial liquid 1 ml (10.15 *102 cfu/ml), directly inoculate in liquid-solid biphasic medium, at this time according to the number of bacteria cultured in 0 h, that is, in biphasic blood agar culture, every millimeter. The concentration of ascending bacterium solution was 1.02 *102/cfu/ml, then the biphasic culture was placed in a mixed gas incubator, and the viable bacteria number was measured on a Brinell blood agar plate at different time [24h-144h].
1.4 The pH value was determined by taking out the strain solution at different time, using the precision pH test paper to determine the pH value of the culture solution, and preparing the digital pH calibration for the test before the determination.
2 Results
2.1 growth characteristics of Helicobacter pylori on solid medium
On solid blood agar plate, cultured in 37 C 48 h Mixed gas, washed with 10% calf serum Brucella soup, the standard turbidimetric tube was prepared with 1.8 billion/ml bacteria. According to 10-fold dilution method, 10-4 tubes were inoculated on solid blood agar. After cultured at 37 C 72 h, the colony count was 1.02 102 cfu/ml and 10-4 was inoculated as well. The number of globular bacteria in the biphasic medium was less than that in the solid medium.
Growth characteristics of 2.2 Hp89404 on biphasic medium
In order to understand the growth characteristics of Helicobacter pylori, the strain Hp89404 isolated from gastric mucosa samples of patients with gastric diseases was cultured in Brinell agar biphasic medium. The cultures were counted at different times and the pH value was determined.
Growth time colony count [cfu / ml] PH value 0.02 * 102 7.2 24 2.50 * 102 6.9 48 4.97 * 102 6.9 72 1.14 * 104 6.8 96 1.24 * 105 6.6 120 2.87 * 105 6.6 144 9.75 * 104 6.4
It can be seen from the appendix that Helicobacter pylori begins to appear turbidity in the blood biphasic medium from 0 to 24 hours after culture, from the upper incubation period to the slow growth period, from 12 hours to 20 hours after culture at 37 degrees C, and from 24 hours to 48 hours after culture, a rapid logarithmic growth period can occur, and lasts to 120 hours, reaching the growth peak. At this time, the maximum number of colonies reaches 2.87 *105cfu/ml, and then after that. With the extension of culture time, the number of colony forming units decreased gradually, and the number of bacteria was 9.75104cfu/ml at 144 H. The morphology of the bacteria was observed under the microscope. The bacteria were found to be curved, screw-like or seagull-like in shape from 24 h to 48 h. The bacteria were polymorphic and globular in shape from 72 h to 96 h. In the determination of pH in liquid medium, 24h - 144H showed that the original 7.2 of pH dropped to 6.4 - 6.9.
3 discussion
The growth and nutrition of Helicobacter pylori are highly demanded. The gastric mucosa culture is cultured in Brucella's blood agar in solid medium for 37 degrees C24h-48h. The bacteria grow slowly. On the solid medium, the fine grey-white and transparent moss with fine colonies can be seen. The morphology of the primary culture is typical seagull spreading wings or S. Campylobacter formosanus, but after subculture, especially after 72 hours, the cultures often become polymorphic, microspheric is particularly common, using biphasic culture liquid uniform turbidity, bacterial growth faster, relatively long preservation time, this is due to the liquid-solid biphasic medium, its nutrition and temperature are conducive to Helicobacter pylori The survival of bacilli needs further study.