不同来源空肠弯曲菌在4℃饥饿条件下能否进入活的但非可培养状态及其形态随时间的改变

发布时间：2018-01-28 18:31

本文关键词： 空肠弯曲菌 VBNC 活性形态 CTC-DAPI　出处：《河北医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：1本实验室保存的不同来源空肠弯曲菌在4℃饥饿条件下在35天的观察期内能否进入活的但非可培养状态。 2观察本实验室保存的不同来源空肠弯曲菌在4℃饥饿条件下在35天的观察期内其革兰染色形态随时间的变化。方法:1空肠弯曲菌菌株的复苏和传代：取自神经病学实验室（河北医科大学第二医院）-80℃冰箱中保存的格林-巴利综合征患者分离培养出的空肠弯曲菌菌株LC株，lulei株，由鸡粪便中分离培养出的chicken株和非GBS源的NCTC11168株，待冻存的菌株自然消融后，接种于哥伦比亚血平板（含7%无菌脱纤维羊血）上，放置于42℃微需氧（10%CO2、5%O2、85%N2）条件的三气培养箱中，所有菌株培养24小时对应的P1代菌株回收，取P1代菌落三区划线接种于含7%无菌脱纤维羊血的哥伦比亚血平板上，放置于三气培养箱，42℃微需氧（10%CO2、5%O2、85%N2）条件下，培养24小时获得各菌株相应的P2代菌株，取P2代菌落三区划线接种法接种于含7%无菌脱纤维羊血的哥伦比亚血平板培养基上，置于三气培养箱，42℃微需氧（10%CO2、5%O2、85%N2）条件下，培养24小时得到各菌株相应的P3代菌株。 2鉴定空肠弯曲菌：革兰染色法显示菌株为革兰阴性杆状菌，，马尿酸水解试验结果显示为阳性。 3收菌用无菌接种环分别刮取4株空肠弯曲菌少量P3代细菌于盛有3ml PBS（PH为7.2+-0.1）液的无菌西林瓶内，振荡器振荡充分摇匀制成菌悬液，每株菌株做4个备份菌悬液并做好记号，所有的菌悬液浓度用细菌浊度仪定量在3*108cfu/ml。 4饥饿状态：将上述盛有菌悬液的无菌西林瓶置于4℃冰箱内，不需要振荡。 5分别在菌悬液置于冰箱内的第0天，第5天，第10天......用CTC-DAPI双染法计数总的细菌数，活细菌数，平板直接计数法（HPC）计数可培养细菌数。分别对每株空肠弯曲菌的一个备份同时计数总细菌数，活细菌数及可培养细菌数。 6平板直接计数法用无菌双蒸水连续稀释的菌悬液样本（0.1ml）一式三份接种到含7%无菌脱纤维羊血的哥伦比亚血平板培养基上。微需氧（10%CO2、5%O2、85%N2）42℃条件下孵育48h，计数在适当的稀释度下的CFUs，并可以计数原来起初的样本中的CFUs。当可培养细胞少于300/ml时，0.1ml水样本涂布在10个含5%马血的哥伦比亚血平板培养皿上。 7CTC-DAPI双染法在相应的时间点，取出来自空肠弯曲菌悬浮液中的样本并根据Cappelier et al.(1997)技术用CTC和DAPI染色细胞。为了激发细胞呼吸，0.5ml脑心浸液和100μl的0.05g/mL丙酮酸溶液加入到要进行分析的0.5ml菌悬液中。CTC用双蒸水稀释到最终浓度即5mmol/L,然后混合液在微需氧42℃下孵育4h。接着细胞会经多聚黑碳膜过滤（0.2μm/孔，直径25mm），并用5μg/mL DAPI液覆盖5min，目的是复染。最后，染色剂会被真空抽滤，在盖玻片盖上前会滴上非荧光的油。405nm的激发滤光片和455nm二色镜的Olympus BX53荧光显微镜来观察，这样可以同时看到这两种染色。随机计数每个滤片的20个显微视野。每个样本，计数4个滤片。呼吸的细胞计数RCCs显示CTC甲腊晶体，总细胞计数TCCs是用DAPI染色的。结果可以换成相应的原始样本中每mL细菌数，并可以计算出RCCs和TCCs的比例。实验一式三份进行。 8根据菌株的状态计数（细菌总数，和培养的菌数的活菌数）绘制随时间变化的曲线，当平板计数（HPC）计数活细胞数不为零时，细菌便进入了VBNC状态。 9相应时间点，取平板计数法后平板上生长的单菌落，进行革兰染色。在相应的时间点，于干净的载玻片上滴一小滴生理盐水，用接种环挑取单菌落于生理盐水中涂布均匀；自然干燥室温；在酒精灯火焰幻灯片固定3~4次，每次1~2S；将载玻片置于支架上，顺次使用结晶紫1min，自来水冲刷，卢戈氏碘液1min，自来水冲洗，95%酒精脱色0.5min，自来水冲洗，稀释复红1min，自来水冲洗（每次自来水充分冲洗）。于室温自然干燥后用1000倍油镜观察。结果：1检测的4株空肠弯曲菌菌株在35天观察期均进入了活的但非可培养（VBNC）状态。 2空肠弯曲菌lulei菌株的VBNC状态在饥饿20天后进入了活的但非可培养（VBNC）状态。 3空肠弯曲菌11168，lc，chicken菌株VBNC状态在25天后进入了活的但非可培养（VBNC）状态。 4被观察的4株空肠弯曲菌显示出了空肠弯曲菌在4℃PBS液中出现不同形态改变。空肠弯曲菌11168,chicken，LC菌株在25天的观察期其形态经历了杆状与球状相互转化，空肠弯曲菌11168菌株在第0天革兰染色呈现出红色杆菌，到第11天可见到大部分革兰染色呈现出红色球菌，第21天开始革兰染色呈现出红色杆菌。空肠弯曲菌Lc菌株在第0天革兰染色呈现出红色杆菌，在第10天开始革兰染色呈现出红色球状，第14天革兰染色呈现出红色短杆状菌，第20天革兰染色呈现出红色杆菌。空肠弯曲菌chicken菌株在第0天革兰染色呈现出红色杆菌，在第15天变革兰染色呈现出红色球菌，第20天杆菌。空肠弯曲菌Lulei菌株在第0天革兰染色呈现出红色杆菌，在第11天革兰染色呈现出紫色球菌。结论：1检测的4株空肠弯曲菌菌株在35天观察期均进入了活的但非可培养（VBNC）状态。其中空肠弯曲菌lulei菌株的VBNC状态在饥饿20天后进入了活的但非可培养（VBNC）状态。空肠弯曲菌11168，lc，chicken菌株VBNC状态在25天后进入了活的但非可培养（VBNC）状态。 2空肠弯曲菌11168,chicken，LC菌株在25天的观察期其形态经历了杆状与球状相互转化，空肠弯曲菌11168菌株在第0天革兰染色呈现出红色杆菌，到第11天可见到大部分革兰染色呈现出红色球菌，第21天开始革兰染色呈现出红色杆菌。空肠弯曲菌Lc菌株在第0天革兰染色呈现出红色杆菌，在第10天开始革兰染色呈现出红色球状，第14天革兰染色呈现出红色短杆状菌，第20天革兰染色呈现出红色杆菌。空肠弯曲菌chicken菌株在第0天革兰染色呈现出红色杆菌，在第15天变革兰染色呈现出红色球菌，第20天杆菌。空肠弯曲菌Lulei菌株在第0天革兰染色呈现出红色杆菌，在第11天革兰染色呈现出紫色球菌。
[Abstract]:Objective: 1 the different sources of Campylobacter jejuni in the laboratory could enter a living but non culturable state during the 35 day observation period of 35 days under the condition of starvation at 4 degrees centigrade.
2 the different sources of Campylobacter jejuni preserved in our laboratory were observed with time during the 35 day observation period of 35 days under the starvation of 4 degrees centigrade.
Methods: 1 strains of Campylobacter jejuni resuscitation and passage: from the neurology Laboratory (the second hospital of Hebei Medical University) -80 C refrigerator Green Barre syndrome patients with isolated Campylobacter jejuni strain LC strain, Lulei strain, chicken strain isolated from chicken feces and non GBS source in NCTC11168 strains. To be frozen after ablation of natural strain, inoculated on Columbia blood agar (sterile and fiber blood containing 7%), placed at 42 DEG C microaerophilic (10%CO2,5%O2,85%N2) three gas conditions in the culture box, all of the strains were cultured for 24 hours, the corresponding P1 strain recovery, Columbia blood agar P1 colony three streak inoculation with 7% aseptic removal on the fiber blood, placed in three gas incubators, 42 C (10%CO2,5%O2,85%N2) micro aerobic conditions, cultured for 24 hours to obtain the corresponding strains of P2 generation strains, P2 colony area three streak Columbia blood agar inoculated with 7% sterile and fiber blood medium, in three gas incubators, 42 C (10%CO2,5%O2,85%N2) micro aerobic conditions, the strains were cultured for 24 hours in P3 generation of strains.
2 Identification of Campylobacter jejuni: gram staining showed that the strains were gram negative bacilli, hippurate hydrolysis test results show positive.
3 bacteria with sterile inoculation loop were scraped from 4 strains of Campylobacter bacteria containing a small amount of P3 generation in 3ml PBS (PH 7.2+-0.1) sterile vial fluid within the oscillator shake made from bacterial suspension, each strains have 4 copies of bacterial suspension and make mark, all of the bacterial suspension the concentration of bacteria in 3*108cfu/ml. quantitative turbidity meter
4 starvation: put the aseptic cicin bottle with the suspension of bacteria in the refrigerator at 4 degrees C, and do not need to oscillate.
5 respectively in the bacterial suspension for zeroth days, was placed in the refrigerator for fifth days, tenth days...... with CTC-DAPI double staining method was used to count the total number of bacteria, the number of live bacteria, flat direct counting method (HPC) to count the number of culturable bacteria. For each strain of Campylobacter jejuni a backup to count the total number of bacteria the number of live bacteria, and the number of culturable bacteria.
The 6 plate direct counting method with sterile double distilled water for dilution of the bacterial suspension sample (0.1ml) three copies of Columbia blood agar was inoculated into sterile and containing 7% fiber blood medium. The micro aerobic (10%CO2,5%O2,85%N2) under 42 DEG C and incubated for 48h counting at the appropriate dilution of the CFUs, and you can count at first, the original samples of CFUs. when cultured cells less than 300/ml, 0.1ml in 10 water samples coated with 5% horse blood Columbia blood agar culture dishes.
7CTC-DAPI double staining method in the corresponding time points, removed from Campylobacter jejuni in suspension samples according to the Cappelier et al. (1997) by CTC and DAPI staining cells. In order to stimulate cell respiration, 0.5ml bacteria 0.05g/mL pyruvate solution 0.5ml brain heart infusion and 100 L was added to the suspension to be analyzed.CTC with double distilled water and diluted to the final concentration of 5mmol/L, and then mixed liquid in micro aerobic incubation at 42 4h. and cells with poly black carbon membrane filtration (25mm diameter 0.2 m/ hole), and 5 g/mL DAPI solution covers 5min, is complex. Finally, dyeing agent by vacuum filtration, on the cover cover will drop on non fluorescent oil.405nm excitation filter and 455nm two color mirror Olympus BX53 fluorescent microscope to observe the forward, so you can see the two staining at the same time. 20 random microscopic field count for each filter. For each sample, counting 4 filter The cell counts of respiration, RCCs, CTC, and total cell count TCCs were DAPI stained. The results could be changed into the number of bacteria per mL in the corresponding original samples, and the ratio of RCCs to TCCs could be calculated. The experiment was carried out in three copies.
8, according to the state count of bacteria (total number of bacteria and the number of viable bacteria), plot the time varying curve. When the count of plate count (HPC) counts the number of live cells is not zero, the bacteria enter the VBNC state.
9 at the same time, the single colony growth plate counting method after plate, Gram stain. At the same time, on the clean glass slides from a small drop of saline, by inoculating loop picking single colony in saline uniform coating; natural drying at room temperature; the alcohol lamp flame slide fixed 3~4 each time, 1~2S; the slide on the support, in order to use crystal violet 1min, tap water flushing, Lugol's iodine solution 1min, tap water, 95% alcohol decolorization of 0.5min, tap water rinse, dilution red 1min, tap water (tap water rinse each fully wash oil 1000 times). Microscopic observation with natural drying at room temperature after.
Results: 1 strains of 4 strains of Campylobacter jejuni were found to be in the live but non culturable (VBNC) state during the 35 day observation period.
2 the VBNC state of strain Lulei of Campylobacter jejuni entered the living but non culturable (VBNC) state after 20 days of starvation.
3 the state of strain 11168, LC, and chicken of Campylobacter jejuni, VBNC, entered the living but non culturable (VBNC) state in 25 days.
Was observed in 4 of 4 strains of Campylobacter jejuni showed at 4 C in PBS fluid of different forms of change. 11168 C.jejuni, chicken, LC in the observation period of 25 days after the morphological transformation and rod-shaped globose, 11168 C.jejuni strains in Gram staining showed zeroth days red bacilli to eleventh days can be seen most of gram staining showed red cocci, twenty-first day Gram staining showed red bacilli. Lc of Campylobacter jejuni strains in zeroth days of Gram staining showed red bacilli, Gram staining in tenth days to start showing a red ball, Gram staining showed fourteenth days red short rod-shaped bacteria, Twentieth Gram staining showed red day. Coli chicken of Campylobacter jejuni strains in zeroth days of Gram staining showed red bacilli in fifteenth days to change blue staining showing red cocci, Twentieth days Lulei of Campylobacter jejuni bacillus. The strain showed red bacilli in zeroth day Gram staining and purple coccus in eleventh days of Gram stain.
Conclusion: 4 strains of Campylobacter jejuni strain 1 Detection on the 35 day observation period into the cultivation of viable but non (VBNC). The Lulei strains of Campylobacter jejuni VBNC in 20 days after entering the hunger culture alive but not (VBNC). 11168 C.jejuni, LC, chicken strain VBNC state in 25 days into the cultivation of viable but non (VBNC).
2 Campylobacter jejuni 11168, chicken, LC in the observation period of 25 days after the morphological transformation and rod-shaped globose, 11168 strains of Campylobacter jejuni in zeroth days Gram staining showing red bacilli to eleventh days can be seen most of Gram staining showed red cocci, twenty-first day Gram staining showed red. Coli Campylobacter jejuni strain Lc on the zeroth day of the gram stain showed red bacilli, Gram staining in tenth days to start showing a red ball, fourteenth days of Gram staining showing red rod-shaped bacteria, Twentieth Gram staining showed red day. Coli chicken of Campylobacter jejuni strains in zeroth days gram stain showing the red rods in fifteenth days, change blue staining showing red cocci, Twentieth days. Coli Campylobacter jejuni strain Lulei on the zeroth day of the gram stain showed red bacilli, Gram staining in eleventh days showing a purple coccus.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741

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