本文關鍵詞： 空腸彎曲菌 VBNC 活性 形態(tài) CTC-DAPI　出處：《河北醫(yī)科大學》2014年碩士論文　論文類型：學位論文

【摘要】：目的：1本實驗室保存的不同來源空腸彎曲菌在4℃饑餓條件下在35天的觀察期內(nèi)能否進入活的但非可培養(yǎng)狀態(tài)。 2觀察本實驗室保存的不同來源空腸彎曲菌在4℃饑餓條件下在35天的觀察期內(nèi)其革蘭染色形態(tài)隨時間的變化。 方法:1空腸彎曲菌菌株的復蘇和傳代：取自神經(jīng)病學實驗室（河北醫(yī)科大學第二醫(yī)院）-80℃冰箱中保存的格林-巴利綜合征患者分離培養(yǎng)出的空腸彎曲菌菌株LC株，lulei株，由雞糞便中分離培養(yǎng)出的chicken株和非GBS源的NCTC11168株，待凍存的菌株自然消融后，接種于哥倫比亞血平板（含7%無菌脫纖維羊血）上，放置于42℃微需氧（10%CO2、5%O2、85%N2）條件的三氣培養(yǎng)箱中，所有菌株培養(yǎng)24小時對應的P1代菌株回收，取P1代菌落三區(qū)劃線接種于含7%無菌脫纖維羊血的哥倫比亞血平板上，放置于三氣培養(yǎng)箱，42℃微需氧（10%CO2、5%O2、85%N2）條件下，培養(yǎng)24小時獲得各菌株相應的P2代菌株，取P2代菌落三區(qū)劃線接種法接種于含7%無菌脫纖維羊血的哥倫比亞血平板培養(yǎng)基上，置于三氣培養(yǎng)箱，42℃微需氧（10%CO2、5%O2、85%N2）條件下，培養(yǎng)24小時得到各菌株相應的P3代菌株。 2鑒定空腸彎曲菌：革蘭染色法顯示菌株為革蘭陰性桿狀菌，，馬尿酸水解試驗結果顯示為陽性。 3收菌用無菌接種環(huán)分別刮取4株空腸彎曲菌少量P3代細菌于盛有3ml PBS（PH為7.2+-0.1）液的無菌西林瓶內(nèi)，振蕩器振蕩充分搖勻制成菌懸液，每株菌株做4個備份菌懸液并做好記號，所有的菌懸液濃度用細菌濁度儀定量在3*108cfu/ml。 4饑餓狀態(tài)：將上述盛有菌懸液的無菌西林瓶置于4℃冰箱內(nèi)，不需要振蕩。 5分別在菌懸液置于冰箱內(nèi)的第0天，第5天，第10天......用CTC-DAPI雙染法計數(shù)總的細菌數(shù)，活細菌數(shù)，平板直接計數(shù)法（HPC）計數(shù)可培養(yǎng)細菌數(shù)。分別對每株空腸彎曲菌的一個備份同時計數(shù)總細菌數(shù)，活細菌數(shù)及可培養(yǎng)細菌數(shù)。 6平板直接計數(shù)法用無菌雙蒸水連續(xù)稀釋的菌懸液樣本（0.1ml）一式三份接種到含7%無菌脫纖維羊血的哥倫比亞血平板培養(yǎng)基上。微需氧（10%CO2、5%O2、85%N2）42℃條件下孵育48h，計數(shù)在適當?shù)南♂尪认碌腃FUs，并可以計數(shù)原來起初的樣本中的CFUs。當可培養(yǎng)細胞少于300/ml時，0.1ml水樣本涂布在10個含5%馬血的哥倫比亞血平板培養(yǎng)皿上。 7CTC-DAPI雙染法在相應的時間點，取出來自空腸彎曲菌懸浮液中的樣本并根據(jù)Cappelier et al.(1997)技術用CTC和DAPI染色細胞。為了激發(fā)細胞呼吸，0.5ml腦心浸液和100μl的0.05g/mL丙酮酸溶液加入到要進行分析的0.5ml菌懸液中。CTC用雙蒸水稀釋到最終濃度即5mmol/L,然后混合液在微需氧42℃下孵育4h。接著細胞會經(jīng)多聚黑碳膜過濾（0.2μm/孔，直徑25mm），并用5μg/mL DAPI液覆蓋5min，目的是復染。最后，染色劑會被真空抽濾，在蓋玻片蓋上前會滴上非熒光的油。405nm的激發(fā)濾光片和455nm二色鏡的Olympus BX53熒光顯微鏡來觀察，這樣可以同時看到這兩種染色。隨機計數(shù)每個濾片的20個顯微視野。每個樣本，計數(shù)4個濾片。呼吸的細胞計數(shù)RCCs顯示CTC甲臘晶體，總細胞計數(shù)TCCs是用DAPI染色的。結果可以換成相應的原始樣本中每mL細菌數(shù)，并可以計算出RCCs和TCCs的比例。實驗一式三份進行。 8根據(jù)菌株的狀態(tài)計數(shù)（細菌總數(shù)，和培養(yǎng)的菌數(shù)的活菌數(shù)）繪制隨時間變化的曲線，當平板計數(shù)（HPC）計數(shù)活細胞數(shù)不為零時，細菌便進入了VBNC狀態(tài)。 9相應時間點，取平板計數(shù)法后平板上生長的單菌落，進行革蘭染色。在相應的時間點，于干凈的載玻片上滴一小滴生理鹽水，用接種環(huán)挑取單菌落于生理鹽水中涂布均勻；自然干燥室溫；在酒精燈火焰幻燈片固定3~4次，每次1~2S；將載玻片置于支架上，順次使用結晶紫1min，自來水沖刷，盧戈氏碘液1min，自來水沖洗，95%酒精脫色0.5min，自來水沖洗，稀釋復紅1min，自來水沖洗（每次自來水充分沖洗）。于室溫自然干燥后用1000倍油鏡觀察。 結果：1檢測的4株空腸彎曲菌菌株在35天觀察期均進入了活的但非可培養(yǎng)（VBNC）狀態(tài)。 2空腸彎曲菌lulei菌株的VBNC狀態(tài)在饑餓20天后進入了活的但非可培養(yǎng)（VBNC）狀態(tài)。 3空腸彎曲菌11168，lc，chicken菌株VBNC狀態(tài)在25天后進入了活的但非可培養(yǎng)（VBNC）狀態(tài)。 4被觀察的4株空腸彎曲菌顯示出了空腸彎曲菌在4℃PBS液中出現(xiàn)不同形態(tài)改變�？漳c彎曲菌11168,chicken，LC菌株在25天的觀察期其形態(tài)經(jīng)歷了桿狀與球狀相互轉化，空腸彎曲菌11168菌株在第0天革蘭染色呈現(xiàn)出紅色桿菌，到第11天可見到大部分革蘭染色呈現(xiàn)出紅色球菌，第21天開始革蘭染色呈現(xiàn)出紅色桿菌。空腸彎曲菌Lc菌株在第0天革蘭染色呈現(xiàn)出紅色桿菌，在第10天開始革蘭染色呈現(xiàn)出紅色球狀，第14天革蘭染色呈現(xiàn)出紅色短桿狀菌，第20天革蘭染色呈現(xiàn)出紅色桿菌�？漳c彎曲菌chicken菌株在第0天革蘭染色呈現(xiàn)出紅色桿菌，在第15天變革蘭染色呈現(xiàn)出紅色球菌，第20天桿菌�？漳c彎曲菌Lulei菌株在第0天革蘭染色呈現(xiàn)出紅色桿菌，在第11天革蘭染色呈現(xiàn)出紫色球菌。 結論：1檢測的4株空腸彎曲菌菌株在35天觀察期均進入了活的但非可培養(yǎng)（VBNC）狀態(tài)。其中空腸彎曲菌lulei菌株的VBNC狀態(tài)在饑餓20天后進入了活的但非可培養(yǎng)（VBNC）狀態(tài)。空腸彎曲菌11168，lc，chicken菌株VBNC狀態(tài)在25天后進入了活的但非可培養(yǎng)（VBNC）狀態(tài)。 2空腸彎曲菌11168,chicken，LC菌株在25天的觀察期其形態(tài)經(jīng)歷了桿狀與球狀相互轉化，空腸彎曲菌11168菌株在第0天革蘭染色呈現(xiàn)出紅色桿菌，到第11天可見到大部分革蘭染色呈現(xiàn)出紅色球菌，第21天開始革蘭染色呈現(xiàn)出紅色桿菌�？漳c彎曲菌Lc菌株在第0天革蘭染色呈現(xiàn)出紅色桿菌，在第10天開始革蘭染色呈現(xiàn)出紅色球狀，第14天革蘭染色呈現(xiàn)出紅色短桿狀菌，第20天革蘭染色呈現(xiàn)出紅色桿菌。空腸彎曲菌chicken菌株在第0天革蘭染色呈現(xiàn)出紅色桿菌，在第15天變革蘭染色呈現(xiàn)出紅色球菌，第20天桿菌�？漳c彎曲菌Lulei菌株在第0天革蘭染色呈現(xiàn)出紅色桿菌，在第11天革蘭染色呈現(xiàn)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.

【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R741

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