細菌鞭毛鍍銀染色法的創新

摘要：    

    目的：發明一種新的細菌鞭毛鍍銀染色方法。

　　方法：染色媒染劑由A、B 2 種溶液組成，A液是酸化的FeCl3溶液，B液是含有甲醛的丹寧酸溶液，A、B 液混合後，微加熱，染塗片50s，洗淨塗片後鍍銀染色。共有19屬34 種228株細菌，每株菌都進行固體培養和液體培養進行鞭毛染色，並采用West氏評分法對鞭毛染色質量進行評價。

　　結果：鞭毛形態及其在菌體的位置極易觀察。228 株細菌獲得良好的鞭毛染色質量，血瓊脂平板培養平均每株菌獲得4.7分，肉湯培養獲得4.6分。與一些腸杆菌株不同,100 株非發酵菌血瓊脂平板培養鞭毛染色均獲得5分,而99 株腸杆菌肉湯培養鞭毛染色獲得4分以上。一些弧菌科細菌鞭毛位置分布因這2種培養方法的不同而有差異。並發現1株非O1群霍亂弧菌有單側毛和亞極端毛。

　　結論：這種鞭毛染色方法操作簡單、快速,試劑穩定，重複性好。由於可靠性好，可以作為常規方法。非發酵菌適合於固體培養進行鞭毛染色獲得最佳效果，液體培養對於一些腸杆菌鞭毛染色更為適合。

　　關鍵詞：細菌； 鞭毛； 鍍銀染色； 媒染劑； 培養基

　Abstract

Objective：To develope a new technique for bacterial flagella staining.

Methods：Reagent A was acidized ferric chloride solution and B was tannic acid containing formalin. The mixture of A and B was heated slightly, the smears were covered with the cooling mixture for 50 sec. Washed gently with distilled water, the smears were stained with silver solution. 228 strains of 19 genera 34 species were demonstrated for flagella. Each culture was incubated into a tube of flagella broth medium and onto a sheep blood agar (SBA) plate. All stained smears were rated by WEST′s method.

 

Results：The flagella and their position on the bacteria were easily discerned under the microscope, 228 strains of organism growing on SBA plates and in broth medium had the highly ratings with the mean of 4.7 and 4.6, each rating of 100 cultures of nonfermentative rods grown on SBA was highly scored 5 different from that of 104 cultures of enterobacteria grown in flagella broth medium with rating score above 4. As to some strains of Vibrioraceae, flagellar arrangement may differ with the two kinds of incubation media. Single lateral flagellum and subterminal flagellum were demonstrated in 1 strain of V. cholerae non-O1.

 

Conclusions：This simple and fast method with the stable mordant was good in reliability. This technique overcomed almost all the difficulties in flagella staining and so can be used as a routine method. Nonfermentative bacilli growing on solid medium and enterobacteria growing in flagella broth were more suitable for flagella-staining.

 

Key words：Bacteria；Flagella；Silver stain；Mordant；Culture

　　細菌側毛作為細菌分類主要依據之一[1]，說明細菌鞭毛染色在細菌鑒定中是很重要的技術。細菌鞭毛染色的方法文獻有很多報道，但基本方法可以歸納為：Leifson法[2]、Gray法[2]、鍍銀法[3]、Ryu法[4]，但這些方法操作複雜或染液不穩定或著色欠佳，盡管科赫(Koch)在一個世紀前就發明了細菌鞭毛染色技術，但至今仍沒有一個穩定而簡易可推行的方法。

　　本文報告一種新的細菌鞭毛鍍銀染色方法，通過19屬34種228株細菌鞭毛染色證實該方法操作簡單、快速，可作為常規方法推廣。

 

　　材料和方法

　　標準菌株(13 株)：

E.coli ATCC25922、P.aeruginosa ATCC27853 (ATCC43088)、L. monocytogenes ATCC15313、V. parahaemolyticus ATCC17802、P. shigelloides ATCC14029、A. hydrophila ATCC7966、A. caviae ATCC15468、V. mimicus ATCC33653、V. vulnificus ATCC27562、B. pickettii ATCC27511、E. cloacae ATCC43091、P. mirabilis ATCC7002。

　　質控菌株(18 株)：

P. penneri、S. maltophilia、S. putrefaciens、A. veronii biovar sobria、P. pseudoalcaligenes、P. stutzeri、S. enterica subsp. arizonae、A. hydrophila、A. caviae、P. pudida、B. cereus、B. cepacia、A.xylosoxidans、S. marcescens、Y. enterocolitica、P. shigelloides、 L. monocytogenes、P. rettgeri。

　　臨床分離菌株(197株)：

P. aeruginosa46株，P.pudida3株，P. pseudoalcaligenes2株，S . putrefaciens4株，P. mirabilis19株，A. hydrophila 13株，A. caviae2株，A. faecalis7株，P. fluorescens10株，E. coli28株，C. freundii8株，S . marcescens4株，E. cloacae12株，P. vulagaris11株，S .typhi 2株，S .arizonae1株，S . maltophilia15株，V. cholerae non2O1 1株，B .pseudomallei3株，M. morganii6株。

　　鞭毛肉湯：參照文獻[5]配製

Tryptose (DIFCO)：10.0g/ L，NaCl：2.5g/L，K2HPO4：1.0g/L，pH：7.0，121℃滅菌15 min。

　　菌株培養：所有菌株均分別劃線接種血瓊脂平板和鞭毛肉湯管2種培養基，30℃培養18～24h。鞭毛肉湯管出現微混濁即在顯微鏡下觀察動力，每株有動力菌分別製備這2種培養物的塗片。

　　塗片製備：血平板培養物：在處理過的潔淨玻片一端加2～3滴蒸餾水，用滅菌過的接種針蘸取蒸餾水後沾取單個菌落，輕輕點於玻片上蒸餾水中，輕輕晃動，使菌體分散於玻片上，室溫風幹或置於35℃溫箱幹燥。2ml鞭毛肉湯培養物加入0.1ml 37%福爾馬林，1200×g離心20min，傾掉上清後加入2ml蒸餾水輕輕晃動使菌體分散，再離心20min，再加入適量蒸餾水，變成微乳混濁，製成塗片[6]。

　　染色液配製：媒染劑A：3.0g FeCl3 6H2O ，100ml0.01mol/ L HCl溶液，室溫存放，長期穩定。媒染劑B：單寧酸(SIGMA)15.0g 溶解於100ml蒸餾水中，加37 %甲醛1.0ml。室溫存放，長期穩定。銀染液C：按文獻[7]。AgNO3 5.0g溶於100ml蒸餾水。取出10.0ml備用，向餘下的90ml硝酸銀溶液緩緩滴加濃氨水，邊加邊搖動直到形成沉澱又漸漸溶解恰好形成澄清溶液，再用備用AgNO3 溶液慢慢回滴形成穩定薄霧狀溶液。取出20ml，餘下染液避光密封，4℃冰箱存放。

　　染色方法：取A 液0.1ml(4滴)加入帶有塞的試管內，再加入B液0.1ml(4滴)，充分混合，用酒精燈火焰輕微緩緩加熱10～20s，稍冷卻。這樣處理過的A、B混合液染40s(30～60s)即可，蒸餾水緩慢衝洗幹淨。A、B混合物不穩定，加熱後10 min內使用，否則影響染色質量。

 

本文報告的方法不同於已發表的銀染法〔3 ,7 ,14 ,15〕，丹寧酸含量略有增加，將媒染分為兩部分，增強了穩定性，氯化鐵的酸化處理使此溶液能長期穩定。Finegan和Smith〔6〕報告根據Porter等人〔15〕改良法使用老化媒染劑增強鞭毛染色，研究證明，使用加熱過的媒染劑能提高染色速度且背景清晰，鞭毛粗大；甲醛必不可少，但硫酸鉀鋁成分對於本試驗是不適用的。

總之，本文報告的鞭毛染色方法具有簡單、快速，重複性好，染色後的鞭毛清晰等多種優點，從而可作為常規方法。

 

　本文作者：穀海瀛《中華微生物學和免疫學雜誌》

海南省人民醫院檢驗科(E-mail: clinmi-crobiollab @cmmail. com)海口570311

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