| 摘要： | ||
| 目的：发明一种新的细菌鞭毛镀银染色方法。 方法：染色媒染剂由A、B 2 种溶液组成，A液是酸化的FeCl3溶液，B液是含有甲醛的丹宁酸溶液，A、B 液混合后，微加热，染涂片50s，洗净涂片后镀银染色。共有19属34 种228株细菌，每株菌都进行固体培养和液体培养进行鞭毛染色，并采用West氏评分法对鞭毛染色质量进行评价。 结果：鞭毛形态及其在菌体的位置极易观察。228 株细菌获得良好的鞭毛染色质量，血琼脂平板培养平均每株菌获得4.7分，肉汤培养获得4.6分。与一些肠杆菌株不同,100 株非发酵菌血琼脂平板培养鞭毛染色均获得5分,而99 株肠杆菌肉汤培养鞭毛染色获得4分以上。一些弧菌科细菌鞭毛位置分布因这2种培养方法的不同而有差异。并发现1株非O1群霍乱弧菌有单侧毛和亚极端毛。 结论：这种鞭毛染色方法操作简单、快速,试剂稳定，重复性好。由于可靠性好，可以作为常规方法。非发酵菌适合于固体培养进行鞭毛染色获得最佳效果，液体培养对于一些肠杆菌鞭毛染色更为适合。 关键词：细菌； 鞭毛； 镀银染色； 媒染剂； 培养基 本文作者：谷海瀛 |
A new silver－plating method for staining flagella
谷海瀛
GU Haiying. Clinical Laboratory Department, Hainan Provincial People’s Hospital, Haikou 570311, P. R. China
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
海南省人民医院检验科(E-mail: clinmi-crobiollab @cmmail. com)海口570311
细菌侧毛作为细菌分类主要依据之一[1]，说明细菌鞭毛染色在细菌鉴定中是很重要的技术。细菌鞭毛染色的方法文献有很多报道，但基本方法可以归纳为：Leifson法[2]、Gray法[2]、镀银法[3]、Ryu法[4]，但这些方法操作复杂或染液不稳定或着色欠佳，尽管科赫(Koch)在一个世纪前就发明了细菌鞭毛染色技术，但至今仍没有一个稳定而简易可推行的方法。
本文报告一种新的细菌鞭毛镀银染色方法，通过19属34种228株细菌鞭毛染色证实该方法操作简单、快速，可作为常规方法推广。
材料和方法
标准菌株(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。
质控菌株(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。
临床分离菌株(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。
鞭毛肉汤：参照文献[5]配制
Tryptose (DIFCO)：10.0g/ L，
NaCl：2.5g/L，
K2HPO4：1.0g/L，
pH：7.0，121℃
灭菌15 min。
菌株培养：所有菌株均分别划线接种血琼脂平板和鞭毛肉汤管2种培养基，30℃培养18～24h。鞭毛肉汤管出现微混浊即在显微镜下观察动力，每株有动力菌分别制备这2种培养物的涂片。
涂片制备：血平板培养物：在处理过的洁净玻片一端加2～3滴蒸馏水，用灭菌过的接种针蘸取蒸馏水后沾取单个菌落，轻轻点于玻片上蒸馏水中，轻轻晃动，使菌体分散于玻片上，室温风干或置于35℃温箱干燥。2ml鞭毛肉汤培养物加入0.1ml 37%福尔马林，1200×g离心20min，倾掉上清后加入2ml蒸馏水轻轻晃动使菌体分散，再离心20min，再加入适量蒸馏水，变成微乳混浊，制成涂片[6]。
染色液配制：媒染剂A：3.0g FeCl3 6H2O ，100ml0.01mol/ L HCl溶液，室温存放，长期稳定。媒染剂B：单宁酸(SIGMA)15.0g 溶解于100ml蒸馏水中，加37 %甲醛1.0ml。室温存放，长期稳定。银染液C：按文献[7]。AgNO3 5.0g溶于100ml蒸馏水。取出10.0ml备用，向余下的90ml硝酸银溶液缓缓滴加浓氨水，边加边摇动直到形成沉淀又渐渐溶解恰好形成澄清溶液，再用备用AgNO3 溶液慢慢回滴形成稳定薄雾状溶液。取出20ml，余下染液避光密封，4℃冰箱存放。
染色方法：取A 液0.1ml(4滴)加入带有塞的试管内，再加入B液0.1ml(4滴)，充分混合，用酒精灯火焰轻微缓缓加热10～20s，稍冷却。这样处理过的A、B混合液染40s(30～60s)即可，蒸馏水缓慢冲洗干净。A、B混合物不稳定，加热后10 min内使用，否则影响染色质量。
本文报告的方法不同于已发表的银染法〔3 ,7 ,14 ,15〕，丹宁酸含量略有增加，将媒染分为两部分，增强了稳定性，氯化铁的酸化处理使此溶液能长期稳定。Finegan和Smith〔6〕报告根据Porter等人〔15〕改良法使用老化媒染剂增强鞭毛染色，研究证明，使用加热过的媒染剂能提高染色速度且背景清晰，鞭毛粗大；甲醛必不可少，但硫酸钾铝成分对于本试验是不适用的。
总之，本文报告的鞭毛染色方法具有简单、快速，重复性好，染色后的鞭毛清晰等多种优点，从而可作为常规方法。
参考文献
1.Holt JG, Krieg NR, Sneath PHA, et al . Bergey′s Manual of determinative
bacteriology(Ninth Edition). William and Wilkins Co. Baltimore, 1994,P190－192.
2.Murray RGE, Doetsch RN, Robinow CF. Determinative and cytological light microscopy. In： Gerhardt P, Murray RGE, Wood WA and Krieg NR. Mehthods for general and molecular bacteriology. American So-ciety for Microbiology. Washington DC, 1994, P35-36.
3.Rhodes ME.The cytology of Pseudomonas spp. as revealed by a silverplating staining method.J Gen Microbiol, 1958, 18: 639－648.
4.Kodaka H, Armfield AY, Lombard GL, et al. Practical procedure for demonstrating bacterial flgella. J Clin Microbiol, 1982, 16: 948－952.
5.Clark WA. A simplified leifson flagella stain. J Clin Microbiol, 1976, 3:632－634.
6.Finegan SM, Smith RA. Enhanced staining of bacterial flagella using aged mordant in the silver stain. Biotechnic &Histochemistry, 1994, 69: 199－202.
7.West M, Burdash NM, Freimuth F. Simplified silver2plating stain for flagella. J Clin Microbiol, 1977, 6: 414－419.
8.Forbes L. Rapid flagella stain. J Clin Microbiol, 1981, 13: 807－809.
9.Toshio SHIMADA, Riichi SAKAZAKI, Kenji SUZUKI. Peritrichous flagella in mesophilic strains of Aeromonas . Japan J Med Biol, 1985, 38:141－145.
10.Kohaku INOUE, Yoshimasa KASAKO, Kenji SUZUKI, et al. Peritrichous flagella in plesiomonas shigelloids strains. Japan J Med Sci Biol,1991, 44: 141－146.
11.Krieg NR, Holt JG, Murray RGE, et al. Bergey′s Manual of systematic bacteriology(Volume1).Williams and Wilkins Co., Baltimore. 1984,P518.
12.Tison DL. Vibrio. In: Murray PR, Baron EJ, Pfaller MA, et al. Manual of Clinical Microbiology (Seven Edition). American Society for Microbiology. Washington DC, 1999, P497.
13.Clarridge JE, Mullins JM. Microscopy and staining. In: Howard BJ,Keiser JF, Weissfeld AS, et al . Clinical and pathogenic microbiology.(Second Edition) Mosby, 1994, P112－113.
14.Blenden DC, Goldberg HS. Silver impregnation stain for leptospira and flagella. J Bacteriol, 1965, 89: 899－900.
15.Porter JR, Thomulka KW, Smith RA. Demonstrating bacterial flagella.The American Biology Teacher, 1992, 54: 108－111.
染色方法：取A 液0.1ml(4滴)加入带有塞的试管内，再加入B液0.1ml(4滴)，充分混合，用酒精灯火焰轻微缓缓加热10～20s，稍冷却。这样处理过的A、B混合液染40s(30～60s)即可，蒸馏水缓慢冲洗干净。A、B混合物不稳定，加热后10 min内使用，否则影响染色质量。
本文报告的方法不同于已发表的银染法〔3 ,7 ,14 ,15〕，丹宁酸含量略有增加，将媒染分为两部分，增强了稳定性，氯化铁的酸化处理使此溶液能长期稳定。Finegan和Smith〔6〕报告根据Porter等人〔15〕改良法使用老化媒染剂增强鞭毛染色，研究证明，使用加热过的媒染剂能提高染色速度且背景清晰，鞭毛粗大；甲醛必不可少，但硫酸钾铝成分对于本试验是不适用的。
总之，本文报告的鞭毛染色方法具有简单、快速，重复性好，染色后的鞭毛清晰等多种优点，从而可作为常规方法。
参考文献
1.Holt JG, Krieg NR, Sneath PHA, et al . Bergey′s Manual of determinative
bacteriology(Ninth Edition). William and Wilkins Co. Baltimore, 1994,P190－192.
2.Murray RGE, Doetsch RN, Robinow CF. Determinative and cytological light microscopy. In： Gerhardt P, Murray RGE, Wood WA and Krieg NR. Mehthods for general and molecular bacteriology. American So-ciety for Microbiology. Washington DC, 1994, P35-36.
3.Rhodes ME.The cytology of Pseudomonas spp. as revealed by a silverplating staining method.J Gen Microbiol, 1958, 18: 639－648.
4.Kodaka H, Armfield AY, Lombard GL, et al. Practical procedure for demonstrating bacterial flgella. J Clin Microbiol, 1982, 16: 948－952.
5.Clark WA. A simplified leifson flagella stain. J Clin Microbiol, 1976, 3:632－634.
6.Finegan SM, Smith RA. Enhanced staining of bacterial flagella using aged mordant in the silver stain. Biotechnic &Histochemistry, 1994, 69: 199－202.
7.West M, Burdash NM, Freimuth F. Simplified silver2plating stain for flagella. J Clin Microbiol, 1977, 6: 414－419.
8.Forbes L. Rapid flagella stain. J Clin Microbiol, 1981, 13: 807－809.
9.Toshio SHIMADA, Riichi SAKAZAKI, Kenji SUZUKI. Peritrichous flagella in mesophilic strains of Aeromonas . Japan J Med Biol, 1985, 38:141－145.
10.Kohaku INOUE, Yoshimasa KASAKO, Kenji SUZUKI, et al. Peritrichous flagella in plesiomonas shigelloids strains. Japan J Med Sci Biol,1991, 44: 141－146.
11.Krieg NR, Holt JG, Murray RGE, et al. Bergey′s Manual of systematic bacteriology(Volume1).Williams and Wilkins Co., Baltimore. 1984,P518.
12.Tison DL. Vibrio. In: Murray PR, Baron EJ, Pfaller MA, et al. Manual of Clinical Microbiology (Seven Edition). American Society for Microbiology. Washington DC, 1999, P497.
13.Clarridge JE, Mullins JM. Microscopy and staining. In: Howard BJ,Keiser JF, Weissfeld AS, et al . Clinical and pathogenic microbiology.(Second Edition) Mosby, 1994, P112－113.
14.Blenden DC, Goldberg HS. Silver impregnation stain for leptospira and flagella. J Bacteriol, 1965, 89: 899－900.
15.Porter JR, Thomulka KW, Smith RA. Demonstrating bacterial flagella.The American Biology Teacher, 1992, 54: 108－111.
