一、R2A/R3A培养基
配方(g/L): R2A R3A
酵母粉 0.5 g 1.0 g
胰蛋白胨 0.5 g 1.0 g
酪蛋白氨基酸(casamino acid) 0.5 g 1.0 g
葡萄糖 0.5 g 1.0 g
可溶性淀粉 0.5 g 1.0 g
磷酸氢二钾 0.3 g 0.6 g
七水硫酸镁 0.05 g 0.1g
丙酮酸钠 0.3 g 0.5g
用磷酸氢二钾或磷酸二氢钾调pH到7.2后,加1L蒸馏水,搅拌溶解,121℃灭菌15min。
特点:
R2A培养基主要用于水的菌落计数,它可以修复被损伤的细菌,支持耐受的微生物的生长。培养要求,低温(20-30℃)长时间培养(5-7天)这样得到的菌落数比常规菌落总数检测的菌落数更符合实际。 R3A培养基主要用于菌株的生化特征描述和鉴定。
参考文献:Reasoner DJ, Geldreich EE. A new medium for the enumeration and subculture of bacteria from potable water. Appl Environ Microbiol. 1985,49(1):1-7.
二、VL55培养基(Sait M et al,2002)(土壤筛菌)
配方(g/L) 1× 2×
2-(N-吗啉基)乙磺酸 1.95 g 3.9 g
MgSO4 0.024 g(0.2mM) 0.048 g(0.4mM)
CaCl2 0.033294 g(0.3mM) 0.066588 g(0.6mM)气体放电灯
(NH4)2HPO4 0.026421 g(0.2mM) 0.052824 g(0.4mM)
亚硒酸盐/钨酸盐溶液 1mL 2mL
微量溶液SL10 1mL 2mL
用0.2mol/L的NaOH和0.1mol/L的KOH混合液调pH到5.5(这与土壤的pH一致)
Sait M等取2×的培养基121℃灭菌15min后,冷却至56℃,加入10mL5%(w/v)的木聚糖溶液,2mL的维生素溶液1,6mL的维生素溶液2。
当量3%洗过的琼脂121℃灭菌15min后,冷却至56℃,与前面培养基混匀后倒平板(Widdel F et al.,1992)。
备注:
亚硒酸盐/钨酸盐溶液(g/L):NaOH 0.5 g,Na2SeO3·5H20 3mg,Na2WO4·2H20 4mg,dd H20 1L,溶解混匀,过滤除菌,4℃备用。(Tschech and 20kv高压直流电源Pfennig, 1984)
微量溶液SL7(g/L):25%HCl 10mL,FeCl2·4H2O 1.5g,CoCl2·6H2O 0.19g,MnCl4·4H2O 0.1g,ZnCl2 0.07g,H3BO3 0.062g,Na2MoO4·2H2O 0.036g,NiCl2·6H2O 0.024g,CuCl2·2H2O 0.017g,dd H20 1L,溶解混匀,过滤除菌,4℃备用。(Widdel F et al.,1981)
微量溶液SL10(g/L):25%HCl 10mL,FeCl2·4H2O 1.5g,CoCl2·6H2O 0.19g,MnCl4·4H2O 0.1g,ZnCl2 0.07g,H3BO3 0.006g,Na2MoO4·2H2O 0.036g,NiCl2·6H2O 0.024g,CuCl2·2H2O 0.002g,dd H20 1L,溶解混匀,过滤除菌,4℃备用。(Widdel F et al.,1983)
维生素溶液1(mg/L):维生素B-x(对氨基苯甲酸)40mg,维生素B7(生物素)10mg,维生素B3(烟酸)100mg,维生素B5(泛酸)50mg,维生素B6(吡哆醇类)150mg,维生素B1(硫胺)100mg,维生素B12(钴胺素)50mg,dd H20 1L,溶解混匀,过滤除菌,4℃备用。(Janssen PH et al.,1997)
维生素溶液2(mg/L):DL-6,8-thioctic acid(硫辛酸)10mg,维生素B2(核黄素)10mg,维生素B9(叶酸)4mg,溶解混匀,过滤除菌,4℃备用。(Janssen PH et al.,1997)
参考文献:
Sait M, Hugenholtz P, Janssen PH. Cultivation of globally distributed soil bacteria from phylogenetic lineages previously only detected in cultivation-independent surveys. Environ Microbiol. 2002,4(11):654-666.
Widdel F, Pfennig N. Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids. I.Isolation of new sulfate-reducing bacteria enriched with acetate from saline environments. Description of Desulfobacter postgatei gen. nov.,sp.nov.Arch Microbiol. 1981,129(5):395-400.
Widdel F, Kohring水净化系统 G, Mayer F. Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids. III. Characterization of the Filamentous Gliding Desulfonema l
imicola gen. nov. sp. nov.,and Desulfonema magnum sp. nov.蓄电池防盗.Arch Microbiol.1983,134(4):286- 294.
Andreas Tschech, Norbert Pfennig. Growth yield increase linked to caffeate reduction in Acetobacterium woodii. Arch Microbiol .1984,137(2)防尘服:163-167.
Janssen PH, Schuhmann A, Mörschel E, Rainey FA. Novel anaerobic ultramicrobacteria belonging to the Verrucomicrobiales lineage of bacterial descent isolated by dilution culture from anoxic rice paddy soil. Appl Environ Microbiol. 1997 ,63(4):1382-1388.
Widdel F, Bak F. Gram-negative mesophilic sulfate-reducing bacteria. In The Prokaryotes, 2nd edn. Balows A., Trüper H.G., Dworkin M., Harder W., Schleifer K.H. (eds). New York: Springer-Verlag, 1992,pp. 3352–3378.
三、改良的VL55培养基(土壤筛菌)(Davis KE et al.,2005)
修改的VL55培养基:凝固剂分琼脂和结冷胶两种;用等量的KH2PO4代替(NH4)2HPO4;(Joseph SJ et al.,2002)
修改的VL55培养基+gellan/琼脂+不同生长底物(Davis KE
et al.,2005) 生长底物1:N-乙酰葡萄糖胺 2mM;(Joseph SJ et al.,2002)
生长底物2:葡萄糖,半乳糖,木糖,阿拉伯糖混合物,每种各0.5mM;(Joseph SJ et al.,2002)
生长底物3:半乳糖醛酸酯;葡萄糖醛酸酯;抗坏血酸;葡糖酸盐,每种各0.5mM;(Joseph SJ et al.,2002)
生长底物4:乙酸盐,苯甲酸盐,乳酸盐,甲醇,每种各0.5mM;(Joseph SJ et al.,2002)程控电压衰减器