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Colony Immunoblotting Assay for Detection of Bacterial Cell-surface or Extracellular Proteins
菌落免疫印迹法检测细菌细胞表面或胞外蛋白   

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Abstract

This simple protocol describes how to detect antigens from agar-grown bacterial colonies transferred to nitrocellulose using specific antibodies. The protocol is well suitable for detection of bacterial proteins exposed on the cell surface or secreted to the extracellular space and it can be modified also for detection of intracellular proteins. The assay can distinguish bacterial clones with different expression rates (high, medium and low) from colonies that do not express target protein. We used this assay for screening of Mat fimbriae-producing Escherichia coli mutants obtained by mini-Tn5 transposon mutagenesis and immunomagnetic separation (Lehti et al., 2013).

Keywords: Protein expression(蛋白的表达), Screening(筛选), Colony(殖民地), Blotting(印迹), Agar plate(琼脂平板)

Materials and Reagents

  1. Bacteria (e.g. Escherichia coli)
  2. Sterile toothpicks or pipette tips
  3. Circular Protran BA85 nitrocellulose membrane, 0.45 μm, 82 mm in diameter (Whatman, catalog number: 10401116 )
  4. Albumin from bovine serum (BSA) (Sigma-Aldrich, catalog number: A7906 )
  5. Tween 20 (Sigma-Aldrich, catalog number: P1379 )
  6. Unconjugated primary antibody/antiserum (e.g. Polyclonal rabbit antiserum against Mat fimbriae of E. coli (Pouttu et al., 2001))
  7. Alkaline phosphatase-conjugated secondary antibody (e.g. Alkaline phosphatase-conjugated swine anti-rabbit immunoglobulins (Dako, catalog number: D0306 ))
  8. 1-Step BCIP/NBT solution (Pierce Antibodies, catalog number: 34042 )
  9. Distilled water
  10. Bacto Tryptone (BD Biosciences, catalog number: 211705 )
  11. Bacto Yeast extract (BD Biosciences, catalog number: 212750 )
  12. Bacto Agar (BD Biosciences, catalog number: 214010 )
  13. 10% (w/v) SDS solution in distilled water
  14. 0.5 M NaOH, 1.5 M NaCl in distilled water
  15. 0.5 M Tris-HCl pH 7.5, 1.5 M NaCl in distilled water
  16. 2x SSC (see Recipes)
  17. LB agar plates(see Recipes)
  18. Phosphate buffered saline (PBS) (see Recipes)
  19. Blocking buffer: 2% (w/v) BSA in PBS (see Recipes)
  20. Antibody dilution buffer: 1% (w/v) BSA in PBS (or the buffer as recommended in the antibody datasheet)(see Recipes)
  21. Washing buffer: 0.05% (v/v) Tween 20 in PBS (see Recipes)

Equipment

  1. 3 mm paper (Whatman 3MM, catalog number: 3030-6185 )
  2. Petri dishes, 90 mm (Sterilin®, catalog number: 101RT )
  3. Beaker, 1,000 ml (Pyrex Corning, catalog number: 1000-1L )
  4. Incubator (Termaks)
  5. Belly dancer laboratory shaker (Stovall Life Science)
  6. Sterile 18-gauge needle (BD Biosciences)
  7. Tweezers (Bochem)

Procedure

  1. Place identical orientation marks in three asymmetric locations on the exterior wall of two fresh agar plates (e.g. LB agar for Escherichia coli). Be sure to include orientation marks on the bottom plate (i.e. not on the lid).
  2. Pick each fresh colony to be tested with a sterile toothpick or pipette tip and make a short streak of about 0.5 cm in length onto the test agar plate and then onto the master agar plate. To keep the colonies in a clear physical order use a grid pattern (see Figure 1) under the plates and streak each colony in an identical position on both plates. A colony density of 100 colonies per plate (9 cm diameter) is optimal and minimizes possibility of cross-contamination between colonies. It is recommended to include a negative and positive control colony onto the plates.


    Figure 1. A grid template for precise orientation of 100 cell colonies on two agar plates. A single grid with 85 mm in diameter is used for a 90 mm Petri dish (the original picture size 19 cm x 10 cm is suitable for printing).

  3. To grow the bacteria, invert the plates and incubate them at an appropriate temperature overnight (e.g. for Escherichia coli at 37 °C). To minimize cross-contamination among neighboring colonies, avoid overgrowth and potential formation of satellite colonies.
  4. Using two tweezers, place a circular nitrocellulose membrane (82 mm in diameter) carefully and evenly on top of the colonies on the test plate for 10-60 sec.
    1. Pre-cool the test plate at 4 °C for approximately 30 min before placing the membrane on its surface so that the agar will not adhere onto the membrane.
    2. To ensure contact with the colonies, take care to avoid introducing air bubbles between the membrane and the agar surface. Hold the membrane at opposite edges with two tweezers and bend it slightly, and then allow the curved membrane to make contact with the center of the plate. Finally, lower the sides gently onto the agar surface. To avoid cross-contamination and smearing of colonies, do not reorientate the membrane once it has been applied.
    3. Mark the membrane orientation according to the three marks on the test plate by stabbing through the membrane with a sterile 18-gauge needle.
    4. Save the master plate in 4 °C for later use.
  5. Using tweezers, peel off the membrane from the agar and transfer the membrane colony-side up to a beaker.
    1. Pour 100 ml of blocking buffer into beaker. Place the beaker on laboratory shaker (Belly dancer) and shake at speed setting 3-4 at room temperature for 5 min to remove excess of colony material.
    2. Decant the blocking buffer.
    3. Repeat steps 5a and 5b two more times.
    4. Pour 100 ml of blocking buffer into beaker and incubate in blocking buffer for 45 min with gently agitation on laboratory shaker (Belly dancer, speed setting 2-3) to ensure complete blocking of unoccupied protein binding sites.
    5. Care should be taken to prevent the membrane from drying out during incubation. Multiple membranes can be placed in a single beaker.
      Note: If working with intracellular proteins, cells should be lysed before blocking. To lyse the cells, transfer the membrane colony-side up from the test plate to a Petri dish containing a sheet of 3 mm (Whatman 3 MM) paper moistened with 2-3 ml of 10% (w/v) SDS solution. Avoid introducing air bubbles. After 5 min incubation, place the membrane on top of the 3 mm paper saturated with 0.5 M NaOH, 1.5 M NaCl for 5 min. Then, neutralize by incubating two times for 5 min on the 3 mm paper soaked with 0.5 M Tris-HCl (pH 7.5), 1.5 M NaCl. Finally, place the membrane on top of the 3 mm paper saturated with 2x SSC for 15 min. Transfer the membrane colony-side up to a beaker and continue with the blocking step 5d.
  6. Prepare a primary antibody dilution in antibody dilution buffer and transfer the membrane to a Petri dish containing the diluted primary antibody (one membrane per Petri dish).
    1. For each 82 mm-diameter membrane, use 7-10 ml of diluted primary antibody. Ensure the membrane is adequately covered with the solution to prevent it from drying out during incubation.
    2. The antibody dilution and incubation conditions depend on your primary antibody; please refer to primary antibody datasheet. It is recommended to start with the same conditions used for Western blotting. For optimal results, perform a titration experiment (e.g. 1:100-1:3,000) and optimize the dilution according to the results.
    3. A serum dilution of 1:500-1:1,000 (5-10 μl in 10 ml) and incubation for 1 hour with gently agitation at room temperature will normally be sufficient.
  7. Wash the membrane three times in 10 ml of washing buffer for 5 min each to remove unbound antibodies.
  8. Prepare a secondary antibody dilution in antibody dilution buffer and replace the washing buffer with the diluted secondary antibody.
    1. Use 7-10 ml of diluted alkaline phosphatase-conjugated secondary antibody per 82 mm-diameter membrane.
    2. Please refer to secondary antibody datasheet for recommended antibody dilution buffer and recommended antibody dilution.
    3. Typically 1:500 to 1:2,000 dilutions of the commercial conjugates are appropriate. Incubate as in step 6.
  9. Wash as in step 7.
  10. To visualize positive colonies reacting with the primary antibodies, replace the washing buffer with 5-10 ml of BCIP/NBT solution and incubate with gentle agitation at room temperature until the desired color develops (usually 2 to 5 min) or background colour begin to appear. The positive control should be dark purple-coloured (see Figure 2).


    Figure 2. A representative colony immunoblotting membrane of Mat fimbriae expression in Escherichia coli MG1655-Rif derivatives. Bacteria subjected to mini-Tn5 transposon mutagenesis and enrichment with immunomagnetic particles coated with anti-Mat antibodies (available from previous work (Pouttu et al., 2001)) were grown overnight on LB agar plates at 37 °C, and colonies transferred onto a nitrocellulose membrane were left to react with anti-Mat antibodies (a dilution of 1:500) and detected with alkaline phosphatase-conjugated secondary antibodies (a dilution of 1:2,000). Colony-represents MG1655-Rif, a negative control for Mat fimbriae expression (Lehti et al., 2013). Colony + indicates IHE3034 matA(A536C) (ptet-matA), a positive control of Mat fimbriae expression (Lehti et al., 2013). Colonies 1 to 4 are candidate Mat-fimbriated MG1655-Rif Tn5 mutants.

  11. Stop the reaction by rinsing the membrane several times in distilled water.
  12. Allow the membrane to air dry in the dark.
  13. Find the desired colonies by matching the colonies on the master plate to the reaction spots in the membrane by using orientation marks. Prepare pure cultures of the reactive colonies onto fresh agar plates.

Recipes

  1. 2x SSC
    0.3 M NaCl, 0.03 M sodium citrate (pH 7.0) in distilled water
  2. LB agar plates (1 L) (approximately 40-45 plates)
    Add to 800 ml of H2O
    10 g Tryptone
    5 g Yeast extract
    5 g NaCl
    15 g Agar
    If needed, adjust pH to 7.1 with NaOH
    Add H2O to final volume of 1 L and sterilize by autoclaving (20 min, 121 °C).
    Cool down to 55 °C and supplement with appropriate antibiotics.
    Dispense approximately 20 ml per 90 mm-diameter Petri dish.
    Store at 4 °C for up to 2 months
  3. Phosphate-buffered saline (PBS) (pH 7.4) (1 L)
    Add to 800 ml of H2O
    8 g of NaCl
    0.2 g of KCl
    1.44 g of Na2HPO4
    0.24 g of KH2PO4
    Adjust the pH to 7.4 with HCl.
    Add H2O to a total volume of 1 L and sterilize by autoclaving (20 min, 121 °C). 
    Store at room temperature
  4. Blocking buffer
    Add 10 g BSA to 500 ml of PBS and mix well.
    Store at 4 °C.
  5. Antibody dilution buffer
    Add 0.2 g BSA to 20 ml of PBS and mix well. Alternatively, mix 10 ml of blocking buffer with 10 ml of PBS to make 1% BSA in PBS.
    Store at 4 °C.
  6. Washing buffer
    Add 50 μl Tween 20 to 100 ml of PBS and mix well.
    Store at room temperature

Acknowledgments

This protocol was modified from Sambrook, J. and Russell, D. (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory). The authors would like to acknowledge University of Helsinki, Viikki Graduate School in Biosciences, the Academy of Finland (ERA-NET PathoGenoMics grant number 118982) and the European Network of Excellence in EuroPathoGenomics EPG (CEE LSHB-CT-2005-512061) for financial support.

References

  1. Lehti, T. A., Bauchart, P., Kukkonen, M., Dobrindt, U., Korhonen, T. K. and Westerlund-Wikstrom, B. (2013). Phylogenetic group-associated differences in regulation of the common colonization factor Mat fimbria in Escherichia coli. Mol Microbiol 87(6): 1200-1222.
  2. Pouttu, R., Westerlund-Wikstrom, B., Lang, H., Alsti, K., Virkola, R., Saarela, U., Siitonen, A., Kalkkinen, N. and Korhonen, T. K. (2001). matB, a common fimbrillin gene of Escherichia coli, expressed in a genetically conserved, virulent clonal group. J Bacteriol 183(16): 4727-4736.

简介

这个简单的协议描述如何使用特异性抗体检测从转移到硝酸纤维素的琼脂生长的细菌菌落的抗原。 该方案非常适合于检测暴露于细胞表面或分泌至细胞外空间的细菌蛋白,并且其还可以被修饰以用于检测细胞内蛋白。 该测定可以区分具有不同表达率(高,中和低)的细菌克隆与不表达靶蛋白的集落。 我们使用该测定法用于筛选通过mini-Tn5转座子诱变和免疫磁性分离获得的产生Mat菌毛的大肠杆菌突变体(Lehti等人,2013)。

关键字:蛋白的表达, 筛选, 殖民地, 印迹, 琼脂平板

材料和试剂

  1. 细菌(例如 大肠杆菌)
  2. 无菌牙签或移液器吸头
  3. 圆形Protran BA85硝酸纤维素膜,0.45μm,直径82mm(Whatman,目录号:10401116)
  4. 来自牛血清(BSA)的白蛋白(Sigma-Aldrich,目录号:A7906)
  5. 吐温20(Sigma-Aldrich,目录号:P1379)
  6. 未结合的一级抗体/抗血清(例如 大肠杆菌Mat菌毛的多克隆兔抗血清(Pouttu等人,2001) >
  7. 碱性磷酸酶缀合的二抗(例如碱性磷酸酶结合的猪抗兔免疫球蛋白(Dako,目录号:D0306))
  8. 1步BCIP/NBT溶液(Pierce Antibodies,目录号:34042)
  9. 蒸馏水
  10. Bacto胰蛋白胨(BD Biosciences,目录号:211705)
  11. Bacto酵母提取物(BD Biosciences,目录号:212750)
  12. Bacto琼脂(BD Biosciences,目录号:214010)
  13. 10%(w/v)SDS蒸馏水溶液
  14. 0.5M NaOH,1.5M NaCl的蒸馏水中洗涤
  15. 0.5M Tris-HCl pH 7.5,1.5M NaCl的蒸馏水溶液
  16. 2x SSC(参见配方)
  17. LB琼脂平板(见配方)
  18. 磷酸盐缓冲盐水(PBS)(见Recipes)
  19. 封闭缓冲液:PBS中的2%(w/v)BSA(参见配方)
  20. 抗体稀释缓冲液:PBS中的1%(w/v)BSA(或抗体数据表中推荐的缓冲液)(参见配方)
  21. 洗涤缓冲液:0.05%(v/v)Tween 20的PBS溶液(参见Recipes)

设备

  1. 3mm纸(Whatman 3MM,目录号:3030-6185)
  2. 培养皿,90mm(Sterilin,目录号:101RT)
  3. 烧杯,1000ml(Pyrex Corning,目录号:1000-1L)
  4. 孵化器(Termaks)
  5. 肚皮舞者实验室摇床(Stovall Life Science)
  6. 无菌18号针(BD Biosciences)
  7. 镊子(Bochem)

程序

  1. 在两个新鲜琼脂平板(例如用于大肠杆菌的LB琼脂)的外壁上的三个不对称位置中放置相同的定向标记。确保在底板上包含方向标记(,而不是盖子)。
  2. 用无菌牙签或移液管吸头挑取待测试的每个新菌落,并在测试琼脂平板上进行长度约0.5cm的短条纹,然后在主琼脂平板上。为了使菌落保持清楚的物理顺序,在板下面使用网格图案(参见图1),并且在每个板上在相同位置划线每个菌落。每个平板100个菌落(9cm直径)的菌落密度是最佳的,并且使菌落之间的交叉污染的可能性最小化。建议在平板上包括阴性和阳性对照菌落。


    图1.用于在两个琼脂平板上精确定向100个细胞集落的网格模板。将直径为85mm的单个网格用于90mm培养皿(原始图片大小为19cm×10 cm适合打印)。

  3. 为了生长细菌,倒置平板并在合适的温度下将它们孵育过夜(例如在37℃下用于大肠杆菌)。为了最小化邻近集落之间的交叉污染,避免 过度生长和潜在形成卫星菌落
  4. 使用两个镊子,将圆形硝化纤维素膜(直径82mm)小心地并均匀地放置在测试板上的菌落顶部10-60秒。
    1. 在将膜放置在其表面上之前,将测试板在4℃下预冷约30分钟,使得琼脂不会粘附在膜上。
    2. 为了确保与菌落的接触,注意避免在膜和琼脂表面之间引入气泡。用两个镊子夹住膜的相对边缘,并稍微弯曲,然后让弯曲的膜与板的中心接触。最后,将两边轻轻地降低到琼脂表面。为了避免菌落的交叉污染和涂片,一旦膜被施用,不重新定向膜。
    3. 根据测试板上的三个标记,通过用无菌18号针刺穿膜来标记膜取向。
    4. 将母板保存在4°C以备以后使用。
  5. 使用镊子,从琼脂上剥离膜并将膜集落侧转移到烧杯。
    1. 将100ml的封闭缓冲液倒入烧杯中。 将烧杯放在实验室摇床(肚皮舞者)上,并在3-4度的速度下在室温下摇动5分钟以除去过量的菌落材料。
    2. 剔除阻塞缓冲区。
    3. 重复步骤5a和5b两次以上。
    4. 将100ml封闭缓冲液倒入烧杯中,在封闭缓冲液中孵育45分钟,在实验室摇床(Belly dancer,速度设置2-3)上轻轻摇动,以确保完全封闭未占据的蛋白质结合位点。
    5. 应注意防止膜在孵育过程中变干。多个膜可以放置在单个烧杯中。
      注意:如果使用细胞内蛋白质,细胞应在阻断前裂解。为了裂解细胞,将膜集落侧从测试板转移到含有用2-3ml 10%(w/v)SDS溶液润湿的3mm片(Whatman 3MM)纸的培养皿中。避免引入气泡。孵育5分钟后,将膜置于用0.5M NaOH,1.5M NaCl饱和的3mm纸的顶部5分钟。然后,通过在用0.5M Tris-HCl(pH 7.5),1.5M NaCl浸泡的3mm纸上孵育两次5分钟来中和。最后,将膜置于用2x SSC饱和的3mm纸的顶部15分钟。将膜菌落一面转移到烧杯,继续阻断步骤5d。
  6. 在抗体稀释缓冲液中制备一抗稀释液,并将膜转移到含有稀释的一抗的培养皿(每个培养皿一个膜)。
    1. 对于每个82mm直径的膜,使用7-10ml稀释的一抗。确保膜被溶液充分覆盖,以防止其在孵育过程中变干
    2. 抗体稀释和孵育条件取决于您的一抗;请参阅一抗抗体数据表。建议从用于Western印迹的相同条件开始。为了获得最佳结果,请进行滴定实验(例如 1:100-1:3,000),并根据结果优化稀释。
    3. 1:500-1:1,000的血清稀释液(5-10ml在10ml中)并在室温下轻轻搅拌孵育1小时通常是足够的。
  7. 在10ml洗涤缓冲液中洗膜三次,每次5分钟,以除去未结合的抗体
  8. 在抗体稀释缓冲液中制备第二抗体稀释液,并用稀释的第二抗体替换洗涤缓冲液
    1. 每82mm直径的膜使用7-10ml稀释的碱性磷酸酶缀合的二级抗体。
    2. 请参考二级抗体数据表中推荐的抗体稀释缓冲液和推荐的抗体稀释液。
    3. 通常商业缀合物的1:500至1:2,000稀释是合适的。 按步骤6进行孵育。
  9. 按照步骤7清洗。
  10. 为了显现与一级抗体反应的阳性菌落,用5-10ml BCIP/NBT溶液替换洗涤缓冲液,并在室温下温和搅拌孵育,直到所需的颜色发展(通常为2至5分钟)或背景颜色开始出现。阳性对照应为深紫色(见图2)。


    图2.在大肠杆菌MG1655-Rif衍生物中表达Mat菌毛的代表性菌落免疫印迹膜。经过mini-Tn5转座子诱变和用包被有抗-TN5抗体的免疫磁性颗粒富集的细菌-Mat抗体(可从以前的工作获得(Pouttu等人,2001))在LB琼脂平板上在37℃下生长过夜,将转移到硝酸纤维素膜上的菌落与抗 - Mat抗体(1:500的稀释),并用碱性磷酸酶缀合的二抗(1:2000的稀释)检测。菌落 - 代表MG1655-Rif,Mat毛毡表达的阴性对照(Lehti等人,2013)。 Colony +表示IHE3034 mat (A536C)( ptet-matA ),Mat fimbriae表达的阳性对照(Lehti等。,2013)。集落1至4是候选的Mat-fimbriated MG1655-Rif Tn5突变体
  11. 通过在蒸馏水中冲洗膜几次来停止反应
  12. 让膜在黑暗中风干。
  13. 通过使用定向标记将母板上的菌落与膜中的反应斑点匹配来找到所需的菌落。 在新鲜的琼脂平板上制备反应性菌落的纯培养物

食谱

  1. 2x SSC
    0.3M NaCl,0.03M柠檬酸钠(pH7.0)的蒸馏水中
  2. LB琼脂平板(1L)(大约40-45平板) 加入到800ml H 2 O中 10克胰蛋白胨
    5克酵母提取物
    5克NaCl
    15克琼脂
    如果需要,用NaOH将pH调节至7.1 加入H 2 O至最终体积为1L,并通过高压灭菌(20分钟,121℃)灭菌。
    冷却至55°C并补充适当的抗生素。
    每90mm直径的培养皿分配约20ml。
    在4°C储存2个月
  3. 磷酸盐缓冲盐水(PBS)(pH 7.4)(1L)
    加入到800ml H 2 O中 8克NaCl
    0.2克KCl
    1.44g的Na 2 HPO 4
    0.24g的KH 2 PO 4 sub/
    用HCl调节pH至7.4。
    加入H 2 O至总体积为1L,并通过高压灭菌(20分钟,121℃)灭菌。
    在室温下贮存
  4. 阻塞缓冲区
    将10克BSA加入500毫升PBS中,充分混合。
    储存于4°C。
  5. 抗体稀释缓冲液
    将0.2g BSA加入20ml PBS中并混合均匀。 或者,将10ml封闭缓冲液与10ml PBS混合,制成1%BSA的PBS溶液。
    储存于4°C。
  6. 洗涤缓冲液
    加入50μlTween 20至100 ml PBS,充分混匀。
    在室温下贮存

致谢

该方案由Sambrook,J。和Russell,D。(2001)Molecular Cloning:A Laboratory Manual(Cold Spring Harbor,NY:Cold Spring Harbor Laboratory)修改。作者要感谢赫尔辛基大学,Viikki生物科学研究生院,芬兰科学院(ERA-NET PathoGenoMics授权号118982)和欧洲优秀的欧洲网络EPG(CEE LSHB-CT-2005-512061)金融支持。

参考文献

  1. Lehti,T.A.,Bauchart,P.,Kukkonen,M.,Dobrindt,U.,Korhonen,T.K.and Westerlund-Wikstrom,B.(2013)。 Phylogenetic group-associated differences in regulation of the common colonization factor Mat fimbria in Escherichia coli /em>。 Mol Microbiol 87(6):1200-1222。
  2. Pouttu,R.,Westerlund-Wikstrom,B.,Lang,H.,Alsti,K.,Virkola,R.,Saarela,U.,Siitonen,A.,Kalkkinen,N.and Korhonen,T.K。(2001)。 是大肠杆菌的常见病毒基因在遗传保守的,强毒的克隆组中表达。细菌 183(16):4727-4736。
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引用:Lehti, T. A. and Westerlund-Wikström, B. (2013). Colony Immunoblotting Assay for Detection of Bacterial Cell-surface or Extracellular Proteins. Bio-protocol 3(17): e888. DOI: 10.21769/BioProtoc.888.
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jakeer hussain
mylan laboratories
How to place the gridded 0.45um membrane on to the agar surface ofter filtration of water sample?
Either the gridded surface of membrane should touch the agar surface or white surface of membrane should touch the agar surface.
11/28/2013 3:58:50 AM Reply