搜索

Testing the Effect of UV Radiation on the Survival of Burkholderia glumae
测试紫外线辐照对水稻细菌性谷枯病菌存活的影响   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Molecular Plant Pathology
Dec 2014

Abstract

Burkholderia glumae (B. glumae) is becoming a serious threat in the major rice producing areas of the world. It was reported that Burkholderia spp., including B. glumae, are adapted to a wide range of ecological niches. Different bacterial strains show different levels of UV tolerance which may be due to the presence of different protection mechanisms. Previously we reported that pigment producing strains of B. glumae are more tolerant to UV radiation than non-pigmented strains. Here, we describe the protocol of UV tolerance assay for B. glumae in different exposure times. Using this protocol, we can calculate the survival rate of B. glumae strains, as well as other bacterial species, in exposure to UV radiation.

Materials and Reagents

  1. Microcentrifuge tubes
  2. Cuvettes (BrandTech Macro Methacrylate Cuvette) (Cole-Parmer, catalog number: 759081D )
  3. Spreaders
  4. Bacterial inoculation loops
  5. A metal inoculation needle or sterile tooth picks
  6. Strains of Burkholderia glumae (or other bacterial species)
  7. Luria-Bertani broth (Sigma-Aldrich, catalog number: L3522 )
  8. Bacteriological agar (Sigma-Aldrich, catalog number: A5306 )
  9. Bacto Peptone (BD, DifcoTM, catalog number: 211677 )
  10. Glucose (Sigma-Aldrich, catalog number: G5400 )
  11. Casamino acid (Thermo Fisher Scientific, catalog number: BP1424 )
  12. Nitrofurantoin (MP Biomedicals, catalog number: 155881 )
  13. Luria-Bertani broth (LB) agar plates (see Recipes)
  14. Casamino acid-peptone-glucose (CPG) agar plates (see Recipes)

Equipment

  1. Laminar hood installed with a UV light bulb (The Baker Company, EdgeGard®)
  2. Spectrophotometer (Thermo Fisher Scientific, model: BioMate3 )
  3. Incubator (GMI, New Brunswick Scientific, model: Innova 4230 )
  4. Germicidal fluorescent bulb, 15 W (General Electric Company, model: G15T8 )

Procedure

Note: For handling bacterial cultures, aseptic techniques must be used and each step should be carried out in a sterile laminar flow hood when practicable.
Day 1

  1. Streak the bacterial cells from glycerol stock on an LB agar plate supplemented with nitrofurantoin (100 µg/ml), an antibiotic to which B. glumae is naturally resistant. 500x stock solution of nitrofurantoin (50 ml/ml) should be prepared by dissolving in dimethylformamide (DMF).
  2. Incubate the LB plate at 37 °C (optimal temperature for bacterial growth) overnight (~ 18 h).

Day 2

  1. Streak the overnight-grown bacterial cells from a single colony on a CPG agar plate, using a metal inoculation needle or a sterile toothpick. Incubate the inoculated CPG agar plate at 30 °C (optimal temperature for the production of pigment, which is a main determinant for bacterial tolerance to UV) for 48 h (Figure 1).



Figure 1. Strains of Burkholderia glumae grown on CPG agar plates. The upper-left and bottom plates: Pigment-producing strains of B. glumae. The upper-right plate: A pigment-deficient strain of B. glumae. Photo was taken after 48 h of incubation at 30 °C.

Day 4

  1. Resuspend the bacterial colonies grown on CPG agar in sterile water in a sterile microcentrifuge tube by flicking with hands.
  2. Adjust the bacterial concentration to OD600 = 0.1 (~0.5 x 107 CFU/ml), using a spectrophotometer.
  3. Dilute the bacterial suspension with 1:10 ratio in sterile dH2O.
  4. Spread 100 µl of the diluted B. glumae cells on a CPG plate.
  5. Expose B. glumae cells to UV light from a G15T8 germicidal fluorescent bulb, 15 W in a laminar hood at the distance of 70 cm with 253.7 nm wave length for various durations (Figure 2). The lid should be off during the UV exposure.


    Figure 2. An image showing the UV irradiation procedure

  6. To determine the number of CFU accurately, serial dilutions up to 1:1,000 should be made and spread on CPG agar plates
  7. Incubate the inoculated plates at 30 °C for 48 h.

Day 6

  1. Count the surviving bacterial colonies (Figure 3).


    Figure 3. An image of bacterial colonies that survived UV exposure. The upper two rows: pigment-producing strains of Burkholderia glumae. The botton row: A pigment-deficient strain of B. glumae. From left to right: Each vertical row represents the CPG agar plates exposed to UV for 0, 10, 20, 40, and 60 sec, respectively.

  2. Convert the data to percentage of survival in different time of exposure to UV radiation.
    Survival rate (%) = # colonies from UV-exposed cells/# colonies from non UV-exposed cells x 100

Recipes

  1. LB Agar plates (1L)
    25 g of LB broth
    18 g of agar
    Add dH2O to 1 L
    Sterilize by autoclaving on liquid cycle
  2. CPG agar plates (1 L)
    1.5 g of Casamino acid
    10 g of Peptone
    5 g of Glucose
    18 g of agar
    Add dH2O to 1 L
    Sterilize by autoclaving on liquid cycle

Acknowledgments

This work was supported by the USDA NIFA (Hatch Project #: LAB93918 and LAB94203), the Louisiana State University Agricultural Center, and the Louisiana Board of Regents (the Research and Development Program: LEQSF(2008-11)-RD-A-02). This protocol was modified from our previous work published in PLoS ONE (Karki et al., 2012).

References

  1. Karki, H. S., Shrestha, B. K., Han, J. W., Groth, D. E., Barphagha, I. K., Rush, M. C., Melanson, R. A., Kim, B. S. and Ham, J. H. (2012). Diversities in virulence, antifungal activity, pigmentation and DNA fingerprint among strains of Burkholderia glumae. PLoS One 7(9): e45376.

简介

Burkholderia glumae ( B。glumae )正在成为世界主要稻米生产地区的严重威胁。 据报道,伯克霍尔德菌属包括 B。 腮腺炎,适应广泛的生态位。 不同的细菌菌株显示不同水平的UV耐受性,这可能是由于存在不同的保护机制。 以前,我们报告了颜色产生菌株的B。 腮腺炎比非色素菌株更耐紫外线辐射。 在这里,我们描述了紫外线耐受性测定的方案。 腮腺炎在不同的暴露时间。 使用这个协议,我们可以计算的生存率。 腮腺炎菌株,以及其他细菌物种,暴露于紫外线辐射。

材料和试剂

  1. 微量离心管
  2. 比色皿(BrandTech Macro Methacrylate Cuvette)(Cole-Parmer,目录号:759081D)
  3. 撒布机
  4. 细菌接种环
  5. 金属接种针或无菌牙签
  6. (或其他细菌物种)的菌株
  7. Luria-Bertani肉汤(Sigma-Aldrich,目录号:L3522)
  8. 细菌琼脂(Sigma-Aldrich,目录号:A5306)
  9. 细菌蛋白胨(BD,Difco TM ,目录号:211677)
  10. 葡萄糖(Sigma-Aldrich,目录号:G5400)
  11. 酪氨酸(Thermo Fisher Scientific,目录号:BP1424)
  12. 呋喃妥因(MP Biomedicals,目录号:155881)
  13. Luria-Bertani肉汤(LB)琼脂平板(见Recipes)
  14. 酪蛋白氨基酸 - 蛋白胨 - 葡萄糖(CPG)琼脂平板(见Recipes)

设备

  1. 安装有UV灯泡的层压罩(The Baker Company,EdgeGard )
  2. 分光光度计(Thermo Fisher Scientific,型号:BioMate3)
  3. 孵育器(GMI,New Brunswick Scientific,型号:Innova 4230)
  4. 杀菌荧光灯,15W(通用电气公司,型号:G15T8)

程序

注意:为了处理细菌培养,必须使用无菌技术,每个步骤都应在无菌层流罩中进行。
第1天

  1. 将来自甘油储备液的细菌细胞在补充有呋喃妥因(100μg/ml)的LB琼脂板上划线,所述硝化呋喃糖素是天牛抗性的抗疟原虫。应通过溶解在二甲基甲酰胺(DMF)中制备500x呋喃妥因(500ml/ml)的储备溶液
  2. 孵育LB板在37°C(细菌生长的最佳温度)过夜(约18小时)。

第2天

  1. 使用金属接种针或无菌牙签在CPG琼脂平板上从单个菌落中划线过夜生长的细菌细胞。孵育接种的CPG琼脂板在30°C(生产色素的最佳温度,这是对紫外线细菌耐受性的主要决定因素)48小时(图1)。



图1.在CPG琼脂平板上生长的伯克霍尔德氏菌菌株。左上和底部平板:的颜料产生菌株。 glumae 。右上图:B的色素缺陷菌株。 glumae 。在30℃孵育48小时后拍照。

第4天

  1. 通过用手轻拂,将在CPG琼脂上生长的细菌菌落在无菌微量离心管中的无菌水中重悬。
  2. 使用分光光度计将细菌浓度调节至OD 600 = 0.1(?0.5×10 7 sup/CFU/ml)。
  3. 在无菌dH 2 O中以1:10的比例稀释细菌悬浮液。
  4. 传播100微升的稀释的B。在CPG板上的glumae 细胞
  5. 公开 B。在来自G15T8杀菌荧光灯的紫外光下,在层流罩中,在70cm的距离,253.7nm的波长下,在不同的持续时间(图2),将帚毛细胞接种到UV光。在UV暴露期间应关闭盖子。


    图2.显示紫外线照射程序的图像

  6. 为了准确地确定CFU的数量,应当制备高达1:1000的系列稀释液并将其在CPG琼脂板上铺开
  7. 将接种的平板在30℃下孵育48小时

第6天

  1. 计数存活的细菌菌落(图3)

    图3.在UV暴露后存活的细菌菌落的图像。上两行: glumae 。从左到右:每个垂直行代表分别暴露于UV 0,10,20,40和60秒的CPG琼脂板。

  2. 将数据转换为暴露于紫外线辐射的不同时间内的存活百分比。
    存活率(%)=来自UV暴露细胞的#菌落/来自非UV暴露细胞的#菌落×100

食谱

  1. LB琼脂平板(1L)
    25g LB肉汤
    18克琼脂
    将dH <2> O添加到1 L

    通过高压灭菌在液体循环中灭菌
  2. CPG琼脂平板(1L)
    1.5克酪蛋白氨基酸
    10g蛋白胨
    5克葡萄糖
    18克琼脂
    将dH <2> O添加到1 L

    通过高压灭菌在液体循环中灭菌

致谢

这项工作得到美国农业部NIFA(Hatch项目#:LAB93918和LAB94203),路易斯安那州立大学农业中心和路易斯安那州理事会(研究与开发项目:LEQSF(2008-11)-RD-A-02 )。该协议从我们之前在PLoS ONE中公开的工作(Karki等人,2012)修改。

参考文献

  1. Karki,H.S.,Shrestha,B.K.,Han,J.W.,Groth,D.E.,Barphagha,I.K.,Rush,M.C.,Melanson,R.A.,Kim,B.S.and Ham,J.H。 Burkholderia glumae菌株的毒力,抗真菌活性,色素沉着和DNA指纹的多样性。 PLoS One 7(9):e45376。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Karki, H. S. and Ham, J. H. (2016). Testing the Effect of UV Radiation on the Survival of Burkholderia glumae. Bio-protocol 6(5): e1755. DOI: 10.21769/BioProtoc.1755.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要谷歌账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。