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Determination of the Predatory Capability of Bdellovibrio bacteriovorus HD100
测定噬菌蛭弧菌HD100的掠食能力   

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Abstract

Bdellovibrio bacteriovorus HD100 is an obligate predator that preys upon a wide variety of Gram negative bacteria. The biphasic growth cycle of Bdellovibrio includes a free-swimming attack phase and an intraperiplasmic growth phase, where the predator replicates its DNA and grows using the prey as a source of nutrients, finally dividing into individual cells (Sockett, 2009). Due to its obligatory predatory lifestyle, manipulation of Bdellovibrio requires two-member culturing techniques using selected prey microorganisms (Lambert et al., 2003). In this protocol, we describe a detailed workflow to grow and quantify B. bacteriovorus HD100 and its predatory ability, to easily carry out these laborious and time-consuming techniques.

Keywords: Bdellovibrio bacteriovorus(噬菌蛭弧菌), Predatory bacteria(掠食细菌), Predatory quantification(掠食定量)

Background

In the last years, Bdellovibrio has attracted the interest of the scientific community and several applications have been developed, such as evolution studies (Davidov and Jurkevitch, 2009), identification of new biocatalysts (Martínez et al., 2012), therapeutic applications (Atterbury et al., 2011), or biotechnological applications using Bdellovibrio as a lytic agent for the recovery of value added intracellular bioproducts (Martínez et al., 2016). Due to the growing interest in Bdellovibrio, different indirect methods to quantify this predatory bacterium have been developed (Mahmoud et al., 2007; Lambert and Sockett, 2008; Van Essche et al., 2009). However, direct quantification of Bdellovibrio via double-layer method is still necessary to thoroughly characterize Bdellovibrio predatory capability. Here, we describe a well-established, reliable, and broadly used method that allows Bdellovibrio cell number quantification in predatory co-cultures.

Materials and Reagents

  1. 0.45 µm sterilization filter (Sartorius, catalog number: 16555-K )
  2. 0.22 µm sterilization filter (Sartorius, catalog number: 16532-K )
  3. 10-ml glass test tubes (Fisher Scientific, catalog number: 15175134 )
  4. Glass microscope slides (76 x 26 mm) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 10143562BEF ) (see Note 1)
  5. Glass microscope coverslips (22 x 22 mm) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3306 )
  6. 10 ml syringe (BD, catalog number: 307736 )
  7. Bdellovibrio bacteriovorus HD100 (ATCC, catalog number: 15356 ) (Stolp and Starr, 1963)
  8. Pseudomonas putida KT2440 (ATCC, catalog number: 47054 ) (Nelson et al., 2002)
  9. Glycerol (EMD Millipore, catalog number: 104094 )
  10. Bacto tryptone (BD, BactoTM, catalog number: 211705 )
  11. Yeast extract (Conda, catalog number: 1702 )
  12. Sodium chlorice (NaCl) (EMD Millipore, catalog number: 106404 )
  13. Sodium hydroxide (NaOH) pellets for analysis (EMD Millipore, catalog number: 106498 )
  14. Agar (BD, BactoTM, catalog number: 214010 )
  15. Nutrient broth (NB) (BD, DifcoTM, catalog number: 234000 )
  16. Calcium chloride dihydrate (CaCl2·2H2O) (EMD Millipore, catalog number: 102382 )
  17. Magnesium chloride hexahydrate (MgCl2·6H2O) (EMD Millipore, catalog number: 105833 )
  18. HEPES buffer (Sigma-Aldrich, catalog number: H3375 )
  19. Lysogeny broth (LB) medium (1 L, pH 7.5) (see Recipes)
  20. LB, 1.5% (w/v) agar (see Recipes)
  21. NB medium (1 L) (see Recipes)
  22. CaCl2 and MgCl2 salts (see Recipes)
  23. Diluted nutrient broth (DNB) medium (1 L, pH 7.4) (see Recipes)
  24. DNB, 0.7% (w/v) agar (see Recipes)
  25. DNB, 1.5% (w/v) agar (see Recipes)
  26. HEPES buffer (see Recipes)

Equipment

  1. Centrifuge (Eppendorf, model: 5810 R )
  2. 100 ml flasks
  3. 30 °C chamber (JP Select, catalog number: 001257 )
  4. 30 °C shaking incubator (250 rpm) (Eppendorf, New BrunswickTM, model: Innova® 44 )
  5. Water bath (JP Select, catalog number: 6000138 )
  6. Phase contrast microscopy (Nikon Instruments, model: OPTIFHOT-2 ) (Note 1)
  7. Spectrophotometer (Shimadzu, model: UV-260 )
  8. Leica DFC345 FX camera (Leica Microsystems, model: DFC345 FX )
  9. Autoclave

Procedure

  1. Culture of Bdellovibrio preying upon a prey (Figures 1A and 1B)
    1. Preparation of prey cell suspension (Figure 1A)
      1. In this example, P. putida KT2440 is used as prey. To obtain cell suspensions, grow the prey in NB medium at 30 °C and 250 rpm for 16 h (see Note 2), centrifuge (30 min, 4000 x g, 4 °C) and resuspend to OD600 = 10 in HEPES buffer (see Note 3).
      2. Prey cells can be stored at 4 °C for up to 2 weeks. Immediately prior to use, dilute the cells to OD600 = 1.
    2. Preparation of the preinocule of Bdellovibrio (two-step cultivation) (Figure 1B)
      1. Recover Bdellovibrio from glycerol stocks stored at -80 °C (see Note 4) by adding 50 µl directly to 10 ml of prey cell suspension prepared in DNB medium at OD600 = 1. Incubate at 30 °C and 250 rpm.
      2. After 24 h of predation, transfer 100-300 µl of the co-culture to 10 ml of prey suspension prepared in HEPES buffer at OD600 = 1 (predator-prey ratio of 1:10). Incubate at 30 °C and 250 rpm for 24 h.
        Note: Set up the co-cultures by adding 10 ml of suspension to 100 ml flasks.
    3. Isolation of Bdellovibrio cells (Figure 1B). After predation, filter co-cultures twice through a 0.45 µm filter to recover B. bacteriovorus.
      Note: Use a new filter for the second filtration step.
    4. Set up the co-cultures of interest: transfer 100-300 µl of Bdellovibrio cells to 10 ml of prey suspension prepared in HEPES buffer at OD600 = 1 (predator-prey ratio of 1:10). Incubate at 30 °C and 250 rpm for 24 h.


      Figure 1. Workflow to set up Bdellovibrio co-cultures. A. Preparation of the prey cell suspension (here, P. putida KT2440). B. Preparation of Bdellovibrio cells. Two-step cultivation of Bdellovibrio is needed to obtain the predatory cells for the experiment. C. Double layer method to quantify the cell number of B. bacteriovorus HD100. D. Development of B. bacteriovorus HD100 on a lawn of prey on DNB agar plates after 2-3 days of incubation at 30 °C. Bdellovibrio cell number can be quantified as plaque-forming-units (pfu/ml).

  2. Calculation of the viability of Bdellovibrio and prey cells (Figure 1C)
    Make serial dilutions of the co-cultures from 10-1 to 10-7 (see Note 5) in DNB medium.
    1. Determine Bdellovibrio viability using the double layer method (see Figure 1C) (Lambert et al., 2003):
      1. Add 4 ml of DNB 0.7% (w/v) agar into a glass tube and keep at 45-50 °C in a water bath (see Note 6).
      2. Add 0.5 ml of prey cell suspension (P. putida KT2440 prepared at OD600 = 10 in HEPES buffer; see Figure 1A).
      3. Add 0.1 ml of the appropriate dilution from the co-cultures.
      4. Vortex the tube gently.
      5. Rapidly, pour the mixture onto a DNB 1.5 % (w/v) agar plate.
      6. Incubate the plates for 2-3 days at 30 °C. Bdellovibrio growth is monitored as plaque-forming units per milliliter (pfu/ml) developing on a lawn of the prey (Figure 1D).
    2. Calculation of prey cell viability
      Place 10 µl of each dilution on LB agar plates (see Note 5) and incubate them at 30 °C. Prey cells are counted as colony-forming units per milliliter (cfu/ml).

Data analysis

A representative graph of the predatory activity of Bdellovibrio is shown below (Figure 2). Predator and prey cell number at the beginning of the experiment (0 h) and after 24 h of predation upon P. putida KT2440 are represented.


Figure 2. Predation profile to study the predatory capability of B. bacteriovorus HD100 upon P. putida KT2440. A. Schematic representation of the co-culture involving B. bacteriovorus HD100 and the control culture of P. putida without predator. B. Viability of the prey and predator cells at the beginning of the experiment (0 h) and after 24 h of incubation. The purple bars represent the viability of the prey cells and the orange bars correspond to the predator viable cell number. Error bars indicate the standard deviation of the mean (n ≥ 3).

Notes

  1. If possible, predation events should be checked using a phase contrast microscope to ensure the success of the following steps (see Figure 3).
  2. Select the culture medium based on the prey cells used to grow Bdellovibrio.
  3. To measure OD600, prepare a 1/10 dilution of the culture of interest. E.g., Add 100 µl of the culture to 900 µl of saline solution and mix.
  4. To store Bdellovibrio at -80 °C, add 0.3 ml of 85 % (w/v) glycerol and 0.7 ml of the co-culture of Bdellovibrio and the prey bacteria to a cryogenic vial, and place the tube directly in the -80 °C freezer. Bdellovibrio cells can be revived by simply scratching the ice of the cryogenic vial with a sterile loop and adding it to the prey suspension. There is no need to de-freeze the cryogenic vial for the inoculation.
  5. Dilute samples further if a high concentration of cells is expected. E.g., Prepare 1:10 serial dilutions by adding 100 µl of the co-culture to 900 µl of DNB medium in Eppendorf tubes and repeat until the desired concentration is obtained.
  6. DNB 0.7 % (w/v) agar tubes can be previously prepared and stored at room temperature. Prior to use, melt the tubes in a water bath at 150-200 °C and keep them at 45-50 °C.
  7. Prepare DNB medium (or HEPES buffer) and the CaCl2 and MgCl2 salts separately. Add the salts immediately prior to use.


    Figure 3. Co-culture of B. bacteriovorus HD100 preying on P. putida KT2440 under the microscope. A. Co-culture at the onset of predation (time zero); B. After 24 h of incubation, only predatory cells can be observed. Cultures are routinely visualized using a 100x phase-contrast objective and images taken with a Leica DFC345 FX camera. 

Recipes

  1. Lysogeny broth (LB) medium (1 L)
    10 g Bacto tryptone
    5 g yeast extract
    10 g NaCl
    Adjust to pH 7.5 using 1 N NaOH
    Autoclave at 121 °C for 20 min
  2. LB, 1.5% (w/v) agar (1 L)
    10 g Bacto tryptone
    5 g yeast extract
    10 g NaCl
    Adjust to pH 7.5 using 1 N NaOH
    15 g agar
    Autoclave at 121 °C for 21 min
  3. NB medium (1 L)
    8 g NB
    Autoclave at 121 °C for 20 min
  4. CaCl2 and MgCl2 salts
    2 mM CaCl2·2H2O
    3 mM MgCl2·3H2O
    Sterilize by filtration through a 0.22 µm filter into a sterile container
  5. Diluted nutrient broth (DNB) medium (1 L) (see Note 7)
    0.8 g NB
    Adjust to pH 7.4 using 1 N NaOH
    Autoclave
    Add CaCl2 and MgCl2 salts (see Recipe 4)
  6. DNB, 0.7% (w/v) agar (1 L) (see Note 7)
    0.8 g NB
    Adjust to pH 7.4 using 1 N NaOH
    7 g agar
    Autoclave at 121 °C for 20 min
    Add CaCl2 and MgCl2 salts (see Recipe 4)
  7. DNB, 1.5% (w/v) agar (1 L) (see Note 7)
    0.8 g NB
    Adjust to pH 7.4 using 1 N NaOH
    15 g agar
    Autoclave at 121 °C for 20 min
    Add CaCl2 and MgCl2 salts (see Recipe 4)
  8. HEPES buffer (see Note 7)
    25 mM HEPES buffer
    Adjust to pH 7.8 using NaOH pellets
    Autoclave at 121 °C for 20 min
    Add CaCl2 and MgCl2 salts (see Recipe 4)

Acknowledgments

This protocol was modified and adapted from a predatory assay previously described (Martínez et al., 2016; Lambert et al., 2003). This work was funded by the EU Seventh Framework Programme under grant agreement No. 311815 (SYNPOL), and by grants from the Comunidad de Madrid (P2013/MIT2807) and the Spanish Ministerio de Economía y Competitividad, (BIO2010-21049, BIO2013-44878-R).

References

  1. Atterbury, R. J., Hobley, L., Till, R., Lambert, C., Capeness, M. J., Lerner, T. R., Fenton, A. K., Barrow, P. and Sockett, R. E. (2011). Effects of orally administered Bdellovibrio bacteriovorus on the well-being and Salmonella colonization of young chicks. Appl Environ Microbiol 77(16): 5794-5803.
  2. Davidov, Y. and Jurkevitch, E. (2009). Predation between prokaryotes and the origin of eukaryotes. Bioessays 31(7): 748-757.
  3. Gophna, U., Charlebois, R. L. and Doolittle, W. F. (2006). Ancient lateral gene transfer in the evolution of Bdellovibrio bacteriovorus. Trends Microbiol 14(2): 64-69.
  4. Jurkevitch, E. and Davidov, Y. (2007). Phylogenetic diversity and evolution of predatory prokaryotes. In: Jurkevitch, E. (Ed.). Microbiol Monogr Predatory Prokaryotes Heidelberg. Springer, pp: 11-56.
  5. Jurkevitch, E., Minz, D., Ramati, B. and Barel, G. (2000). Prey range characterization, ribotyping, and diversity of soil and rhizosphere Bdellovibrio spp. isolated on phytopathogenic bacteria. Appl Environ Microbiol 66(6): 2365-2371.
  6. Lambert, C., Smith, M. C. and Sockett, R. E. (2003). A novel assay to monitor predator-prey interactions for Bdellovibrio bacteriovorus 109 J reveals a role for methyl-accepting chemotaxis proteins in predation. Environ Microbiol 5(2): 127-132.
  7. Lambert, C. and Sockett, R. E. (2008). Laboratory maintenance of Bdellovibrio. Curr Protoc Microbiol 9:7B.2.1-7B.2.13.
  8. Mahmoud, K. K., McNeely, D., Elwood, C. and Koval, S. F. (2007). Design and performance of a 16S rRNA-targeted oligonucleotide probe for detection of members of the genus Bdellovibrio by fluorescence in situ hybridization. Appl Environ Microbiol 73(22):7488.
  9. Martínez, V., de la Peña, F., García-Hidalgo, J., Mata, I., García, J. L., and Prieto, M. A. (2012). Identification and biochemical evidence of a medium-chain-length polyhydroxyalkanoate depolymerase in the Bdellovibrio bacteriovorus predatory hydrolytic arsenal. Appl Environ Microbiol 78: 6017-6026.
  10. Martínez, V., Herencias, C., Jurkevitch, E. and Prieto, M. A. (2016). Engineering a predatory bacterium as a proficient killer agent for intracellular bio-products recovery: the case of the polyhydroxyalkanoates. Sci Rep 6: 24381.
  11. Nelson, K. E., Weinel, C., Paulsen, I. T., Dodson, R. J., Hilbert, H., Martins dos Santos, V. A., Fouts, D. E., Gill, S. R., Pop, M., Holmes, M., Brinkac, L., Beanan, M., DeBoy, R. T., Daugherty, S., Kolonay, J., Madupu, R., Nelson, W., White, O., Peterson, J., Khouri, H., Hance, I., Chris Lee, P., Holtzapple, E., Scanlan, D., Tran, K., Moazzez, A., Utterback, T., Rizzo, M., Lee, K., Kosack, D., Moestl, D., Wedler, H., Lauber, J., Stjepandic, D., Hoheisel, J., Straetz, M., Heim, S., Kiewitz, C., Eisen, J. A., Timmis, K. N., Dusterhoft, A., Tummler, B. and Fraser, C. M. (2002). Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol 4(12): 799-808.
  12. Sockett, R. E. (2009). Predatory lifestyle of Bdellovibrio bacteriovorus. Annu Rev Microbiol 63: 523-539.
  13. Stolp, H. and Starr, M. P. (1963). Bdellovibrio Bacteriovorus gen. et sp. n., a predatory, ectoparasitic, and bacteriolytic microorganism. Antonie Van Leeuwenhoek 29: 217-248.
  14. Van Essche, M., Sliepen, I., Loozen, G., Van Eldere, J., Quirynen, M., Davidov, Y., Jurkevitch, E., Boon, N and Teughels, T. (2009). Development and performance of a quantitative PCR for the enumeration of Bdellovibrionaceae. Environ Microbiol Rep 1(4):228-33.

简介

HDD是一种专有捕食者,可以用于各种革兰氏阴性菌。 Bdellovibrio的双相生长周期包括自由游泳攻击阶段和胞内生长期,其中捕食者复制其DNA并使用猎物作为营养源,最终分成单个细胞(Sockett ,2009)。由于其必要的掠夺性生活方式,使用选择的猎物微生物(Lambert等人,2003)的双重培养技术的操作需要Bdellovibrio 。在这个协议中,我们描述了一个详细的工作流程来增长和量化。细菌性HD100及其掠夺能力,轻松实施这些费时费力的技术。

在过去几年中,Bdellovibrio 已经吸引了科学界的兴趣,并且已经开发了若干应用,如进化研究(Davidov和Jurkevitch,2009),鉴定新的生物催化剂(Martínezet al。 (2012)),治疗应用(Atterbury等人,2011),或使用Bdellovibrio的生物技术应用作为回收附加值的溶解剂细胞内生物产物(Martínez等人,2016)。由于对Bdellovibrio 的兴趣日益增加,已经开发了用于量化这种掠食性细菌的不同间接方法(Mahmoud等人,2007; Lambert和Sockett,2008; Van Essche et al。,2009)。然而,通过双层方法直接量化B。o is is is is is is is is is to to to to to to to to to to。to。to to。to。。。。。。。。在这里,我们描述了一种成熟,可靠和广泛使用的方法,允许在掠夺性共培养中进行细胞数量化。

关键字:噬菌蛭弧菌, 掠食细菌, 掠食定量

材料和试剂

  1. 0.45μm灭菌过滤器(Sartorius,目录号:16555-K)
  2. 0.22μm消毒过滤器(Sartorius,目录号:16532-K)
  3. 10 ml玻璃试管(Fisher Scientific,目录号:15175134)
  4. 玻璃显微镜载玻片(76 x 26 mm)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:10143562BEF)(见注1)
  5. 玻璃显微镜盖玻片(22×22mm)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:3306)
  6. 10ml注射器(BD,目录号:307736)
  7. HDD(ATCC,目录号:15356)(Stolp和Starr,1963)
  8. 恶臭假单胞菌KT2440(ATCC,目录号:47054)(Nelson等,2002)
  9. 甘油(EMD Millipore,目录号:104094)
  10. Bacto胰蛋白胨(BD,Bacto TM ,目录号:211705)
  11. 酵母提取物(Conda,目录号:1702)
  12. 氯化钠(NaCl)(EMD Millipore,目录号:106404)
  13. 用于分析的氢氧化钠(NaOH)颗粒(EMD Millipore,目录号:106498)
  14. 琼脂(BD,Bacto TM ,目录号:214010)
  15. 营养肉汤(NB)(BD,Difco TM,目录号:234000)
  16. 氯化钙二水合物(CaCl 2·2H 2 O)(EMD Millipore,目录号:102382)
  17. 氯化镁六水合物(MgCl 2·6H 2 O)(EMD Millipore,目录号:105833)
  18. HEPES缓冲液(Sigma-Aldrich,目录号:H3375)
  19. 溶菌酵母(LB)培养基(1L,pH7.5)(参见食谱)
  20. LB,1.5%(w/v)琼脂(参见食谱)
  21. NB培养基(1L)(参见食谱)
  22. CaCl 2和MgCl 2盐(参见食谱)
  23. 稀释的营养肉汤(DNB)培养基(1L,pH7.4)(参见食谱)
  24. DNB,0.7%(w/v)琼脂(参见食谱)
  25. DNB,1.5%(w/v)琼脂(参见食谱)
  26. HEPES缓冲区(见配方)

设备

  1. 离心机(Eppendorf,型号:5810 R)
  2. 100ml烧瓶
  3. 30°C室(JP选择,目录号:001257)
  4. 30℃振荡培养箱(250rpm)(Eppendorf,New Brunswick TM,型号:Innova 44)
  5. 水浴(JP选择,目录号:6000138)
  6. 相位显微镜(Nikon Instruments,型号:OPTIFHOT-2)(注1)
  7. 分光光度计(Shimadzu,型号:UV-260)
  8. 徕卡DFC345 FX相机(Leica Microsystems,型号:DFC345 FX)
  9. 高压灭菌器

程序

  1. 掠夺猎物的Bdellovibrio 的文化(图1A和1B)
    1. 捕食细胞悬液的制备(图1A)
      1. 在这个例子中,恶臭 KT2440用作猎物。为了获得细胞悬浮液,在NB培养基中在30℃和250rpm下将猎物生长16小时(见注2),离心(30分钟,4000×g,4℃)并重悬于OD HEPES缓冲区中的 600 = 10(见注3)。
      2. 猎物细胞可以在4℃下储存长达2周。在使用之前,将细胞稀释至OD 600以上。
    2. (二步培养)的制备(图1B)
      1. 从储存在-80℃(参见附注4)的甘油储备物中回收乙二醇二乙醇胺(参见附注4),直接加入到在DDN培养基中制备的10毫升OD 600中的10毫升猎物细胞悬浮液中, 1.在30℃和250rpm下孵育。
      2. 在捕食24小时后,将100-300μl的共培养物转移到在OD 600(1 = 10)的捕食 - 猎物比为1:10的HEPES缓冲液中制备的10毫升猎物悬浮液中。在30℃和250rpm下孵育24小时。
        注意:通过在100ml烧瓶中加入10ml悬浮液来设置共培养物。
    3. 分离Bdellovibrio 细胞(图1B)。捕食后,通过0.45μm过滤器过滤共同培养两次以恢复细菌。
      注意:对第二个过滤步骤使用新的过滤器。
    4. 设置感兴趣的共培养物:将100-300μl的细胞扩散细胞转移到10ml OD 600的HEPES缓冲液中制备的10毫升猎物悬浮液(捕食者 - 猎物比例为1:10)。在30℃和250rpm下孵育24小时。


      图1.设置共同培养的Bdellovibrio 的工作流程。 A.捕食细胞悬浮液的制备(这里,恶臭假单胞菌KT2440)。 B.Bdellovibrio 细胞的制备。需要两步培养Bdellovibrio 以获得实验的捕食细胞。 C.双层方法来量化B细胞数。细菌 HD100。 D.发展B。在30℃下孵育2-3天后,在DNB琼脂平板上的猎物草坪上的细菌抗真菌HD100。细胞数可以定量为斑块形成单位(pfu/ml)。

  2. 计算Bdellovibrio和猎物细胞的活力(图1C)
    在DNB培养基中连续稀释共培养物,从10 -1 至10 -7 (见注5)。
    1. 使用双层方法(参见图1C)(Lambert等人,2003)确定生物活性Bdellovibrio
      1. 将4ml DNB 0.7%(w/v)琼脂加入玻璃管中,在水浴中保持在45-50℃(见注6)。
      2. 在HEPES缓冲液中加入0.5ml在OD 600下制备的猎物细胞悬浮液(恶臭假单胞菌KT2440;参见图1A)。
      3. 从共培养物中加入0.1ml适当的稀释液。
      4. 轻轻旋转管。
      5. 迅速将混合物倒入DNB 1.5%(w/v)琼脂平板上
      6. 在30℃下将板孵育2-3天。生长被监测为斑块形成单位/毫升(pfu/ml)在猎物的草坪上发育(图1D)。
    2. 计算猎物细胞活力
      在LB琼脂平板上放置10μl稀释液(见注5),并在30℃下孵育。猎物细胞计数为每毫升(cfu/ml)的集落形成单位。

数据分析

下面显示了Bdellovibrio 的掠夺活动的代表性图表(图2)。在实验开始时(0小时)和捕食24小时后的捕食者和猎物细胞数。恶臭 KT2440表示。


图2.用于研究B的掠夺能力的掠夺概况。细菌 HD100。恶臭 KT2440。 A.涉及B的共同文化的示意图。菌株HD100和对照培养物。没有食肉动物的恶臭。 B.在实验开始(0小时)和培养24小时后,猎物和捕食者细胞的活力。紫色条代表猎物细胞的活力,橙色条对应于捕食者活细胞数。误差条表示平均值的标准偏差(n≥3)。

笔记

  1. 如果可能,应使用相差显微镜检查捕食事件,以确保以下步骤的成功(见图3)。
  2. 根据用于生长Bdellovibrio 的猎物细胞选择培养基。
  3. 为了测量OD 600,制备目标培养物的1/10稀释度。例如,将100μl培养物加入到900μl盐水溶液中并混合。
  4. 为了在-80℃下储存Bdellovibrio,加入0.3ml 85%(w/v)甘油和0.7ml Bdellovibrio共培养物和猎物细菌至一个低温小瓶,并将管直接放在-80°C冰箱中。可以通过简单地用无菌回路刮伤低温小瓶的冰并将其添加到猎物悬浮液中来恢复细胞。不需要冷冻用于接种的低温小瓶。
  5. 如果预期高浓度的细胞,则进一步稀释样品。例如,通过向Eppendorf管中加入100μl的共培养物至900μl的DNB培养基中制备1:10的连续稀释液并重复直至获得所需的浓度。
  6. DNB 0.7%(w/v)琼脂管可以预先制备并在室温下储存。使用前,将管子在150-200℃的水浴中熔化,并保持在45-50°C
  7. 分别制备DNB培养基(或HEPES缓冲液)和CaCl 2和MgCl 2盐。使用前立即加入盐。


    图3.共同文化B。杀菌剂 恶臭,KT2440在显微镜下。 A.捕食开始时的共培养(时间零); B.孵育24小时后,只能观察到捕食性细胞。使用100x相位对比度物镜和用徕卡DFC345 FX相机拍摄的图像,可以常规地显示文化。 

食谱

  1. 溶菌酵母(LB)培养基(1L)
    10克Bacto胰蛋白胨
    5克酵母提取物
    10克NaCl
    使用1N NaOH调节至pH 7.5 在121℃高压灭菌20分钟
  2. LB,1.5%(w/v)琼脂(1L)
    10克Bacto胰蛋白胨
    5克酵母提取物
    10克NaCl
    使用1N NaOH调节至pH 7.5 15克琼脂
    在121℃高压灭菌21分钟
  3. NB中(1L)
    8 g NB
    在121°C高压灭菌20分钟
  4. CaCl 2和MgCl 2盐
    2mM CaCl 2·2H 2 O O
    3mM MgCl 2·3H 2 O
    通过0.22μm过滤器过滤灭菌至无菌容器中
  5. 稀释营养液(DNB)培养基(1L)(见附注7)
    0.8 g NB
    使用1N NaOH调节至pH 7.4 高压灭菌器
    加入CaCl 2和MgCl 2盐(参见配方4)
  6. DNB,0.7%(w/v)琼脂(1L)(见注7)
    0.8 g NB
    使用1N NaOH调节至pH 7.4 7克琼脂
    在121℃高压灭菌20分钟
    加入CaCl 2和MgCl 2盐(参见配方4)
  7. DNB,1.5%(w/v)琼脂(1L)(见附注7)
    0.8 g NB
    使用1N NaOH调节至pH 7.4 15克琼脂
    在121℃高压灭菌20分钟
    加入CaCl 2和MgCl 2盐(参见配方4)
  8. HEPES缓冲区(见注7)
    25 mM HEPES缓冲液
    使用NaOH颗粒调节至pH 7.8 在121℃高压灭菌20分钟
    加入CaCl 2和MgCl 2盐(参见配方4)

致谢

该方案被修改并根据先前描述的掠夺性测定(Martínez等人,2016; Lambert等人,2003)进行了修改。这项工作由欧盟第七框架计划资助,授权协议第311815号(SYNPOL),以及马德里组合(P2013/MIT2807)和西班牙经济贸易大臣(BIO2010-21049,BIO2013-44878)的赠款-R)。

参考文献

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引用:Herencias, C., Prieto, M. and Martínez, V. (2017). Determination of the Predatory Capability of Bdellovibrio bacteriovorus HD100. Bio-protocol 7(6): e2177. DOI: 10.21769/BioProtoc.2177.
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