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Culturing Bacteria from Caenorhabditis elegans Gut to Assess Colonization Proficiency
利用秀丽隐杆线虫肠道培养细菌以评估其定植能力   

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Nature Communications
Jan 2017

Abstract

Determining an accurate count of intestinal bacteria from Caenorhabditis elegans is one critical way to assess colonization proficiency by a given bacteria. This can be accomplished by culturing appropriate dilutions of worm gut bacteria on selective or differential agarized media. Because of the high concentration of bacteria in gut worm, dilution is necessary before plating onto growth media. Serial dilutions can reduce the concentration of the original intestinal sample to levels low enough for single colonies to be grown on media plates, allowing for the calculation of the initial counts of bacteria in the intestinal sample.

Keywords: Caenorhabditis elegans (秀丽隐杆线虫), Bacillus subtilis (枯草芽孢杆菌), Escherichia coli (大肠埃希杆菌), Culture (培养)

Background

Animals rarely live in isolation but rather exist in association with microorganisms. The more characteristic host-microbe interaction in nature is the symbiotic relationship between host and intestinal microbiota (Rosenberg and Zilber-Rosenberg, 2011). In mammals, host-microbe symbiotic interactions mainly occur along mucosal surfaces, with the most important one being the intestinal mucosa. When freshly isolated from the wild, C. elegans often harbours a diverse bacterial flora in its gut lumen, reminiscent of the microbial communities of higher organisms (Duveau and Felix, 2012; Bumbarger et al., 2013). By contrast, in the laboratory C. elegans is typically maintained in the presence of single bacterial strain (Brenner, 1974). Most often, this is the Gram-negative bacterium Escherichia coli. However, other species are sometimes used, such as the Gram-positive Bacillus subtilis (Garsin et al., 2003). An adult worm contains approximately 10,000 bacterial cells, a number 10-times greater than that of host worm somatic cells (Portal-Celhay and Blaser, 2012): perhaps coincidentally, this microbiota-to-host cell ratio is similar to that found in humans.

Different strategies are being used to measure the colonization proficiency of C. elegans gut by a bacterium such as fluorescein isothiocyanate (FITC)-labelled bacteria, bacteria expressing a fusion reporter (green fluorescent protein [GFP] or β-galactosidase). However, the more accurate method is to measure the number of CFU isolated from the worm intestine. In this protocol, we show how to isolate and count E. coli and B. subtilis strains from C. elegans gut. In the case of B. subtilis, we also show how to distinguish vegetative forms from highly resistant spores formed by this bacterium inside the C. elegans gut.

Materials and Reagents

  1. Pipette tips 2-200 µl Eppendorf® epT.I.P.S. (Eppendorf, catalog number: 022492039 )
  2. Pipette tips 50-1,000 µl Eppendorf® epT.I.P.S. (Eppendorf, catalog number: 022492055 )
  3. Petri dishes 60 x 15 mm 500/cs (Fisher Scientific, catalog number: FB0875713A )
  4. Petri dishes 35 x 10 mm 500/cs (Fisher Scientific, catalog number: FB0875711YZ )
  5. Corning® 15 ml centrifuge tubes (Corning, catalog number: 430791 )
  6. Eppendorf® Safe-Lock 1.5 ml microcentrifuge tubes (Eppendorf, catalog number: 022363204 )
  7. Toothpick
  8. 99.95 % Platinum, 0.05 % Iridium wire (3 ft/pk) (Tritech Research, catalog number: PT-9901 )
  9. OP50 E. coli bacteria (University of Minnesota, C. elegans Genetics Center, MN)
  10. Experimental and control C. elegans strains (University of Minnesota, C. elegans Genetics Center, MN)
  11. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  12. Bacto peptone (BD, BactoTM, catalog number: 211677 )
  13. Agar (Sigma-Aldrich, catalog number: A1296 )
  14. Hypochlorite (Sigma-Aldrich, catalog number: 13440 )
  15. Commercial Bleach 60 g/L (DROGUERÍA INDUSTRIAL SAN JUAN, http://www.sanjuandrogueria.com)
  16. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  17. Luria broth (Sigma-Aldrich, catalog number: L3522 )
  18. Luria broth with agar (Sigma-Aldrich, catalog number: L2897 )
  19. Lysozyme from chicken egg white (Sigma-Aldrich, catalog number: L6876 )
  20. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S3264 )
  21. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  22. Cholesterol (Sigma-Aldrich, catalog number: C8667 )
  23. 100% ethanol (Sigma-Aldrich, catalog number: E7023 )
  24. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: M1880 )
  25. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C3881 )
  26. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P2222 )
  27. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045 )
  28. Levamisole hydrochloride (Sigma-Aldrich, catalog number: L0380000 )
  29. Nematode growth medium (NGM) (see Recipes)
  30. M9 buffer (see Recipes)
  31. 5 mg/ml cholesterol (see Recipes)
  32. 1 M MgSO4 (see Recipes)
  33. 1 M CaCl2 (see Recipes)
  34. Phosphate buffer (see Recipes)
  35. 1 N NaOH (see Recipes)
  36. 25 mM Levamisole (see Recipes)

Equipment

  1. Erlenmeyer flask (Fisher Scientific, catalog number: FB5006000 )
  2. Pipettor (Gilson, catalog number: F167300 )
  3. Worm pick. Worm picks can either be purchased (Genesee Scientific, catalog number: 59-AWP ) or made in the lab as described in Wollenberg et al., 2013
  4. Pasteur glass pipette (Fisher Scientific, catalog number: 22-378893 )
  5. Autoclave (Tuttnauerusa, model: 6690 )
  6. Stirring hotplate (Corning, catalog number: 6795-620 )
  7. Centrifuge (Eppendorf, model: 5430 )
  8. Tabletop centrifuge (Eppendorf, model: 5424 )
  9. Pellet pestle (Kimble Chase Life Science and Research Products, catalog number: 7495211590 )
  10. Refrigerated incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: HerathermTM General Protocol Microbiological Incubators , catalog number: 51028064)
  11. Bunsen burner (Humbolt, catalog number: H-5870 )
  12. Dissecting stereomicroscope (Olympus, model: SMZ645 )
  13. Incubators for stable temperature (AQUA® LYTIC incubator 20 °C)
  14. Freezers (-20 °C; So-Low Environmental Equipment) (Siemens, model: C85-22 )
  15. Water bath

Procedure

  1. Preparation of a synchronized nematode population
    1. For NGM plate’s preparation, mix 3 g NaCl, 2.5 g Bacto peptone and 17 g agar in Erlenmeyer flask. Add to 1 L of dH2O. Autoclave 121 °C for 20 min. Cool flask in 55 °C water bath for 15 min. Add 1 ml 1 M CaCl2, 1 ml 5 mg/ml cholesterol in ethanol, 1 ml 1 M MgSO4 and 25 ml 1 M KPO4 (Phosphate) buffer (see Recipes). Swirl to mix well. Using sterile procedures dispense the NGM solution into 60 x 15 mm Petri plates. Fill plates 2/3 full of agar.
    2. Pick 10 young adults onto an E. coli OP50 seeded 60 mm NGM plate (see Recipes). Allow the worms to grow 2-3 days to ensure at least 100 gravid adult worms and an adequate number of eggs on the plate.
    3. Once reached this worm number, pour 3 ml of M9 buffer (see Recipes) onto the plate and gently swirl it to dislodge the worms. Repeat this procedure one more time using 2 ml of M9 buffer. One plate is enough for bleaching.
    4. Using a pipette, transfer the worms to a 15 ml conical tube.
    5. Centrifuge for about 1 min at 252 x g to pellet the worms.
    6. Aspirate most of the M9 without disturbing the worm pellet.
    7. Add about 1 ml of 3% hypochlorite solution and 2.5 ml of 1 N NaOH (see Recipes) to the tube.
    8. Vortex the tube for approximately 5 min or until you see a decrease in the number of intact adult worms. Do not bleach for much longer than this or you will kill the eggs.
    9. Once most of the bodies have dissolved, neutralize the alkaline hypochlorite by adding M9 buffer until 15 ml of the conical tube and centrifuge at 252 x g for 1 min.
    10. Aspirate most of the supernatant solution without disturbing the egg pellet.
    11. Add about 15 ml of M9 to the tube and mix well.
    12. Centrifuge again at 252 x g for 1 min.
    13. Aspirate most of the M9 without disturbing the egg pellet.
    14. Repeat steps A11-A13 at least one more time.
    15. Add about 5 ml of fresh M9 and agitate to resuspend the egg-enriched pellet. Incubate overnight at room temperature with gentle rocker stirrer. The eggs will hatch and the animals will arrest as starved L1 larvae.
    16. Transfer between 100 and 200 starved L1 larvae to a 60 mm NGM plate with E. coli OP50 cells (1 x 105 cells per plate) or spores (1 x 105 spores per plate) (see Note 2) of each B. subtilis strain and allow the worms to grow 2 days until they reach L4/adult stage.

  2. Preparation of nematode samples for intestinal bacterial counting
    1. Use an eyebrow hair and collect 50 adult animals in M9 buffer in a 1.5 ml tube (see Note 1).
    2. Use the same number of animals (worms) fed on each bacterial strain during sample preparation.
    3. Treat the worms with 0.25 mM levamisole to induce temporal paralysis.
    4. Superficially sterilize with 3% commercial Bleach for 5 min (see Note 5).
    5. Wash at least three times with M9 buffer.
    6. After the worms were surface sterilized, worms devoid of outside bacteria are disrupted using a pellet pestle and 1% Triton X-100 (see Note 6).
    7. Centrifuge at 14,800 x g for 10 min, remove the supernatant and resuspend the pellet of each tube in 500 µl M9 buffer (see Note 4).

  3. Preparation of sample dilutions (Figure 1)
    1. To begin the procedure, take 50 µl of the cell suspensions and add to 450 µl of deionized water. Shake the suspension well, and label as ‘10-1’.
    2. Before the sample settles, remove 50 µl of the cell suspension with a sterile pipette and transfer it to a 450 µl-deionized water blank. Vortex the sample, and label as ‘10-2’.
    3. Repeat this dilution step, using 50 µl of the previous suspension and a 450 µl-deionized water blank. Label these sequentially as tubes ‘10-3’.


      Figure 1. Diagram of how to prepare bacterial dilutions. Serial dilution of an initial culture to obtain solutions that are 1/10th, 1/100th and 1/1,000th the concentration of the initial sample (10-1, 10-2 and 10-3 dilutions, respectively). For each dilution, tubes initially have 450 µl of water in them so that the final volume will be 500 µl after the addition of 50 µl of culture.

  4. Making spread plates for bacterial culture
    1. To get bacterial colonies from worm gut isolated E. coli and vegetative B. subtilis (see Figure 2A), take three LB agar plates (see Note 3) and label them as A, B, and C. Vortex samples 10-1, 10-2, and 10-3, and pipette 50 µl onto each plate. This increases the dilution value further, by a factor of ten (A = 10-2, B = 10-3, C = 10-4) (see Figure 1).


      Figure 2. The micrographs show the vegetative cells (A) and spores (B) of Bacillus subtilis. Bar size = 5 µm.

    2. Next, dip a glass spreader into ethanol. Place the spreader in a flame for a few seconds to ignite and burn off the ethanol. This will sterilize the spreader.
    3. Hold the spreader above the first plate until the flame is extinguished. Open the plate quickly, holding the lid close by. Touch the spreader to the agar away from the inoculum (Inoculum = cells used to begin a culture) to cool, and then spread the drop of inoculum around the surface of the agar until traces of free liquid disappear.
    4. Re-flame the spreader and repeat the process with the next plate, working quickly so as not to contaminate the agar with airborne organisms.
    5. Incubate the bacteria plates at 37 °C for 16-18 h. Make sure the plates are inverted during the incubation to prevent drops of moisture from condensation from falling onto the agar surface.

  5. Making spread plates for spore culture
    1. Heat-treat the tubes with B. subtilis samples labeled as 10-1, 10-2, and 10-3 for 20 min at 80 °C to kill vegetative cells. Vortex spores samples (see Figure 2B) and pipette 50 µl onto each plate. This increases the dilution value further, by a factor of ten (A = 10-2, B = 10-3, C = 10-4).
    2. Next, dip a glass spreader into ethanol. Place the spreader in a flame for a few seconds to ignite and burn off the ethanol. This will sterilize the spreader.
    3. Hold the spreader above the first plate until the flame is extinguished. Open the plate quickly, holding the lid close by. Touch the spreader to the agar away from the inoculum to cool, and then spread the drop of inoculum around the surface of the agar until traces of free liquid disappear.
    4. Re-flame the spreader and repeat the process with the next plate, working quickly so as not to contaminate the agar with airborne organisms.
    5. Incubate the bacteria plates at 37 °C for 16-18 h. Make sure the plates are inverted during the incubation to prevent drops of moisture from condensation from falling onto the agar surface.

  6. Bacterial counts
    1. After incubation, examine all of the bacteria plates carefully, and note differences in colony size and shape.
    2. Count and record the number of bacterial colonies. Only count and use for calculation, plates with 30-300 colonies per plate. The number of colonies per plate bellow 30 is not statistically significant whereas a number higher than 300 colonies per plate is hard to count, and it is possible a colony arise from more than one cell.
    3. The number of CFU/worm is calculated as follows:
      Number of CFU/worm in plate A = (number of colonies x 102)/50
      Number of CFU/worm in plate B = (number of colonies x 103)/50
      Number of CFU/worm in plate C = (number of colonies x 104)/50

Data analysis

  1. For each strain or condition, use at least 50-100 animals to obtain results that are more accurate.
  2. For each experiment, the same number of nematodes should be examined for each strain and condition.
  3. Each assay should be run in triplicate and repeated at least three times (Duveau and Felix, 2012).
  4. Use the Student’s t-test with a significance cut-off level of P < 0.05 for comparisons between two groups.
  5. Use the one-factor (ANOVA) variance analysis and correct by the post hoc Bonferroni test for multiple comparisons.

Notes

  1. Take a toothpick and glue an eyebrow hair to the tip of it. Let it dry at room temperature. Then, use this tool to pick nematodes. Before using the eyebrow hair always, sterilize it by using ethanol.
  2. To obtain pure spores, the heat-treated culture (20 min at 80 °C to kill vegetative cells in a water bath) was treated three times with lysozyme, washed each time with cold water and centrifuged until 100% of the culture consisted of phase-bright spores.
  3. For the LB plates used to seed Bacillus subtilis, we strongly recommend drying plates at 45 °C for 20 min or 37 °C for 40 min before to drop the dilutions onto each plate to avoid the sliding movement of this bacterium in solid surface with water drops from condensation.
  4. Samples should be used immediately or store for 1 week at -20 °C.
  5. To surface sterilize, resuspend 50 worms in 500 µl M9 buffer, add 0.25 mM levamisole (5 µl of 25 mM levamisole stock solution) and 3 % commercial Bleach (stock solution). Mix samples by gentle inversion for 15 min. Then, wash the worms with M9 buffer at least 3 times.
  6. After the worms are surface sterilized, pour the 200 µl worm suspension in M9 buffer in a 1.5 ml microcentrifuge tube. Add the extraction solution of choice (1% Triton X-100), and the pestle is used to grind the sample 10 times or until a creamy white to yellow suspension is obtained. After centrifugation, cellular debris and bacteria remain firmly in the bottom of the conical tube, and the supernatant is easily pipetted out.

Recipes

  1. Nematode growth medium (NGM)
    Dissolve 3 g NaCl, 2.5 g Bacto peptone and 17 g agar to 1 L of dH2O
    Autoclave
    Store at room temperature
  2. M9 buffer
    Dissolve 5 g NaCl, 6 g Na2HPO4 and 3 g KH2PO4 to 1 L of dH2O
    Autoclave
    Store at room temperature
  3. 5 mg/ml cholesterol
    Dissolve 0.25 g of cholesterol in 50 ml of 100% ethanol
    Do not autoclave
    Store at room temperature
  4. 1 M MgSO4
    Dissolve 6 g MgSO4 heptahydrate in 50 ml of dH2O
    Autoclave
    Store at room temperature
  5. 1 M CaCl2
    Dissolve 5.55 g CaCl2 dihydrate in 50 ml of dH2O
    Autoclave
    Store at room temperature
  6. Phosphate buffer
    Dissolve 10.7 g K2HPO4 and 32.5 g KH2PO4 to 300 ml of dH2O
    Adjust pH to 6.0
    Autoclave
    Store at room temperature
  7. 1 N NaOH
    Dissolve 2 g NaOH in 50 ml of dH2O
  8. 25 mM levamisole
    Dissolve 0.3 g levamisole in 50 ml of dH2O

Acknowledgments

This work was supported by CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas) and FONCyT (Fondo para la Investigación Científica y Tecnológica) with the aid of the Pew Latin-American Program in Biological Sciences (Philadelphia, PA, USA), the Fulbright Committee (Washington, DC, USA) and former Fundación Antorchas (Buenos Aires, Argentina). We modified the media and NGM plate’s preparation from Stiernagle T. Maintenance of C. elegans. WormBook. 2006 11:1-11. We modified C. elegans synchronization from Montserrat Porta-de-la-Riva et al. (2012). Basic Caenorhabditis elegans Methods: Synchronization and Observation. Journal of Visualized Experiments; 64, e4019, 1-9.

References

  1. Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77(1): 71-94.
  2. Bumbarger, D. J., Riebesell, M., Rodelsperger, C. and Sommer, R. J. (2013). System-wide rewiring underlies behavioral differences in predatory and bacterial-feeding nematodes. Cell 152(1-2): 109-119.
  3. Duveau, F. and Felix, M. A. (2012). Role of pleiotropy in the evolution of a cryptic developmental variation in Caenorhabditis elegans. PLoS Biol 10(1): e1001230.
  4. Garsin, D. A., Villanueva, J. M., Begun, J., Kim, D. H., Sifri, C. D., Calderwood, S. B., Ruvkun, G. and Ausubel, F. M. (2003). Long-lived C. elegans daf-2 mutants are resistant to bacterial pathogens. Science 300(5627): 1921.
  5. Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A. and Cerón, J. (2012). Basic Caenorhabditis elegans methods: synchronization and observation. J Vis Exp 64: e4019.
  6. Portal-Celhay, C. and Blaser, M. J. (2012). Competition and resilience between founder and introduced bacteria in the Caenorhabditis elegans gut. Infect Immun 80(3): 1288-1299.
  7. Rosenberg, E. and Zilber-Rosenberg, I. (2011). Symbiosis and development: the hologenome concept. Birth Defects Res C Embryo Today 93(1): 56-66.
  8. Stiernagle, T. (2006). Maintenance of C. elegans. WormBook 11:1-11.
  9. Wollenberg, A. C., Visvikis, O., Alves, A. F. and Irazoqui, J. E. (2013). Staphylococcus aureus killing assay of Caenorhabditis elegans. Bio-protocol 3(19): e916.

简介

从秀丽隐杆线虫确定肠道细菌的准确计数是评估给定细菌的定殖能力的关键方法之一。 这可以通过在选择性或差异琼脂培养基上培养适当稀释的蠕虫细菌来实现。 由于肠蠕虫中细菌的浓度高,因此稀释必须在生长培养基上。 连续稀释可以将原始肠样品的浓度降低到足够低的浓度,使单个菌落生长在培养基平板上,从而计算肠样品中细菌的初始计数。
【背景】动物很少分离生活,但与微生物有关。主要与微生物之间的共生关系是Rosentberg和Zilber-Rosenberg在2011年的共生关系。在哺乳动物中,宿主 - 微生物共生相互作用主要发生在粘膜表面,最重要的是肠粘膜。当从野生新鲜分离时,线虫通常在其肠腔内容纳多种细菌菌群,使人联想到高等生物的微生物群落(Duveau和Felix,2012; Bumbarger等,2013)。相比之下,在实验室中,秀丽隐杆线虫通常保持在单一细菌菌株的存在下(Brenner,1974)。大多数情况下,这是革兰氏阴性细菌大肠杆菌。然而,有时使用其他物种,例如革兰氏阳性枯草芽孢杆菌(Garsin等,2003)。成虫蠕虫含有大约10,000个细菌细胞,比宿主蠕虫体细胞数量高10倍(Portal-Celhay和Blaser,2012):或许巧合的是,这种微生物群与宿主细胞的比例与人类发现的相似。
正在使用不同的策略来测量秀丽隐杆线虫的细菌如异硫氰酸荧光素(FITC)标记的细菌,表达融合报告基因的细菌(绿色荧光蛋白[GFP]或β-半乳糖苷酶))的定殖能力。然而,更准确的方法是测量从蠕虫肠分离出的CFU的数量。在本协议中,我们展示了如何分离和计数秀丽隐杆线虫的大肠杆菌和枯草芽孢杆菌菌株。在枯草芽孢杆菌的情况下,我们还展示了如何区分营养形式与秀丽隐杆线虫内部这种细菌形成的高度抗性孢子。

关键字:秀丽隐杆线虫, 枯草芽孢杆菌, 大肠埃希杆菌, 培养

材料和试剂

  1. 移液器吸头2-200μlEppendorf ® epT.I.P.S. (Eppendorf,目录号:022492039)
  2. 移液器吸头50-1,000μlEppendorf ® epT.I.P.S. (Eppendorf,目录号:022492055)
  3. 培养皿60 x 15 mm 500/cs(Fisher Scientific,目录号:FB0875713A)
  4. 培养皿35 x 10 mm 500/cs(Fisher Scientific,目录号:FB0875711YZ)
  5. Corning ® 15ml离心管(Corning,目录号:430791)
  6. Eppendorf ® Safe-Lock 1.5 ml微量离心管(Eppendorf,目录号:022363204)
  7. 牙签
  8. 99.95%铂,0.05%铱丝(3ft/pk)(Tritech Research,目录号:PT-9901)
  9. OP50 E。大肠杆菌细菌(明尼苏达大学,秀丽隐杆线虫遗传学中心,MN)
  10. 实验和控制。线虫菌株(明尼苏达大学,线虫,遗传学中心,MN)
  11. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  12. 细菌蛋白胨(BD,Bacto TM ,目录号:211677)
  13. 琼脂(Sigma-Aldrich,目录号:A1296)
  14. 次氯酸盐(Sigma-Aldrich,目录号:13440)
  15. 商业漂白剂60克/升(DROGUERÍAINDUSTRIAL SAN JUAN, http://www.sanjuandrogueria.com
  16. Triton X-100(Sigma-Aldrich,目录号:X100)
  17. Luria肉汤(Sigma-Aldrich,目录号:L3522)
  18. 用琼脂培养肉汤(Sigma-Aldrich,目录号:L2897)
  19. 来自鸡蛋白的溶菌酶(Sigma-Aldrich,目录号:L6876)
  20. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S3264)
  21. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  22. 胆固醇(Sigma-Aldrich,目录号:C8667)
  23. 100%乙醇(Sigma-Aldrich,目录号:E7023)
  24. 硫酸镁七水合物(MgSO 4·7H 2 O)(Sigma-Aldrich,目录号:M1880)
  25. 氯化钙二水合物(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C3881)
  26. 磷酸氢二钾(K 2/2 HPO 4)(Sigma-Aldrich,目录号:P2222)
  27. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:S8045)
  28. 盐酸左旋咪唑(Sigma-Aldrich,目录号:L0380000)
  29. 线虫生长培养基(NGM)(见食谱)
  30. M9缓冲区(见配方)
  31. 5 mg/ml胆固醇(见食谱)
  32. 1 M MgSO 4(参见食谱)
  33. 1 M CaCl 2 (见配方)
  34. 磷酸盐缓冲液(见配方)
  35. 1 N NaOH(参见食谱)
  36. 25mM左旋咪唑(参见食谱)

设备

  1. 锥形瓶(Fisher Scientific,目录号:FB5006000)
  2. Pipetor(Gilson,目录号:F167300)
  3. 蠕虫选择可以购买蠕虫选择(Genesee Scientific,目录号:59-AWP),或者在实验室中制作,如Wollenberg等人,2013所述。
  4. 巴斯德玻璃移液器(Fisher Scientific,目录号:22-378893)
  5. 高压灭菌器(Tuttnauerusa,型号:6690)
  6. 搅拌电热板(康宁,目录号:6795-620)
  7. 离心机(Eppendorf,型号:5430)
  8. 台式离心机(Eppendorf,型号:5424)
  9. 颗粒杵(Kimble Chase生命科学研究产品,目录号:7495211590)
  10. 冷藏培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heratherm TM General Protocol Microbiological Incubators,catalog number:51028064)
  11. 本生灯(Humbolt,目录编号:H-5870)
  12. 解剖立体显微镜(Olympus,型号:SMZ645)
  13. 温度稳定的孵化器(AQUA ® LYTIC培养箱20°C)
  14. 冷冻机(-20°C;低温环境设备)(西门子,型号:C85-22)
  15. 水浴

程序

  1. 准备同步的线虫群体
    1. 对于NGM板的制备,将3g NaCl,2.5g细菌蛋白胨和17g琼脂混合在锥形瓶中。加入1L dH 2 O。高压灭菌121°C 20分钟。在55℃水浴中冷却烧瓶15分钟。加入1ml 1M CaCl 2,1ml 5mg/ml胆固醇的乙醇溶液,1ml 1M MgSO 4和25ml 1M KPO 4, (磷酸盐)缓冲液(参见食谱)。旋转混匀。使用无菌方法将NGM溶液分配到60×15mm培养皿中。填充2/3满的琼脂。
    2. 选择10名年轻成年人到E。大肠杆菌 OP50接种60mm NGM板(参见食谱)。允许蠕虫生长2-3天,以确保至少100只妊娠成虫和足够数量的鸡蛋在板上。
    3. 一旦达到这个蠕虫数,将3毫升M9缓冲液(见食谱)倒入盘子上,轻轻地旋转,以便除虫。再次使用2 ml M9缓冲液重复此过程。一块板足以漂白。
    4. 使用移液管将蠕虫转移到15 ml锥形管中
    5. 离心约252分钟离心约1分钟,以使颗粒蠕动。
    6. 吸引大多数M9,而不会干扰蠕虫颗粒。
    7. 将约1ml 3%次氯酸盐溶液和2.5ml 1N NaOH(见食谱)加入管中。
    8. 旋转管约5分钟,直到看到完整的成年蠕虫数量减少。不要比这更长时间漂白,否则你会杀死鸡蛋。
    9. 一旦大部分物体溶解,通过加入M9缓冲液中和碱性次氯酸盐,直到15ml锥形管,并以252×g离心1分钟。
    10. 吸出大部分上清液,不会干扰蛋沉淀。
    11. 将约15ml的M9加入管中,并充分混合
    12. 再次离心在252 x g 1分钟。
    13. 吸引大部分M9而不打扰蛋沉淀。
    14. 再次重复步骤A11-A13。
    15. 加入约5ml新鲜的M9并搅拌以重悬富含蛋白的颗粒。在室温下用温和的摇臂搅拌器孵育过夜。鸡蛋会孵化,动物将被捕获为饥饿的L1幼虫。
    16. 将100和200个饥饿的L1幼虫转移到具有E的60mm NGM板上。大肠杆菌OP50细胞(每板1×10 5个细胞)或孢子(每个板1×10 5个孢子)(见注2) > B中。枯草芽孢杆菌菌株,并允许蠕虫生长2天,直到达到L4 /成人阶段
  2. 制备肠道细菌计数的线虫样品
    1. 使用眉毛,并在M9缓冲液中收集50只成年动物的1.5 ml管(见注1)
    2. 在样品制备过程中使用与每种细菌菌株相同数量的动物(蠕虫)
    3. 用0.25mM左旋咪唑处理蠕虫,引起瘫痪
    4. 用3%商业漂白剂表面灭菌5分钟(见注5)
    5. 用M9缓冲液洗涤至少三次。
    6. 蠕虫表面消毒后,使用颗粒杵和1%Triton X-100破坏外部细菌的蠕虫(见附注6)。
    7. 以14,800 x g离心10分钟,取出上清液,将每个管的沉淀重悬于500μlM9缓冲液中(见注4)。

  3. 样品稀释液的制备(图1)
    1. 要开始该步骤,取50μl细胞悬液,加入450μl去离子水。摇匀悬挂,标签为'10 -1 '。
    2. 在样品沉降之前,用无菌移液管取出50μl细胞悬浮液,并将其转移到450μl去离子水中。旋转样品,并标记为'10 -2 '。
    3. 重复该稀释步骤,使用50μl先前的悬浮液和450μl去离子水空白。将这些顺序标记为管'10 -3 '。


      图1.如何制备细菌稀释的图。初始培养物的连续稀释以获得初始样品浓度的1/10,1/100和1/100的溶液(10 < sup> -1 ,10 -2 和10 -3 稀释液)。对于每种稀释液,管最初在其中含有450μl的水,使得在加入50μl培养物后,最终体积将为500μl。

  4. 制作扩散板用于细菌培养
    1. 从分离大肠杆菌的蠕虫肠获得细菌菌落和营养B。 (参见图2A),取三个LB琼脂平板(参见注3),并将其标记为A,B和C.涡旋样品10 ,10 2 和10 -3 ,并将50μl移液到每个平板上。这进一步增加了稀释值10倍(A = 10 -2 ,B = 10 -3 ,C = 10 -4 )(见图1)

      图2.显微照片显示枯草芽孢杆菌的营养细胞(A)和孢子(B)。条尺寸= 5μm。

    2. 接下来,将玻璃吊具浸入乙醇中。将吊具放在火焰中几秒钟以点燃和燃烧乙醇。这将对吊具进行消毒。
    3. 将吊具放在第一块板上方,直到火焰熄灭。快速打开盖子,靠近盖子。将接触器上的琼脂(接种物=用于开始培养的细胞)放在琼脂上,然后将琼脂接种到琼脂表面附近,直到痕量的游离液体消失。
    4. 重新点燃撒布机,并用下一个板块重复该过程,迅速工作,以免污染带有空气中的生物体的琼脂。
    5. 将细菌板在37℃孵育16-18小时。在孵化期间确保板倒置,以防止水分从冷凝水滴落到琼脂表面上
  5. 制作扩散板用于孢子文化
    1. 用B进行热处理。在80℃下标记为10 -1 ,10 -2 和10 -3 20分钟的枯草芽孢杆菌样品杀死营养细胞。漩涡样品(参见图2B),并将50μl移液到每个平板上。这进一步增加了稀释值10倍(A = 10 -2 ,B = 10 -3 ,C = 10 -4 )。
    2. 接下来,将玻璃吊具浸入乙醇中。将吊具放在火焰中几秒钟以点燃和燃烧乙醇。这将对吊具进行消毒。
    3. 将吊具放在第一块板上方,直到火焰熄灭。快速打开盖子,靠近盖子。将撒布机从接种物上接触琼脂,以冷却,然后将接种物滴在琼脂表面附近,直到痕量的游离液体消失。
    4. 重新点燃撒布机,并用下一个板块重复该过程,迅速工作,以免污染带有空气中的生物体的琼脂。
    5. 将细菌板在37℃孵育16-18小时。在孵化期间确保板倒置,以防止水分从冷凝水滴落到琼脂表面上
  6. 细菌计数
    1. 孵化后,仔细检查所有细菌盘,并注意菌落大小和形状的差异
    2. 计数并记录细菌菌落的数量。只计算和使用计算,盘每30-300个殖民地每板。每个板块下方的30个菌落不具有统计学意义,而每盘高于300个菌落的数量难以计数,并且可能由多个细胞产生菌落。
    3. CFU /蠕虫的数量计算如下:
      板中CFU /蠕虫数A =(菌落数x 10 2 )/50
      板块CFU /蠕虫数B =(菌落数×10 3 )/50
      盘中CFU /蠕虫数C =(菌落数×10 4 )/50

数据分析

  1. 对于每个菌株或病症,使用至少50-100只动物获得更准确的结果
  2. 对于每个实验,应对每个菌株和条件检查相同数量的线虫。
  3. 每个测定应该运行一式三份,重复至少三次(Duveau和Felix,2012)
  4. 使用学生的 t 测试,具有 P 的重要性截止级别0.05用于两组之间的比较
  5. 使用单因素(ANOVA)方差分析,并通过事后Bonferroni检验进行多次比较。

笔记

  1. 拿一个牙签,将眉毛粘在头上。让它在室温下干燥。然后,使用此工具选择线虫。在使用眉毛之前,请用乙醇对其进行消毒
  2. 为了获得纯净的孢子,将经热处理的培养物(80℃下杀灭水浴中的营养细胞20分钟)用溶菌酶处理三次,每次用冷水洗涤并离心至100%的培养物为相-bright孢子。
  3. 对于用于种子枯草芽孢杆菌的LB板,我们强烈建议在45℃干燥20分钟或37℃40分钟,然后将稀释液放在每个板上以避免滑动的这种细菌在固体表面与冷凝水滴
  4. 应立即使用样品或在-20°C储存1周。
  5. 为了表面消毒,将50个蠕虫重悬于500μlM9缓冲液中,加入0.25mM左旋咪唑(5μl25mM左旋咪唑储备溶液)和3%商业Bleach(储备溶液)。通过轻轻倒置混合样品15分钟。然后用M9缓冲液清洗蠕虫至少3次。
  6. 蠕虫表面灭菌后,将200μl蠕虫悬浮液倒入1.5 ml微量离心管中的M9缓冲液中。加入选择的提取溶液(1%Triton X-100),杵用于研磨样品10次,或直至获得乳白色至黄色悬浮液。离心后,细胞碎片和细菌牢固地保留在锥形管的底部,上清液很容易吸出。

食谱

  1. 线虫生长培养基(NGM)
    将3g NaCl,2.5g细菌蛋白胨和17g琼脂溶解在1L dH 2 O中, 高压灭菌器
    在室温下存放
  2. M9缓冲区
    将5g NaCl,6g Na 2 HPO 4和3g KH 2 PO 4溶解至1L的dH 2 O
    高压灭菌器
    在室温下存放
  3. 5 mg/ml胆固醇
    将0.25g胆固醇溶于50ml的100%乙醇中 不要高压
    在室温下存放
  4. 1 M MgSO 4
    将6g硫酸镁四水合物溶解在50ml dH 2 O中,// 高压灭菌器
    在室温下存放
  5. 1 M CaCl 2
    将5.55g CaCl 2二水合物溶解在50ml dH 2 O中, 高压灭菌器
    在室温下存放
  6. 磷酸盐缓冲液
    将10.7g K 2 N 2 HPO 4和32.5g KH 2 PO 4溶解至300ml dH 3 2 O
    调节pH至6.0
    高压灭菌器
    在室温下存放
  7. 1 N NaOH
    将2g NaOH溶解在50ml的dH 2 O中
  8. 25 mM左旋咪唑
    将0.3g左旋咪唑溶解在50ml的dH 2 O中

致谢

这项工作得到了CONICET(国际考察委员会)和FONCyT(Fondo para laInvestigaciónCientíficayTecnológica)的支持,在拉丁美洲生物科学计划(Philadelphia,PA,USA),富布莱特委员会(华盛顿特区,美国)和前基辅安那府(阿根廷布宜诺斯艾利斯)。我们修改了Stiernagle T的媒体和NGM版的准备工作。线虫。 WormBook。 2006 11:1-11。我们修改了C。来自Montserrat Porta-de-la-Riva的线虫同步等。 (2012年)。基本的秀丽隐杆线虫方法:同步和观察。可视化实验杂志; 64,e4019,1-9。

参考

  1. Brenner,S.(1974)。  遗传学< Caenorhabditis elegans 。 Genetics 77(1):71-94。
  2. Bumbarger,DJ,Riebesell,M.,Rodelsperger,C.and Sommer,RJ(2013)。  系统范围的重新布线是捕食和细菌饲养线虫的行为差异的基础。细胞 152(1-2):109-119。 >
  3. Duveau,F。和Felix,MA(2012)。  多效性在秀丽隐杆线虫中的隐秘发育变化的演变中的作用。 10(1):e1001230。
  4. Garsin,DA,Villanueva,JM,Begun,J.,Kim,DH,Sifri,CD,Calderwood,SB,Ruvkun,G。和Ausubel,FM(2003)。< a class ="ke-insertfile"href = "http://www.ncbi.nlm.nih.gov/pubmed/12817143"target ="_ blank">长寿命C. elegans daf-2突变体对细菌病原体具有抗性。 300(5627):1921。
  5. Porta-de-la-Riva,M.,Fontrodona,L.,Villanueva,A.和Cerón,J.(2012)。基本 Caenorhabditis elegans 方法:同步和观察 J Vis Exp 64:e4019 。
  6. Portal-Celhay,C. and Blaser,MJ(2012)。  秀丽隐杆线虫肠道中的创始人和引入的细菌之间的竞争和弹性。 80(3):1288-1299。
  7. Rosenberg,E.和Zilber-Rosenberg,I。(2011)。  维护℃。电影。 WormBook 11:1-11。
  8. Wollenberg,AC,Visvikis,O.,Alves,AF和Irazoqui,JE(2013)。  金黄色葡萄球菌杀伤细线虫的杀伤试验。生物方案3(19):e916 。
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引用:Rodriguez Ayala, F., Cogliati, S., Bauman, C., Leñini, C., Bartolini, M., Villalba, J. M., Argañaraz, F. and Grau, R. (2017). Culturing Bacteria from Caenorhabditis elegans Gut to Assess Colonization Proficiency. Bio-protocol 7(12): e2345. DOI: 10.21769/BioProtoc.2345.
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