बीच पारस्परिक अध्ययन<em> Xenorhabdus</em> बैक्टीरिया और<em> Steinernema</em> नेमाटोड तरीकों, बैक्टीरियल उपस्थिति और नेमाटोड भीतर स्थान पर नजर रखने के लिए विकसित किए गए. प्रयोगात्मक दृष्टिकोण है, जो अन्य प्रणालियों के लिए लागू किया जा सकता है, इंजीनियरिंग बैक्टीरिया हरी फ्लोरोसेंट प्रोटीन और visualizing व्यक्त करने के लिए पारदर्शी निमेटोड भीतर प्रतिदीप्ति माइक्रोस्कोपी बैक्टीरिया का उपयोग करने की जरूरत पर जोर देता है.
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode’s infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.
यहाँ वर्णित प्रोटोकॉल एक निमेटोड मेजबान (1 चित्रा) के भीतर बैक्टीरिया के ऑप्टिकल पता लगाने के लिए एक तरीका प्रदान करता है. इस विधि नेमाटोड पारदर्शिता ऑप्टिकल और fluorescently लेबल बैक्टीरिया की क्षमता क?…
The authors have nothing to disclose.
लेखकों के लिए उनके योगदान के लिए Eugenio Vivas कर्ट Heungens, एरिक Martens, चार्ल्स Cowles, Darby चीनी, एरिक Stabb, और टोड Ciche इस प्रोटोकॉल और उपकरणों का इस्तेमाल विकास के लिए शुक्रिया अदा करना चाहता हूँ. केईएम और जेएमसी राष्ट्रीय स्वास्थ्य संस्थान (एनआईएच) राष्ट्रीय अनुसंधान सेवा T32 पुरस्कार (AI55397 "स्वास्थ्य और रोग में रोगाणुओं") द्वारा समर्थित थे. जेएमसी एक राष्ट्रीय विज्ञान फाउंडेशन (NSF) ग्रेजुएट रिसर्च फैलोशिप द्वारा समर्थित किया गया था. यह काम राष्ट्रीय विज्ञान फाउंडेशन (IOS ०९२०६३१ और IOS-0,950,873) से अनुदान द्वारा समर्थित किया गया.
Name of the reagent | Company | Catalogue number | Comments (optional) |
Lipid Agar (sterile) |
8 grams nutrient broth, 15 grams agar, 5 grams yeast extract, 890 ml water, 10 ml 0.2 g/ml MgCl2. 6H20, 96 ml corn syrup solution*, 4 ml corn oil* Stir media while pouring plates *add sterile ingredient after autoclaving |
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Corn Syrup Solution (sterile) |
7 ml corn syrup, 89 ml water mix and autoclave |
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Egg Solution | 16.6 ml 12% sodium hypochlorite, 5 ml 5M KOH, 80 ml water | ||
Lysogeny Broth (sterile) |
5 grams yeast extract, 10 grams tryptone, 5 grams salt, 1 L water mix and autoclave |
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Microfuge | Fisher | 13-100-675 | Any microfuge that holds microfuge tubes will work |
Centrifuge | Beckman | 366802 | Large table top centrifuge that holds 15 ml and 50 ml conical tubes |
Sterile 60 mm X 15 mm Petri Dish | Fisher | 0875713 | |
50 ml centrifuge tubes | Fisher | 05-539-6 | |
15 ml centrifuge tubes | Fisher | 05-531-6 | |
Sterile 100 mm X 20 mm Petri Dish | Fisher | 0875711Z | Deeper than standard Petri dishes |
24-well plate | Greiner Bio-One | 662000-06 | |
Microscope | The microscope needs florescent capabilities compatible with your fluorophore | ||
Paraformaldehyde | Electron Microscopy Sciences | 15710 | |
PBS (sterile) |
8 g NaCL 0.2 g KCL 1.44 g Na2HPO4 0.24 g KH2PO4 1 L water Adjust to a pH of 7.4 and water to 1 L and autoclave |
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Microfuge tubes | Fisher | 05-408-138 | 2 ml or 1.5 ml tubes |
Shaker | Any shaker that causes the liquid to gently move will work | ||
Diaminopimelic acid | Sigma | D-1377 | If needed, supplement media to a concentration or 1 mM |