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 JoVE Biology

Extracting DNA from the Gut Microbes of the Termite (Zootermopsis Angusticollis) and Visualizing Gut Microbes

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1Department of Environmental Science and Engineering, California Institute of Technology - Caltech

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    Summary

    This video illustrates the technique for extracting DNA from the species of microbes resident in the termite hindgut. The preparation of a wet mount slide, which is useful for visualizing the gut microbial community is also illustrated, and a tour through the species-rich gut environment is given.

    Date Published: 5/28/2007, Issue 4; doi: 10.3791/195

    Cite this Article

    Matson, E., Ottesen, E., Leadbetter, J. Extracting DNA from the Gut Microbes of the Termite (Zootermopsis Angusticollis) and Visualizing Gut Microbes. J. Vis. Exp. (4), e195, doi:10.3791/195 (2007).

    Abstract

    Termites are among the few animals known to have the capacity to subsist solely by consuming wood. The termite gut tract contains a dense and species-rich microbial population that assists in the degradation of lignocellulose predominantly into acetate, the key nutrient fueling termite metabolism (Odelson & Breznak, 1983). Within these microbial populations are bacteria, methanogenic archaea and, in some ("lower") termites, eukaryotic protozoa. Thus, termites are excellent research subjects for studying the interactions among microbial species and the numerous biochemical functions they perform to the benefit of their host. The species composition of microbial populations in termite guts as well as key genes involved in various biochemical processes has been explored using molecular techniques (Kudo et al., 1998; Schmit-Wagner et al., 2003; Salmassi & Leadbetter, 2003). These techniques depend on the extraction and purification of high-quality nucleic acids from the termite gut environment. The extraction technique described in this video is a modified compilation of protocols developed for extraction and purification of nucleic acids from environmental samples (Mor et al., 1994; Berthelet et al., 1996; Purdy et al., 1996; Salmassi & Leadbetter, 2003; Ottesen et al. 2006) and it produces DNA from termite hindgut material suitable for use as template for polymerase chain reaction (PCR).

    Protocol

    Procedural summary for termite whole-gut DNA extraction:

    1. Chill termites on ice, remove gut using sterile tweezers and stabilize gut samples in buffer.
    2. Homogenize samples in PVPP/SDS/phenol buffer.
    3. Extract and purify DNA from crude lysate using Qiagen DNeasy columns.

    Protocol:

    1. On ice, remove the guts from worker caste termites using sterile forceps.
    2. Immediately transfer the guts and contents to a sterile, nuclease-free tube containing 50 μL ice-cold 1x molecular biology grade TE buffer (1 mM Tris-HCl, 0.1 mM EDTA, pH 8.0).  Freeze samples at -20°C, or proceed directly with homogenization. 
    3. Transfer the gut samples and buffer to a sterile, nuclease-free 2 ml screw capped tube pre-loaded with 500 mg of sterile zirconia/silica beads (0.1 mm) and 700 μl of 1x TE buffer containing 1% w/v polyvinylpolypyrrolidone (PVPP). 
    4. Add 50 μl of 20% Sodium dodecyl sulfate (SDS) and 500 μl of phenol to the samples.
    5. Homogenize (bead beat) on the highest setting using three cycles of 30 sec homogenization and 30 sec of chilling on ice.
    6. Sediment insoluble material for 1 min at 8,000x g.
    7. Purify 300-μl aliquots of the aqueous (uppermost) layer with Qiagen DNeasy columns using the method described for crude lysate purification.
    8. Quantify nucleic acid content and freeze samples at -20°C for later use.

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    Discussion

    In our experience, DNA extracted from the microbial communities of wood-feeding termite species like Zootermopis nevadensis is sufficiently pure for PCR template after one round of extraction and purification. However, some termites such as litter-feeding and soil-feeding species may have a higher concentration of humic acids in their gut contents and may require additional purification of gut microbial DNA. The total DNA yield from the guts of 5 Z. nevadensis workers is in the range of 10-30 μg. For termite species significantly smaller or larger than this species, more or fewer specimens may be needed to obtain a similar amount of DNA.

    This method can easily be adapted to allow RNA extraction. For RNA extraction, substitute 1x RNAprotect Bacteria reagent from Qiagen (catlog no. 76506) for the ice-cold TE buffer described in step 2 of the protocol. Qiagen RNeasy reagents and columns (catlog no. 74104) should be used in place of the DNeasy purification procedure described above. As DNA can be purified from RNAprotect-stabilized samples and only 300 μL of the approx. 700 μL aqueous layer retrieved in step 7 is required for nucleic acid purification, this method can be used to retrieve both DNA and RNA in parallel from a single sample.

    These techniques depend on the extraction and purification of high-quality nucleic acids from the termite gut environment. The extraction technique described in this video is a modified compilation of protocols developed for extraction and purification of nucleic acids from environmental samples (Moré et al., 1994; Berthelet et al., 1996; Purdy et al., 1996; Salmassi & Leadbetter, 2003; Ottesen et al. 2006) and it produces DNA from termite hindgut material suitable for use as template for polymerase chain reaction (PCR).

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    Disclosures

    Materials

    Name Type Company Catalog Number Comments
    PVPP/SDS/phenol Buffer homogeneization buffer
    DNeasy Tissue Kit Kit Qiagen 77607 Used according to the protocol for isolation of genomic DNA from crude lysates (Appendix H, product manual version: July 2003)
    TE Buffer Sigma-Aldrich T9285 1x buffer (1 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) from 100x concentrate
    zirconia/silica beads Supplies Biospec Products 11079101z 0.1 mm
    PVPP Reagent Sigma-Aldrich P6755 1% w/v polyvinylpolypyrrolidone prepared from dry reagent as a 1x suspension in TE buffer
    Zootermopsis nevadensis Animal Termites
    SDS Reagent Sigma-Aldrich L4390 Sodium dodecyl sulfate 20% soln. in water from dry reagent
    Phenol Reagent Sigma-Aldrich 77607 TE-saturated, ~73%
    MiniBeadbeater-8 Tool Biospec Products 963
    BSS Buffered Salt Solution, pH 7.2 Formulation per liter: 2.5 g K2HPO4, 1.0 g KH2PO4, 1.6 g KCl, 1.4 g NaCl, 0.075 g CaCl, 1 g MgCl, and 10 mL of a 1M soln. of NaHCO3
    AxioPlan-2 Microscope Carl Zeiss, Inc. Outfitted with 40x objective, 1.6x optivar and 10x ocular lenses. Samples were viewed using phase contrast illumination

    References

    1. Berthelet, M., Whyte, L. G., Greer, C. W. Rapid, Direct Extraction of DNA from Soils for PCR Analysis using Polyvinylpolypyrrolidone Spin Columns. FEMS Microbiol. Lett. 138, 17-22 (1996).
    2. Kudo, T., Ohkuma, M., Moriya, S., Noda, S., Ohtoko, K. Molecular Phylogenetic Identification of the Intestinal Anaerobic Microbial Community in the Hindgut of the Termite, Reticulitermes Speratus, without Cultivation. Extremophiles. 2, 155-161 (1998).
    3. Moré, M. I., Herrick, J. B., Silva, M. C., Ghiorse, W. C., Madsen, E. L. Quantitative Cell Lysis of Indigenous Microorganisms and Rapid Extraction of Microbial DNA from Sediment. Appl. Environ. Microbiol. 60, 1572-1580 (1994).
    4. Odelson, D. A., Breznak, J. A. Volatile Fatty Acid Production by the Hindgut Microbiota of Xylophagous Termites. Appl. Environ. Microbiol. 45, 1602-1613 (1983).
    5. Ottesen, E. A., Hong, J. W., Quake, S. R., Leadbetter, J. R. Microfluidic Digital PCR Enables Multigene Analysis of individual Environmental Bacteria. Science. 314, 1464-1467 (2006).
    6. Purdy, K. J., Embley, T. M., Takii, S., Newdell, D. B. Rapid Extraction of DNA and rRNA from Sediments by a Novel Hydroxyapatite Spin-Column Method. Appl. Environ. Microbiol. 62, 3905-3907 (1996).
    7. Salmassi, T. M., Leadbetter, J. R. Analysis of Genes of Tetrahydrofolate-Dependent Metabolism from Cultivated Spirochaetes and the Gut Community of the Termite Zootermopsis Angusticollis. Microbiology. 149, 2529-2537 (2003).
    8. Schmitt-Wagner, D., Friedrich, M. W., Wagner, B., Brune, A. Phylogenetic Diversity, Abundance, and Axial Distribution of Bacteria in the Intestinal Tract of Two Soil-Feeding Termites (Cubitermes spp). Appl. Environ. Microbiol. 69, 6007-6017 (2003).
    9. Tsai, Y. L., Olson, B. H. Rapid Method for Separation of Bacterial DNA from Humic Substances in Sediments for Polymerase Chain Reaction. Appl. Environ. Microbiol. 58, 2292-2295 (1992).

    Comments

    10 Comments

    can you tell me the resolution power of the microscope at which you have shown the video and the make of the mcroscope used.
    Reply

    Posted by: AnonymousSeptember 17, 2007, 12:56 AM

    I would also like to know the magnification used to view the microbes and if any phase contrast was used with the microscope setup. Thanks.
    Reply

    Posted by: AnonymousDecember 20, 2007, 6:24 PM

    As indicated in the updated protocol, a Zeiss AxioPlan-² Imaging microscope was used to aquire the images of termite gut microorganisms using Phase contrast illumination. Our particular set up allows magnification of samples up to 1,600X. This is accomplished using a 100X oil immersion objective lens + a 1.6X optivar + a 10X ocular lens. However, the images in the video use a 40X hi-dry phase contrast objective lens + the 1.6X optivar. To make the video, the videographer replaced the eyepiece with an adapter and attached the camera to that. Because we did not use a scale bar in these videos I do not know the magnification at which the camera recorded so the final magnification will depend on that factor + the window size used to view the video. I will say that the resolution is approximately consistent with what I observe at 640X magnification.
    Reply

    Posted by: Eric M.January 14, 2008, 1:37 AM

    hello sir sir i am a project fellow in IIIM in india i want to know manual method how to isolate total DNA from termite and beetle gut(wood borer).sir kindly if you can provide me protocol for this i will be very thankful to you as i am in very need of it. thanking you sir and waiting for your reply
    Reply

    Posted by: AnonymousJuly 17, 2008, 8:18 AM

    Hello,sir.I am interest in the manual DNA extraction methods from organic tissue,and acquire the best concentraction of DNA, can you provide me for this protocol, thank you.
    Reply

    Posted by: AnonymousSeptember 20, 2009, 6:21 AM

    The information should appear below the video on the JOVE website, and a PDF of the protocol should be downloadable, however, I notice that the link dŒs not currently work. An alternative path to the document can be found by searching the article on PubMed (www.ncbi.nlm.nih.gov/sites/entrez) and choosing the full-text article from PubMed central. I've contacted JOVE to restore the file on their website. Several strategies for purifying insect gut-community DNA exist. A crucial step in any extraction procedure is to remove PCR-inhibiting substances if present. We have found that 1% w/v polyvinylpolypyrrolidone works well on DNA derived from termite gut contents.
    Reply

    Posted by: AnonymousOctober 5, 2009, 3:14 AM

    good job! very cool!
    Reply

    Posted by: jacqui s.December 12, 2009, 7:28 AM

    Hi! I'm korean student. I want to see this full video.then what can i do?
    Reply

    Posted by: Hyun Y.October 31, 2014, 9:47 AM

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