I am looking for a very simple approach to identify bacterial diversity in bacterial cultures. I have unselectively cultivated fecal samples based on a dilution series, so that only one single bacterial cell is contained per well at the end. Now I'd like to get a quick an easy overview about how big the diversity of the cultures is, e.g. getting to know how many different bacterial cultures I have. At that point I don't want to identify which bacteria I have actually cultivated. I just want to know how many DIFFERENT bacteria I obtained to select samples for further identification. I thought of a simple approach based on 16S rRNA PCR (maybe primers 27F & 1492R) followed by restriction digestion and simple agarose gel visualisation to get an overview about how many different restriction patterns I have. Something similar to T-RFLP fingerprinting but not as complex as I don't need a very high resolution. Is that practicable and has anyone done something similar? Any thought on protocol/primer/restriction enzyme/literature recommendations? Thanks in advance!
T-RFLP analysis is producing less "complex" fingerprinting patterns compared to restriction digestion of PCR products as only the terminal fragment is fluorescently labelled and will be detected. Nowadays, I would not wrestle with fingerprints but go straight for the sequencing of your PCR products using Sanger sequencing, e.g. using the service of a company; the cost for sequencing is really low, especially when you consider that you also need a purified PCR product, restriction enzyme(s) and analytical gel electrophoresis for a restriction based assay. When you suspect that you do not have a pure culture in the last well of your dilution series, you could still tell that it is a mixed culture based on overlain chromatograms from sequencing.
T-RFLP analysis is producing less "complex" fingerprinting patterns compared to restriction digestion of PCR products as only the terminal fragment is fluorescently labelled and will be detected. Nowadays, I would not wrestle with fingerprints but go straight for the sequencing of your PCR products using Sanger sequencing, e.g. using the service of a company; the cost for sequencing is really low, especially when you consider that you also need a purified PCR product, restriction enzyme(s) and analytical gel electrophoresis for a restriction based assay. When you suspect that you do not have a pure culture in the last well of your dilution series, you could still tell that it is a mixed culture based on overlain chromatograms from sequencing.
Alena Marie Bubeck Previously (in late 1990s) we used Single Strand Confirmation polymorphism (SSCP) analysis or Denaturing / Temperature gradient Gel Electrophoresis for such purpose. It worked well but needed some skills.
Schwieger F, Tebbe CC. A new approach to utilize PCR–single-strand-conformation polymorphism for 16S rRNA gene-based microbial community analysis. Appl. Environ. Microbiol.. 1998 Dec 1;64(12):4870-6.
Muyzer G, Smalla K. Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie van Leeuwenhoek. 1998 Jan 1;73(1):127-41.
Dear Alena,
While I agree that you could use 16S rDNA sequencing or T-RFLP approaches to assess strain diversity, you could also do some old-fashioned microbiology and simply plate out your strains onto a variety of different plates (complex or limited nutrients; different sugars; pH dye, etc.) and test growth at different temperatures, etc. to look at phenotype diversity (we test any phenotype that is cheap and easy to do). We often use this approach (as plating out is inexpensive; we don't have money for external sequencing; we have students) and examine the data using Hierarchical cluster analysis to look at similarities (and biological replicates) within our collections of bacteria.
Regards, Andrew
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