Standards for bacterial identification by fluorescence In situ hybridization within eukaryotic tissue using ribosomal rRNA-based probes.

To the Editor: The article BMolecular Characterization of Rectal Mucosa-associated Bacterial Flora in Inflammatory Bowel Disease[ by Maria Mylonaki, Neil B. Rayment, David S. Rampton, Barry N. Hudspith, and Jonathan Brostoff published in the journal (2005;11[5]:481Y487) is not scientifically sound. The authors performed fluorescent in situ hybridization (FISH) using a set of 16s RNA-based Cy5 probes and found high concentrations of Bifidobacteria (mean 15 bacteria/1-mm epithelial surface) in normal control subjects and increased intracellular Escherichia coli and Clostridia in inflammatory bowel disease patients. The authors assure us that accurate morphometric measurements were made of each biopsy, that Bthe total mucosal area was calculated. [ Individual bacterial numbers were expressed as numbers of bacteria per millimeter of epithelial surface. Actually, the Cy5 fluorochrome that the authors used is dark red and cannot be perceived with the human eye. Because the only possible way to monitor the fluorescence is with a camera, the microscopy with Axioplan 2 must be performed Bblindly,[ and morphology can be evaluated only after pictures are taken. However, Cy5 fades quickly, which makes simultaneous searching for optimal location within the biopsy and taking usable pictures difficult. The weak signal of Cy5 makes it impossible to perform orienting shots at low (G 400) magnifications to obtain an overview of the biopsy. An inevitable random focusing over the biopsy surface does not allow any morphometric measurements. The following evaluation of microphotographs depends on photoprocessing software. For each setting of photographs, the investigator must choose individually the brightness, contrast, and so forth at which he or she can best evaluate the findings. This adjustment necessarily manipulates the results. The counting of edited signals by 2 observers does not change anything. In fact, none of the figures presented by the authors shows signals that should be identified as bacteria. Figure 1A shows $100 irregular fluorescent clouds that do not have a bacterial morphology and are not DAPI counterstained. The range the authors give for Bifidobacteria is 4 to 56 cells/1-mm biopsy surface. That means that maximally 25 Bifidobacteria can be seen within a single microscopic field at a magnification of 400. It should be easy for the authors to use arrows to point out what they regard as bacteria in Figure 1A. The negative control, Figure 1B, shows nothing and therefore cannot be interpreted at all. Normally, each FISH probe, especially Cy5-labeled probes, produces a marked background staining by binding nonspecifically to human tissues. This unspecific background fluorescence makes the human tissue morphology clearly perceivable at fluorescence microscopy and eases the orientation. The phenomenon is general, and the unspecific background fluorescence contrasting human tissues is obvious in Figures 1C through I. In Figures 1A and B, the background fluorescence of the human tissue is absent. The authors do not explain why. Because epithelial morphology cannot be perceived, it is impossible to say fromwhat area Figures 1A and B were taken and how the microphotographs were made. Clearly, they were not made from the biopsy tissue or its surrounding area. All of the other panels claiming to present intracellular bacteria (C, E, G, H, I) demonstrate typical biases of unspecific Cy5 binding to inflammatory cells. Unlike hybridization signals specific for bacteria, these signals cannot be washed off at any temperature, even at