Cindy McAllister wrote: > An investigator here is hoping to sort bacteria from rat blood. He would >like to dilute the blood in citrated buffer and throw it on the sorter and >go. I told him the sort would be much quicker and cleaner if he removes >RBC's, WBC's etc. before sorting. I have searched the Purdue archives and >Pub Med. and have not found any articles dealing with this issue. My >question is, is anyone sorting bacteria from blood? If so, would you be so >kind as to send a protocol or indicate a reference where I can look up a >procedure? Many years ago, Mansour et al described using flow cytometry to detect bacteria in blood(Mansour JD, Robson JA, Arndt CW, Schulte TH: Detection of Escherichia coli in blood using flow cytometry. Cytometry 6:186, 1985). In septicemia, a blood culture can be positive if it contains 1 organism per 10 ml of blood; Mansour et al, using a fairly harsh lysing procedure to get rid of the cellular elements in blood, were able to detect 100 organisms/ml after staining with ethidium bromide. What levels of bacteremia does your investigator expect to encounter in rat blood? Recent events have reminded us that, in fulminant anthrax, there can be enough bugs in the blood for them to be readily visualized under the microscope, but patients or animals at that stage of the disease are not likely to be keeping their blood circulating much longer. And, in most other septicemias, it is not all that common for bacteria to be easily seen on a blood smear. A million bacteria/ml should be fairly easy to see; a hundred bacteria/ml won't be. And, if the objective is to sort bacteria from blood, and you're dealing with 100/ml or 1000/ml, you can expect sorting any significant number of organisms to take a long time. Finally, what organisms are we talking about? Gram-positive? Gram-negative? There are some vital staining techniques that will increase the likelihood of detecting the organisms, but nothing works on all species and strains. It is unlikely that you will be able to "dilute the blood in citrated buffer and throw it on the sorter and go", but there may be a way of making the project work, given more specific information. -Howard
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