Is their any easy method by which I could separate microbial cells from saliva samples of human. It would be really helpfull if someone can share related protocol.
Dear Shubhashish Chakraborty,
Among the methods used to separate bacterial cells from human cells are the one described by Wu Z et al. entitled " Soft inertial microfluidics for high throughput separation of bacteria from human blood cells":
Lab Chip. 2009 May 7;9(9):1193-9. doi: 10.1039/b817611f. Epub 2009 Feb 13.
Soft inertial microfluidics for high throughput separation of bacteria from human blood cells.
Wu Z1, Willing B, Bjerketorp J, Jansson JK, Hjort K.
Abstract
We developed a new approach to separate bacteria from human blood cells based on soft inertial force induced migration with flow defined curved and focused sample flow inside a microfluidic device. This approach relies on a combination of an asymmetrical sheath flow and proper channel geometry to generate a soft inertial force on the sample fluid in the curved and focused sample flow segment to deflect larger particles away while the smaller ones are kept on or near the original flow streamline. The curved and focused sample flow and inertial effect were visualized and verified using a fluorescent dye primed in the device. First the particle behaviour was studied in detail using 9.9 and 1.0 microm particles with a polymer-based prototype. The prototype device is compact with an active size of 3 mm(2). The soft inertial effect and deflection distance were proportional to the fluid Reynolds number (Re) and particle Reynolds number (Re(p)), respectively. We successfully demonstrated separation of bacteria (Escherichia coli) from human red blood cells at high cell concentrations (above 10(8)/mL), using a sample flow rate of up to 18 microL/min. This resulted in at least a 300-fold enrichment of bacteria at a wide range of flow rates with a controlled flow spreading. The separated cells were proven to be viable. Proteins from fractions before and after cell separation were analyzed by gel electrophoresis and staining to verify the removal of red blood cell proteins from the bacterial cell fraction. This novel microfluidic process is robust, reproducible, simple to perform, and has a high throughput compared to other cell sorting systems. Microfluidic systems based on these principles could easily be manufactured for clinical laboratory and biomedical applications.
http://www.ncbi.nlm.nih.gov/pubmed/19370236
I believe that this method is applicable for saliva samples taken from human.
Another method which can be used is Kinetically limited differential centrifugation:
Kinetically limited differential centrifugation as an inexpensive and readily available alternative to centrifugal elutriation
http://www.biotechniques.com/BiotechniquesJournal/2012/August/Kinetically-limited-differential-centrifugation-as-an-inexpensive-and-readily-available-alternative-to-centrifugal-elutriation/biotechniques-333838.html?service=print
Hoping this will be helpful,
Rafik
Dear Subhashish,
Check if FACS (Fluorescence Activated Cell Sorter) can be of any use in this case by focussing on some specific marker proteins and labelling them.
All the best.
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