[Cytometry] To Those Sorting Bacteria

Moss, Delynn M. (CDC/OID/NCEZID) dmm3 at cdc.gov
Mon Apr 29 07:49:14 EDT 2013


Hi,

We have been using flow (AriaIII) as a glorified pipettor to sort specific numbers of Escherichia coli for PCR detection limits.  The E. coli is grown in trypticase/soy/broth (TSB) overnight at 37 C with gentle shaking and results in post log-phase growth; only requires 1 - 2 ul suspension/ml of fresh TSB to obtain about 1000 events/sec with a flow rate setting of 1.0.  Most end-users ask, "How do you know the instrument sorted the requested bacteria numbers?", and I reply with fluorescence scope data from Syto 9 DNA stain of the E. coli, which is sorted onto a 1" x 3 " slide; 9 locations with 1 E. coli, 9 with 5, and 9 with 10.  For more than 95% of the time, the fluorescence scope data matches the sort request, indicating good quantitative sorts.  For the less than 5% of the time, where data does not match, dried salts likely obscured the fluorescence of the Syto 9 DNA stain, but on rare occasions, I see multiple nucleic acid Syto 9 stain per one event.

Recently, Vibrio chlolerae and  Salmonella typhimurium were sorted. These bacteria were grown in similar manner but in nutrient broth, and after overnight incubation, they were not in post log-phase growth; had to use about 10 ul of suspension/ml of fresh nutrient broth to obtain 1000 events/sec with a flow rate setting of 1.0.  Unlike the E. coli, observation by brightfield scope showed many highly active organisms in chain formation.  For about 25% of the time, fluorescence scope Syto 9 data did not match sort request, the former higher than the later.  This indicates that some events "seen" by the instrument as a single event are actually multiple organisms, despite the use of Doublet Discrimination, which, I belive, cannot discriminate doublets on a portion of the sorted population that are orientated at the least cross-sectional area to the laser at laser interrogation.

Although less frequent, multiple organisms per event likely occur in all post log-phase cultures.  Is there a way to avoid or minimize this?

Thanks

Delynn Moss
Water, Sanitation and Hygiene Laboratory
Centers for Disease Control and Prevention
National Center for Emerging and Zoonotic Infectious Diseases
Waterborne Disease Prevention Branch
1600 Clifton Road, NE
Mailstop D-66
Atlanta, GA 30329
Phone:  404-718-4136





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