Epifluorescence

Afonso Souza souza at ocean.ocean.fsu.edu
Fri Feb 11 13:58:06 EST 2000


Hi Sussanne,
I received your question through my lab-mate. I work with fluorescent dyes specifically to determine sediment bacterial number and activity. I assume you have a microscope suitable for epifluorescence.

1. Total counts
    The most common way of determining total number is by DAPI staining. This dye works on the UV range of the spectrum absorbing at 358nm and emitting at 461nm . Acridine Orange ( 500nm650nm) also works, but to my purposes DAPI is the dye of choice.

2. Live or dead
    Nowadays, there are a suite of dyes capable of identifying cells with compromised menbranes ( dead ) and cells with intact ones (live). Live/Dead BacLight is a good kit. It has two dyes: SYTO9 and propidium iodine to identify total and intact cells and compromised-membrane cells, respectively. It is sold by Molecular Probes. I have seen works that showed other combinations of dyes and protocols. You can buy only propidium iodine and make a double stain with DAPI. Other way is washing a DAPI stain with ethanol, the residual being live cells.

3. Bacterial activity
    Activity can be determined with CTC, a tetrazodium dye, which is reduced by the bacterium via ETC.The only problem is the long incubation time (4hrs). Otherwise, it is very good technique. The dye is stable and the cells are easily identified. But it seems that your bugs are quite fast growers, I would suggest one of the live/dead stains.
    There is another way to access activity. Respiration rates obtained by CO2 evolution. Radio-labeled carbon is given as food source and CO2 is captured and measured through scintillation counts. The assay can be as short as 15-30 minutes.

4.References
    .Marine pollution bulletin 36(9):739-741
    .Journal of microbiological methods 32:225-236.
    
I hope it helps and contact me anytime.
Regards
.
Afonso Souza
Department of Oceanography
Florida State University
Tallahassee, FL 32306
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