I'm not certain I understand your question, but if you are asking why
differences exist between smaples stained and then examined every five
minutes vs. samples stained only at the end (how long might that be?) of
the experiment, I could offer the following hypothesis:

Acridine orange is a potent carcinogen and could well be toxic to the
bacteria.  Although we have observed that many bacterial species can
continue to grow and appear healthy with stains such as DAPI and Syto
hanging on their nucleic acids, we have not looked at AO nor at the effect
of repeated laser illumination on bacteria stained with
nucleic-acid-binding dyes.  It sounds as though your bacterial cells could
be killed during the visualization process, thereby affecting the
acidification.  I would use some other stain to visualize the bacteria.

Rob


>        Dear Confocalists,
>
>        We are following the acidification of milk with a bacterial culture
>by Confocal Microscopy. The bacteries are stained with Orange Acridine,
>which allows also to vizualise the protein network. We have compared the
>structure of the network after two protocols:
>                1: We follow the kinetics of structuration, taking a view of
>the sample in the same confocal plane every 5 minutes.
>                2: We only look at the structure at the end of the
>acidification.
>       In the second type of experiment, we see that the structuration is
>clearly evidenced and  we clearly observe the bacteries, which is not true
>in the first experiment.
>        Could someone please give us any information on the effect of
>illumination by the laser on the bacterial growth? Could it change the
>metabolism of the bacterial culture?
>        Thank you very much in advance.
>
>        Catherine
>
>
>
>**********************************
>Dr Catherine Garnier
>INRA-LPCM
>Rue de la Geraudiere
>BP 71627
>44 316 Nantes Cedex 03
>France
>Phone: Int-33-2-40-67-50-45
>Fax:   Int-33-2-40-67-50-43
>**********************************

Robert J. Palmer Jr., Ph.D.
Natl Inst Dental Craniofacial Res - Natl Insts Health
Oral Infection and Immunity Branch
Bldg 30, Room 310
30 Convent Drive
Bethesda MD 20892
ph 301-496-2088
fax 301-402-0396