Below a few references regarding the issue. We have sorted rod shaped E.coli
that give a type of figure of eight distribution in the elite (and the old
ortho cytofluorograph) to show that the double cluster they present is a
question of orientation through the beam. However, you get much bigger
variations when crystals form. If bugs form insoluble formazan from tetrazolium
salts and do not change your scatter gates you will actually miss your cells as
the crystal significantly changes light scatter of the cell. This can actually
be used as a trick for triggering or as a non-fluorescent measurement parameter.
Regards
Gerhard
ANOMALOUS BEHAVIOR OF FORWARD AND PERPENDICULAR LIGHT-SCATTERING OF A
CYANOBACTERIUM OWING TO INTRACELLULAR GAS VACUOLES
DUBELAAR GBJ, VISSER JWM, DONZE M
CYTOMETRY
8: (4) 405-412 JUL 1987
Heterogeneity of cyanobacterial gas-vesicle volume and metabolic activity
Brookes JD, Ganf GG, Oliver RL
JOURNAL OF PLANKTON RESEARCH
22: (8) 1579-1589 AUG 2000
Abstract:
The literature suggests that relative gas-vesicle volume and metabolic activity
differ between individuals within natural
populations of cyanobacteria. We demonstrate, using flow cytometry and
fluorcscein diacetate (FDA) conversion rates, that
individual relative gas-vesicle volumes differed by 6.7- and 4.6-fold, and
individual cell photosynthetic rates ranged from 0.026
to 0.127 and from 0.075 to 2.415 fmol O-2 cell(-1) min(-1) within
phosphorus-starved and phosphorus-replete Microcystis
aeruginosa populations, respectively. The observation that there is
considerable heterogeneity in gas-vesicle volume and cell
metabolic activity in cultured populations suggests that this could also
contribute to heterogeneity in buoyancy of field
populations.
SINGLE-CELL LIGHT SCATTER AS A PROBE OF REFRACTILE BODY FORMATION IN
RECOMBINANT ESCHERICHIA-COLI
WITTRUP KD, MANN MB, FENTON DM, TSAI LB, BAILEY JE
BIO-TECHNOLOGY
6: (4) 423-426 APR 1988
Linear correlation between bacterial overexpression of recombinant peptides and
cell light scatter
LavergneMazeau F, Maftah A, Cenatiempo Y, Julien R
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
62: (8) 3042-3046 AUG 1996
Abstract:
Fusion of multiple copies of a test peptide leads to insoluble inclusion
bodies. Their presence within bacteria increases either
forward-angle light scattering or, to lesser extent, right-angle light
scattering. A linear correlation has been established between
cell forward-angle light scattering and the level of overexpression of atrial
natriuretic peptide. The correlation is valid only for
unlysed cells and is protein product specific.
QUANTITATIVE SINGLE-CELL DETECTION OF POLY(BETA-HYDROXYBUTYRATE) ACCUMULATION
IN RHIZOBIUM-MELILOTI BY FLOW-CYTOMETRY
FOUCHET P, JAN S, COURTOIS J, COURTOIS B, FRELAT G, BARBOTIN JN
FEMS MICROBIOLOGY LETTERS
126: (1) 31-35 FEB 1 1995
Abstract:
The ability of flow cytometry to detect intracellular accumulation of
poly(beta-hydroxybutyrate) (PHB) in individual cells of
Rhizobium meliloti was investigated using light scatter parameters. PHB
accumulation during stationary phase was shown to
form inclusion bodies inside cells which were detected by forward angle light
scatter (FALS) parameter. A linear relationship
was found between FALS intensity and percentage of PHB (w/w) of cells,
demonstrating that flow cytometry allowed a
quantitative analysis of intracellular PHB accumulation. In addition, results
showed the ability of flow cytometry to quantify
heterogeneity of rhizobia population in fermentation according to PHB
production and accumulation.
FLOW-CYTOMETRY STUDIES OF RECOMBINANT ESCHERICHIA-COLI IN BATCH AND CONTINUOUS
CULTURES - DNA AND RNA CONTENTS, LIGHT-SCATTER PARAMETERS
FOUCHET P, MANIN C, RICHARD H, FRELAT G, BARBOTIN JN
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
41: (5) 584-590 JUL 1994
Abstract:
Flow cytometry has been used to study the contents of macromolecular compounds
and light-scatter parameters in batch and
continuous cultures of a recombinant Escherichia coli strain that forms protein
inclusion bodies. Changes in relative DNA and
RNA contents and cell mass as estimated by forward-angle light scatter were
detected and tightly correlated in batch culture. In
addition, heterogeneity of wide-angle light scatter (WALS), which we related to
the presence of cellular inclusion bodies, was
observed. In contrast, the relative RNA content and cell mass did not change
during continuous culture, and homogeneity of
WALS was found. In addition, unexpected changes in relative DNA content were
observed after 67 h of culture, indicating a
change in bacterial physiology.
-----Original Message-----
From: Elaine Kunze [SMTP:mek4@PSU.EDU]
Sent: Tuesday, October 10, 2000 8:02 PM
To: Cytometry Mailing List
Subject: bacteria inclusion bodies
Has anyone ever detected/sorted bacteria with inclusion bodies using
scatter measurements? Is it possible to detect this?
****************************************************************************
Elaine Kunze
Flow Cytometry.....Image Analysis...
Life Sciences Consortium
8B Althouse Laboratory (814-863-2762)
Penn State University
University Park, PA 16802
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