Key problem in non-ratiometric counting methods are system dead times and=
flow =
inconsistency. Make sure your reference particles are not out of scale in=
any =
signal as that can fool your electronics. =
Absolute counts for beads can be calculated accurately from % solids and=
=
diameter and polymer density
(0.5u latex at 2.5% solids come at 3.64x10^11/ml)
Absolute counts with the Coulter XL work very well as long as the acquisi=
tion =
rate is below 1300 events per second. When counting 100ul (five 20ul cavi=
ties) A
display of time versus count tells you about clogging and excess flow rat=
e (>300
on 300 second timescale). Unfortunately versionII software cuts of too ea=
rly, so
only the data for 80ul can be used.
Counting methods
- Fixed volume counting or volume integration :
This method is used in most haematology analysers. The volume measurement=
is =
achieved by two electrodes acting as level sensors in a known geometric s=
et-up =
(Partec CAII) or by loading a cavity in a ceramic valve (Coulter XL) or a=
loop =
made of tubing as done in high pressure liquid chromatography instrument=
s. In =
all cases all events within the volume are measured. =
- Time integration :
This approach is based on the assumption of a constant volume flow over t=
ime. It
is best achieved by (syringe) pumps delivering the sample. Such systems a=
re =
implemented in the Ortho Cytron absolute and the former Skatron Argus flo=
w =
cytometer now Bio-Rad Bryte HS.
- Spiking with reference particles (ratiometric counting) :
This method can be used with all flow cytometers. It corrects for system =
dead =
times and the variations in flow rates that can occur in instruments that=
use =
differential pressure to deliver their samples. An example of bacterial c=
ounting
using a bead standard is given in Figure 5. The tight cluster of beads al=
so =
serves as an on line alignment control, particularly important when measu=
ring =
environmental samples that are more likely to block the flow path. =
=
Cell disaggregation
Single cell suspensions are essential for any form of accurate count=
ing. =
Aggregates only give rise to one single colony or event. If for example o=
ne cell
in a triplet is positive for a dead cell marker the whole aggregate is =
registered dead but will grow when sorted onto agar plates. Skin sample a=
re far =
worth as hundreds of bacteria can be attached to a single squame.
Cells can be dissaggregated by either chemical or mechanical meth=
ods. =
Mechanical methods have a broader application spectrum but can lead to pr=
oblems =
with filamentous organisms. Shearing by needles leads to problems with c=
logging
and is very tedious. Shearing with homogenisers is difficult with small v=
olumes =
and causes problems with foaming and sample carryover. =
Ultrasonic treatment is the most convenient method, but it is im=
portant
to apply reproducible energy levels. The geometry of the set-up and the m=
aterial
of the sample container has to be taken into consideration. When using a =
probe, =
energy loss can occur by coupling to ice cold water surrounding the sampl=
e =
container or by airbubbles trapped at the bottom of pointed vessels. =
Transmittable energy in an ultrasonic waterbath is sensitive to the leve=
l of =
water, its temperature and dissolved gas as well as its cleanliness. Soft=
=
container materials like polypropylene do absorb the energy in both syste=
ms.
some points of discussion
The high number of counts achieved by flow cytometry make the tec=
hnique =
superior to others with the regard of counting accuracy. By careful pipet=
ting =
technique and using 0.05% tween 20 to avoid cell sticking we could achiev=
e =
counting variations within 1% of the expected numerical counting error. =
The detection sensitivity of optical systems is limited by the =
statistical abundance of an event and the signal intensity separating the=
event =
from background noise. To identify a cell cluster it is desirable to have=
at =
least 100 cells in it. Thus if there is one organism per =B5l in the fina=
l sample =
volume, 100=B5l have to be measured. Relative frequency is another limit =
to the =
measurement. To detect a log 3 reduction equivalent to an event frequency=
of =
0.1% 100.000 events need to be screened to see 100 wanted cells. Good sig=
nal to =
noise ratio is therefore important, as, at lower relative frequency, the =
labelled cells are end up within the standard deviation of the unlabelled=
=
events. =
Signal to noise ratio is also limiting the speed of measurement, =
as with
increased sample / volume throughput the variations broaden but in partic=
ular =
the background fluorescene increases due to free fluorochrome. We current=
ly run =
flow rates below 10=B5l/min which leaves us with a practical sensitivity =
of =
approximately 10^3 within 10 minutes. Lower concentrations require pati=
ence or
pre-enrichment by physical or biological means.
Key problem in other counting methods are system dead times and flow =
inconsisteny. Make sure your reference particles are not out of scale in =
any =
signal as that can fool your electronics.
Gerhard Nebe-v.Caron
Unilever Research, Colworth,
Sharnbrook, Bedfordshire
GB - MK44 1LQ
Tel: +44(0)1234-222066
FAX: +44(0)1234-222344
gerhard.nebe-von-caron@unilever.com =
______________________________ Reply Separator __________________________=
_______
Subject: absolute counts
Author: novod@muss.cis.mcmaster.ca at INTERNET
Date: 26/02/97 22:37
Hello Everyone,
I am trying to do absolute counts of bacteria. I have been using a
kit but its about $100 for 100 samples. My problem is that the kit keeps
dissapearing faster than the bottle of vodka I keep on the lab shelf for
days that experiments don't work. I am wondering if anyone has a
cheaper/longer lasting suggestion for absolute counting of cells on the
FACScan..
Thanks for your help.
I will compile the suggestions and post them for future reference.
Dave
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I have nothing to put here that anyone would care about. But since
everyone has one of these, I figured I should too . . . . .
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