Re: absolute counts

Gerhard Nebe-von-Caron (
28 Feb 1997 12:32:35 Z

A number of cents from my end (see purdue CD2 for further information)

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=
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=
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=
to apply reproducible energy levels. The geometry of the set-up and the m=
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=

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 =

______________________________ Reply Separator __________________________=
Subject: absolute counts
Author: 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

Thanks for your help.
I will compile the suggestions and post them for future reference.


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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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the Purdue University Cytometry Laboratories and distributed free of charge as an educational service to the cytometry community. If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL, Purdue University, West Lafayette, IN 47907. Phone: (765)-494-0757; FAX(765) 494-0517; Web , EMAIL