Microcyte¢ç Cytometer

The Microcyte¢ç is a specially developed flow cytometer or microbiological analysis. Flow cytometry (FCM) is a technique for rapid counting and analysis of biological cells and other microscopic particles in a liquid. In contrast to standard microbiological, genetic or biochemical techniques, FCM provides information on individual cells, and not just average values for the population. The ability to analyse individual cells gives access to information that cannot be obtained in any other way.

FCM is widely used in cell biology and immunology e.g. for analysis of white blood cells, but its introduction into microbiology has been slow despite its obvious potential. One reason for this may be as pointed out by Jaernes and Steen that "Flow cytometry of bacteria is still in its infancy, mainly because most instruments currently available must be pushed to their very limit of performance in order to yield data of adequate quality". Optoflow AS has developed the MICROCYTE¢ç with outstanding sensitivity and stability for use in routine microbiological analysis.

Recently, Davy and Kell wrote in a review article: "Indeed, a portable (10kg) laser-diode based flow cytometer (the MICROCYTE¢ç) has recently been developed... The instrument has been designed primarily for the analysis of microorganisms and can detect and provide accurate and absolute counts of particles as small as 0.4 ¥ìm or lower". Similarly, Porter et. al. stated as a concluding remark of a mini review: "Other developments include prodution of cytometers specifically designed to detect microorganisms, including a battery-operated, portable FCM, utilizing a diode laser and fixed optics, thus eliminating machine set-up and focusing, and enabling field use (reference to MICROCYTE¢ç). Advances such as these should help to establish FCM where it belongs- as a necessary routine instrument in microbiology.

Simple to operate
The MICROCYTE¢ç may be operated from PC (Microsoft Windows 95 or NT) or from the front panel of the instrument itself. There are no adjustments to be made prior to analysis. Just turn on the instrument and push "RUN".

Patented optical design
A patented design facilitates enhanced sensitivity and stability. All optical components like lenses, filters, light source, detectors and flow cell are mounted in one solid aluminum block. The light source is a long life diode laser emitting light at 635 nm. For detection of scattered and fluorescent light (650-900 nm) state of the art solid state photo detectors are used. The light scatter sensitivity allows measurement of particles size between 0.4 and 15 ¥ìm.

Low background fluorescence
Excitation at 635 nm reduces the problem with auto fluorescence from most bacteria. Auto fluorescence from algae may be used to analyse these microorganisms without the use of dyes.

Special software
Optional software facilitates data registrations and presentation. The blue histogram shows total registrations (light scatter) and the red part of the histogram shows the fraction of cells having fluorescence (gated values). From the PC it is possible to select four different modes of data registrations, setting of regions of interest, export of recorded data to Microsoft Excel, and other user friendly features.

Counting microorganisms
Rapid enumeration of microorganisms is achieved with MICROCYTE¢ç due to a pressure regulator which ensures that the sample is analysed at a constant flow rate. Counting volumes may be preset to 1, 10 or 100 ¥ìl, making it possible to count as low as 10 cells per ml. Counting may be performed in two independent regions of interest.

Transportable
MICROCYTE¢ç has small physical dimensions and weighs only 12 kg and is run on a 12 V battery pack. Except from an optional PC, everything is inside the chassis, including the water supply with recirculation. This facilitatestransportation and field use. For stationary laboratory use, optional external water supply and pump is available.

Operating modes
Selection between forward light scatter (function of particle size) and fluorescence, threshold setting, log/linear amplification and registration of gated values only, is done on a panel hidden behind the front cover. This makes it ideal for routine applications. Once the running conditions have been determined, the operator may not accidentally change the settings.

Detection limit
MICROCYTE¢ç has optimal working range for particle size analysis (light scatter) from 0.5 to 10 ¥ìm. For measurement of fluorescence the optimal number of molecules of e.g. Cy-5 is from 1 000 to 100 000 per cell.
Detection limit is determined by how carefully the background level in the sample is controlled. Fluorescent particles may be detected at a level below 10 per ml.

Cell counts
Rapid enumeration of microorganisms and animal cells is achieved with MICROCYTE¢ç within few minutes. The correlation with traditional methods is shown to the right and below.

Rapid enumeration of microorganisms
By selecting appropriate fluorescent dyes, different cell counts may be obtained.
? Total cell count may be achieved with cell permeant nucleic acid stains like e.g. SYTO 62 (Molecular Probes, Inc).
? Dead cell counts may be achieved with cell impermeant nucleic acid stains like e.g. TO-PRO-3 (Molecular Probes, Inc) which preferentially stains cells with permeabilised membrane (i.e. dead cells).
? Live cell counts may be achieved with a stain that becomes fluorescent after activation by cellular enzymes like e.g. carboxynaphtofluorescein diacetate (CNFDA) that is converted to carboxynaphtofluorescein (CNF) by cellular esterases.

Starter cultures
Starter cultures may be analysed on MICROCYTE¢ç in order to achieve rapid enumeration of organisms and a measurement of the homogeneity of the culture.

Detection of contaminating microorganisms in food and beverages
Contaminating microorganisms may be detected directly on MICROCYTE¢ç. In some cases a pre incubation in selective medium is recommended in order to detect cell growth at an early stage.

Detection of microorganisms or somatic cells in urine
Urine may be analysed on MICROCYTE¢ç in order to obtain a rapid count of microorganisms or somatic cells.

Water analysis
Water is the simplest medium for flow cytometric analysis. Direct measurements of particles, bacteria and algae may be performed on MICROCYTE¢ç including:
? Municipal water plants
? Polluted water
? Bulk water
? Swimming pools
? Water purification plants
? Sea farms

Analysis of algae in seawater
Algae have natural fluorescence and may be analysed directly on MICROCYTE¢ç.
Ecological cultures of algae are used for feeding larvae in sea farms. An easy way to count and analyse the cultures without any staining is achieved with MICROCYTE¢ç.

Light source: 5 mW laser diode, nominal wavelength 635 nm.
Light scatter detector: Avalanche photo diode, wavelength 636 nm +/-5 nm.
Light scatter sensitivity: 0.4-15 ¥ìm, measured with mono disperse polymer particles.
Light scatter resolution: CV: 5%
Fluorescence detector: Avalanche photo diode, wavelength 650-900 nm.
Fluorescence sensitivity: 1000-100 000 molecules equivalents of Cy-5.
Fluorescence resolution: CV: 5%.
Suitable dyes: TOTO-3, TO-PRO-3, APC, Cy-5, CNFDA, NFDA, SYTO-60-63.
Flow cell: Closed flow channel, 0.25 x 0.25 mm.
Sample: 100¥ìl-1ml in micro centrifuge tubes.
Concentration range: 1x102 -1x107 particles per ml. Detection limit: 10 particles per ml.
Analysing volume: 1, 10 or 100 ¥ìl. (corresponding to analysing time of 2, 20 or 200 seconds, respectively).
Functions: Selection of the following settings:
- Light scatter/fluorescence
- Log/linear amplification
- Threshold
- Gating
Power supply: 12-18 VDC, 2 A, from battery pack, mains adapter (100-240 VAC 50/60 Hz) or car battery (12V).
Battery pack: Internal 12 V, 2.5 Ah, rechargeable NiCd battery for 2-3 hours use.
Connections: - One 2.5 mm jack for DC supply.
- Light scatter signal outlet, BNC plug (female).
- Fluorescence signal outlet, BNC plug (female).
- RS232C, 9 pin D-type (male).
- RCA phono connector for external pump unit.
Dimensions: 430x160x330 mm.
Weight: 12 kg.
Software: Optional software for instrument control and data registrations. Minimum requirements:
- Microsoft Windows NT 3.51/ Windows95 or later.
- Pentium 100 or later.
- Serial port for connection to MICROCYTE¢ç.
- Color monitor, minimum 256 colors, 800x600 resolution.
- Minimum 8 MB RAM.
Trademarks: MICROCYTE is a registered trademark of Optoflow AS.
TO-PRO-3, TOTO-3 and SYTO 60-63 are trade marks of Molecular Probes, Inc.
Cy-5 is a registered trade mark of Nycomed Amersham, Ltd