BioDETECT: Frequently Asked Questions


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Frequently Asked Questions

Questions 1 to 7 are fully written by Hazel Davey

General

What is cytometry?
Cytometry refers to the measurement (-metry) of cells (cyto-). These measurements may be of the cell's physical properties (length, volume, etc.) or of its biochemical properties (protein content, lipid content etc.) Traditionally these measurements have been made by light or fluorescence microscopy. Microscopy is a labour-intensive task, prone to error and operator fatigue. It is also slow to perform and consequently conclusions are drawn from measurements of, at best, a few hundred cells. An alternative method of measuring protein content for example, would be to perform bulk biochemical measurements on a population of cells. However in doing this one makes the assumption that all of the cells in a population are behaving in a similar manner. The extent to which this assumption is true will vary with the cell type under investigation but in many cases the heterogeneity of a population of cells will be large, not least because of changes that occur as the cells progress through the cell cycle.
What is flow cytometry?
The term flow cytometry refers to these same physical and biochemical measurements however, the measurements are made as the cells flow past an array of detectors. In microscopy a sample is placed on a microscope slide and the objective lens ("detector") is moved over the sample; in contrast in the flow cytometer the detectors are fixed in position and the sample is moving. Typically, the cell sample is introduced into the centre of a stream of sheath fluid. The sheath fluid is pumped much more quickly than the sample and so the cells are constrained to the centre of the sheath fluid. This process, known as hydrodynamic focusing, allows the cells to be delivered reproducibly to the centre of the measuring point. At the measuring point (Fig 1) the stream of cells intersects a beam of light. In most flow cytometers the light source is a laser or an arc lamp. In the Microcyte a laser diode is used; this reduces the size, weight and cost of the flow cytometer. When cells interact with the light from the laser diode some of the light is scattered out of the beam. A detector in the Microcyte collects light scattered in the forward direction. The amount of scattered light in this region gives a measure of the size of the scattering particle. In addition to light scattering, the Microcyte also measures particle-associated fluorescence. Fluorescent stains that bind specifically to a cellular component (e.g. DNA) may be added before analysis. Alternatively stains that are excluded by living cells but taken up by dead cells may be used. In this way two parameters can be measured simultaneously.
What are the advantages of flow cytometry?
Flow cytometry offers several advantages over conventional methods. The problem of sample heterogeneity was mentioned above. In the flow cytometer measurements are made on individual cells and so sample heterogeneity can be quantified. This means that under some circumstances it may be possible to identify subpopulations of cells or to detect contaminants. Flow cytometry is a rapid technique; measurements are typically made at rates of 1000 cells.s-1. This means that many thousands of cells can be measured in a realistic time scale.

Haemocytometer counts are laborious to perform, lead to operator fatigue and are prone to error. For a count of n organisms the percentage error may be represented as %, i.e. ±1% for a count of 10,000 counts. Rapid sample analysis by flow cytometry permits analysis of >10,000 cells per sample, in most situations it is unlikely that more than 1000 cells would be counted by haemocytometry (>3% error).

Plate counts, although the "gold standard" in microbiology, detect only those cells capable of growing under the conditions provided (pH, carbon source, incubation temperature etc.). As such they give an underestimate not only of the total count but also of the true viable count. The Microcyte detects all particles based on their light scattering properties. There is no requirement that the cells will grow on a designated medium or even that they be alive. This total count can be combined with a viability stain (see below) where a measure of the viable microbial load is required.

Most of the advantages here are related to those given under haemocytometer counts above. In addition, a suitable fluorescent stain may be added to the sample prior to analysis enabling simultaneous determination of both total and viable counts.

A common method of particle counting is to use systems based on the Coulter principle. However the Microcyte has been designed with microorganisms in mind and, unlike the Coulter counter, has a large dynamic range and in the same sample can count (and size) particles from as low as 0.4 um diameter to 15 um. The Coulter counter can determine the concentration of appropriately sized particles but unlike the Microcyte it cannot simultaneously monitor the viability of the sample. Where viability is not an issue the fluorescence parameter of the Microcyte may be used to make other physiological measurements such as determining the DNA content of the particles - this may be useful for distinguishing between biological and non- biological particles.

The majority of flow cytometers are designed primarily for the analysis of mammalian cells (~10 um diameter) and do not function optimally with bacteria (~1 um diameter); in some instruments bacteria are on the limit of detection and difficult to distinguish from background noise. The Microcyte has been designed to count and size particles between 0.4 and 15 um, and the background has been reduced to allow bacteria to be detected easily.

The Microcyte is much smaller than any other commercially available flow cytometer. With dimensions of just 330 x 430 x 160 mm and a weight of just 10 kg the instrument is fully portable. It can be powered from its own rechargeable internal batteries for several hours or by an external 12V D.C. source making it ideal for both field and laboratory applications.

The optical components of the Microcyte are enclosed in a solid aluminium block and so do not need to be aligned (daily alignment is necessary in most other flow cytometers). Internal feedback ensures the stability of both light source and detector - no photomultiplier voltages need to be adjusted and there are no lamp deterioration effects. This makes operation of the Microcyte much simpler, the operator simply switches on the instrument and it is ready for use.

The price of the Microcyte compares extremely favourably with that of other flow cytometers.

What sort of samples can be run?
A wide variety of samples are amenable to flow cytometry. However, the operator must ensure that there are no large clumps of cells present in the sample as these could block the flow cell. If reliable measurements are to be made then the sample must be presented as monodisperse cells, rather than as clumps. Two cells that are stuck together will appear as one large cell to the flow cytometer.

For reliable counting the particle concentration should be between 1 x 103 and 1 x 107 / ml. With the higher concentrations the count is done on 1 ul, while for lower concentrations the measurement time can be increased so that the number of particles in 5 or more microlitres are counted. For more information on counting particles with the Microcyte.

The operator must ensure that no large clumps of particles are present for the reasons described above. The sample must not be too concentrated nor too dilute to allow reliable counting without coincident events. If the sample is too concentrated the Microcyte will display a warning message on the screen, while if it is too dilute then no particles will be detected. If a fluorescent stain is required then the operator must add this before analysing the sample. To run the sample on the Microcyte an Eppendorf-type tube containing at least 0.2 ml is simply placed into the sample holder and the run button is pressed.

Particles in the size range 0.4 to 15 um can be counted. The Microcyte has been tested with bacteria, yeast, animal cells and latex beads.

What is meant by light scattering?
Light scattering measurements have found many applications in biology, ranging from the assessment of bacterial concentration in a suspension to resolution of the fine structure of the cell. Optical density measurement is a common method for the estimation of microbial biomass. When light interacts with a cell suspension some of that light is scattered out of the incident beam, while some is absorbed. The more concentrated the suspension, the more light is absorbed and scattered and thus biomass concentration can be estimated by measuring the amount of light that is transmitted through the suspension. As mentioned earlier, however, the special power of flow cytometry is its ability to make measurements on single cells rather than on populations of cells. When a single cell intersects the light beam of a flow cytometer some of the light is scattered out of the beam. The amount of light that is scattered by a cell is a complex function of its size, shape and refractive index. The sensitivity of light scattering to each of these factors is dependent upon the range of angles over which the scattered light is collected. Light scattered at small angles (i.e. forward light scatter as detected in the Microcyte) is most dependent upon the size of the scattering particle.
What is fluorescence?
When a compound absorbs light electrons are raised from the ground state to an excited state. The electrons return to the ground state via a variety of routes; some processes such as the loss of the energy by heat do not result in fluorescence, but certain molecules also lose energy by a process of radiative transition (fluorescence). Fluorescence is always of a lower energy, and hence longer wavelength, than the exciting light, and this separation in wavelength is known as the Stokes shift. The Stokes shift enables the exciting and emitted light to be separated by optical filters and thus the amount of fluorescence can be quantified. While every cell in a population will scatter light not all cells will necessarily fluoresce significantly; therefore light scattering, together with fluorescence measurements are useful to give a measure of the percentage of fluorescent cells in a population.
What stains can be used with the Microcyte?
A range of stains that can be excited at 635 nm can be found in the following table. Those shown in bold type in the table below have been tested successfully.
Stain,Excitation,Emission,Reference(s),Determinand

TOTO-3 iodide 642 660 MP 17 / T-3604 Nucleic acid
TO-PRO-3 iodide 642 661 MP 25 / T-3605 Nucleic acid / viability
Oxazine 750 673 691 1, 2 DNA
LD700 / rhodamine 700 1 DNA
rhodamine 800 685 700 1, 2 DNA
Cy5 3
Napthofluorescein 594 663 MP 16 / N-650 Enzyme substrate
Allophycocyanin 650 660 MP 14 / A-803 Antibody label
polymethine cyanine dyes
1,1'-diethyl-3,3,3',3'-indodicarbocyanine iodide (DilC2(5)) 636 657
MP 23/D-1124 Membrane / membrane energisation 3,3'-diethyl-thiadicarbo-cyanine iodide (DiSC2(5)) 649 671 MP 23 / D-304, see also D-324, D-306, H-380 Membrane energisation
Oxonol-V 610 639 MP 23 / O-266 Membrane energisation
5-(and-6)-carboxy-napthofluorescein, succinimidyl ester 600 672 MP 5 / C-653
1,9-dimethylmethylene blue 650 674 MP 29 / D-665
DMOTC 682 718 2
Oxazine 720 618 640 2
Nile blue 640 672 2 Lipid

NOTES: MP = Molecular Probes catalogue (Haugland, R.P. (1992). Molecular probes handbook of fluorescent probes and research chemicals (5th edition). ed. Larison, K.D. Molecular Probes Inc.), Molecular Probes Ltd, PO Box 22010, Eugene, Oregon 97402, USA). Set number and catalogue number are given. Excitation and Emission wavelengths for some of the dyes in this table may be as used in the cited papers rather than their l(max). 1. Shapiro, H.M. and Stephens, S. (1986). Cytometry 7, 107-110 2. Rahavendran, S.V. and Karnes, H.T. (1993). Pharmaceutical Research 10, 328-334. 3. Mujumdar, R.B. et al. (1993). Amer. Chem. Soc. 4, 106-111.
What are the key applications of a MICROCYTE^®?
Specially developed for microbiological analysis, sensitive, stable, minimal space requirements, portable, robust, price.
Which end users are our main targets?
Anyone with a need to detect, survey, count and/or identify microorganisms rapidly.

Applications

What are the most common applications?
Fermentation; enumeration and viability analysis, water analysis, beer analysis, microbiological research and bio surveillance of air or water.
How are cell counts separated from general particle counts?
Cells are stained with fluorescent dyes to separate them from inorganic particles.
Which stains are suitable to use with a MICROCYTE^® instruments?
SYTO-62^TM (total cell count) and TOPRO-3^TM (dead cell count).
How can you achieve separate counts for a mixture of two bacteria when they are the same size?
By immunofluorescence and rRNA probes (recommended), labeled with Cy-5 or APC.

Technical

How does the instrument maintain its constant flow rate?
Pressure regulated pump system.
Which factors influence the flow rate?
Temperature, viscosity and the liquid level of the sheath fluid tank and the sample tube. Also the microvalve adjustments and any biofilm or clogging in the flow system.
How do you calibrate the instrument?
By the 17-mins test and with our calibration kit.
What is the purpose of the LS and FL control kits?
To check the performance of a MICROCYTE^® every week and after transportation.
What is the purpose and contents of the sheath fluid?
It is the driving force for the sample and also helps keeping the flow system clean, physically and chemically. It is therefore useful to add a disinfectant to the fluid, and keep the pH at ca. 3.5 to inhibit microbial growth.
When is it necessary to adjust the two screws on the flow cell?
When transport damage has distorted the alignment of the flow cell.
Is the power supply suitable for use in all countries?
Yes, it switches automatically to the local voltage.
For how long can you run the instrument on its internal battery?
2.2 Ah, which at 0.7 A gives a theoretical "life span" of 3.1 hrs.
When is it advantageous to apply linear amplification rather than logarithmic?
When two peaks overlap in the "large region" of the display, i.e. over 1 mm, switching to linear mode can help separating these peaks. Useful feature for DNA-analysis in FL mode and analysis of blood cells of similar sizes.
Which special features of the optics allow simultaneous detection of light scatter and fluorescence?
Two detectors, dichroic filters, long pass filter, quarter wave plate.
Why do you use a red diode laser?
It's small, uses little power, has long lifetime (20000 hrs), low noise and a wavelength that eliminates undesired autofluorescence from bacteria.

Software

What kind of software is used with a MICROCYTE^®?
Two software programs are available; a histogram software (MC2000) and a dotplot software (MC2200).
What are the most important features of a MICROCYTE^® 2000 and 2200 softwares?
Windows-based, allows data storage and more sophisticated presentation of results.
How do you get a license number for the software?
Upon purchase of the software, report your serial hard drive number to BioDETECT AS, who will send you the license number as soon as possible.
Which file formats are available for Save?
.mcf (default format MC2000; MICROCYTE^® file), .xls (Excel) and .bmp (Bitmap), .fcs (default format MC2200).
Why is there not an exact correlation between counting cycles and counting time?
One counting cycle is always either 2, 20 or 200 seconds. The rate at which the display is updated depends on how fast the PC is.

Do you have other questions regarding BioDETECT or the MICROCYTE^®? Please, do not hesitate to contact us.