01
INTRODUCTION
* Research
The microorganisms are the most successful group of all living species occupying each habitat in water, soil, plants and animals including humans with enormous success. This leads to a fundamental impact on all research areas in modern biology and medicine.
MAIN BENEFITS
Precision _ High sensitivity flow cytometers for low signal intensity applications Versatility _ Any microbial FCM application can be performed on Partec instruments Comfort _ Quick enumeration of total cell count within minutes Safety _ Any cell type can be detected and identified by our flow cytometers Costs _ Optimization of large scale production processes saves time and money
* Industrial Applications
Biotechnologically designed and employed microorganisms for applications in food industry, chemistry and pharmacy significantly increase the importance. Because of their small size sophisticated technology is required for detection and characterization. Partec offers dedicated instruments and reagents for the analysis of microorganisms.
02
OVERVIEW
Partec provides the complete range of flow cytometry technology for characterization of microorganisms Research
Microorganisms have desirable properties which make them analyzed on flow cytometers. Due to an instrument set-up predominant model organisms for genome analysis, detec- being optimized for low signal intensity applications the tion of regulatory and metabolic pathways, cell division and Partec instruments are commonly preferred for viral cell-cycle studies and many others. Easy handling and culti- detection and characterization. vation procedures thereby reaching an unlimited number of cells as well as highly developed cell biological and genetic cells. Flow cytometry as the major methodology for cellular analysis supports all research oriented applications by its high potential of analysing cellular properties. Not only cells but also virus particles are successfully being
Image: Dennis Kunkel Microscopy, Inc. Image: Dennis Kunkel Microscopy, Inc.
Quality control of food products is an issue with growing importance. Partec provides valuable support because virtually any cellular contamination can be detected on our flow cytometers. Protocols for general detection of any contaminant or individual species detection based on DNA specific probes are available.
Industrial Applications
Industrial production steps often include the removal of particles by filtering or chemical treatment. This can either occur during the process or further downstream during waste water treatment. Partec flow cytometers allow the detection and hence quantification of virtually any particle with a size smaller than 200 m and therefore function as valuable tools for process optimization.
03
OVERVIEW
The high potential of flow cytometry for microbial analysis conditions the nearly endless versatility. A range of applications is displayed on the following pages, designated Partec instruments and reagents can be found in section 14. Research
04 _ Research in Biology and Medicine 05 _ Virus detection
Industrial Applications
12 _ Particle counting, Paper industry
04
Research
APPLICATIONS
MAIN BENEFITS
_ Superior sensitivity and resolution _ Large selection of excitation light sources _ Modular design of Partec instruments for highest flexibility
Suitable instruments for this application: _ CyFlow SL _ CyFlow space _ CyFlow ML For details please see section 14.
FL3-dead cells
Lactobacillus spec.
SSC
FL1-stain
FL1-viable cells
Staphylococcus spec.
FL3-mitochondrial activity
FSC
FSC
FL1
Seperation of differently sized organisms during a single measurement in a scatter plot: Staphylococcus spec. Lactobacillus spec. Saccharomyces cerevisiae. Subsequent analysis: Mitochondrial activity measurement of Staphylococcus, DNA stain of Lactobacillus, Viability measurement of Saccharomyces.
05
Research
APPLICATIONS
VIRUS DETECTION
Detection of virus particles is very demanding for both the sensitivity of the instrument and the sample preparation conditions because low sensitivities can be expected for both scatter and fluorescence signals. Therefore, technical specifications of the instrument need to be pushed to a limit. Partec has always been working on the leading edge of FCM technology and incorporated the latest developments of available laser technologies for the modern line of Partec instrumentation. Consequently, Partec was the first developer and manufacturer of flow cytometers offering laser excitation power far beyond the well established standards. In addition, Partec flow cytometers can be optimized for low signal intensity measurements. Virus particles in most cases can only be detected by staining of the viral nucleic acids.
MAIN BENEFITS
_ Highest sensitivity by advanced laser power _ Measurements at the leading edge of technology
Suitable instruments for this application: _ CyFlow SL _ CyFlow space _ CyFlow ML For details please see section 14.
SSC
FSC
SSC
FSC
FSC SSC plot of pure buffer (Fig. 2) and virus particles diluted in the same buffer (Fig. 3). Under the preparation conditions phages tend to form aggregates , thus explaining the elongated tail of virus particles in the gated region.
06
APPLICATIONS
MAIN BENEFITS
_ Cell counting within minutes _ Straight forward and simple staining procedures _ Measurement of functional assays
Suitable instruments for this application: _ CyFlow SL _ CyFlow space _ CyFlow ML For details please see section 14.
Comparison of cell counting technologies: flow cytometry against conventional methods CONVENTIONAL METHODS
Result: Cultivable cell count
FLOW CYTOMETRY
Result: Total cell count Viable cell count Identification of species Physiological parameters
Fig. 4
Cell Growth
Analysis by FCM
Typical flow cytometric result of cell counting: cells can easily be identified based on their scatter signals (Fig. 4) or after DNA staining (Fig. 5).
PARTEC FLOW CYTOMETRY MORE THAN 120 TIMES FASTER THAN CONVENTIONAL METHODS
Conventional methods Flow Cytometry 10 minutes 12 48 hours
SSC
07
APPLICATIONS
MAIN BENEFITS
_ Cell counting within minutes _ Assessing biological hazard potential of chemical substances _ Replacement of animal test procedures
Suitable instruments for this application: _ CyFlow SL _ CyFlow space _ CyFlow ML For details please see section 14.
_1
_2
_3
1_ 2_ 3_
Replicates >
Growth of monitor organisms in multi-well plates at time point 72 hours (Fig. 6) without testing component (upper row) or after addition of the testing component at various concentrations (bottom rows). Samples can directly be analyzed from the incubation plates by using the Multi-well plate autoloader RobbyWell. Cell count versus time yields the LC50 for each compound (Fig. 7).
08
APPLICATIONS
MAIN BENEFITS
_ Sequence specific detection of contaminants _ Easy quantification of contaminants _ Quality control of food products
Suitable instruments for this application: _ CyFlow SL _ CyFlow space _ CyFlow ML For details please see section 14.
Count
Count
SSC
FL1
FL1
FSC
SSC
FSC
Detection of Lactobacillus acidophilus cells with a labelled RNA specific probe (detectable in the green fluorescence channel). Histogram of green fluorescence of a control sample (Fig. 8) and a hybridized sample (Fig. 9). Cells labelled with the fluorescent probe show a strong signal in FL1.
Two FSC-SSC plots of the hybridized samples. Labelled cells in RN 1 of Fig. 9 are backgated to Fig. 11 (highlighted in green).
09
APPLICATIONS
MAIN BENEFITS
_ Monitoring of fermentation _ Rapid measurement of cell viability _ Physiological growth parameters
Suitable instruments for this application: _ CyFlow SL _ CyFlow space _ CyFlow ML For details please see section 14.
Fig. 14
Trehalose content of yeast cells
FL3-dead cells
Cells / l
Saccharomyces cerevisiae stained with the Partec Yeast Control Viability kit. The ratio of living and dead cells can be obtained 10 minutes after taking the sample from the fermenter (Fig. 12).
Fluorescence measurement of yeast Trehalose content. Staining with Partec Yeast Control Trehalose at time points 0 (left) and 12 hours (right) after inoculation (Fig. 13).
Trehalose content of growing yeast cells at various stages after inoculation (Fig. 14).
10
APPLICATIONS
MAIN BENEFITS
_ Monitoring of fermentation _ Rapid measurement of cell viability _ Physiological growth parameters
The major area of application is the microbial quality control in food, food additives and beverage production, in the pharmaceutical industry and in production of drinking water and in waste water treatment. Process control in bioreactors cultivating bacteria, yeast or higher cells require constant control of fermentation conditions. Lactobacillus spec. is the most commonly employed bacteria in food
industry. Fermentation success directly influences product quality because slightest variations in taste, colour, smell or stability of the final product are recognized by the consumer. Year-long constancy of the product can only be reached by experience and sophisticated detection methods. Partec instruments contribute significantly by providing essential information of cell count, viability and other physiological growth parameters. Suitable instruments for this application: _ CyFlow SL _ CyFlow space _ CyFlow ML For details please see section 14.
Quick determination of total cell number of Lactobacillus directly taken from the fermenter
Fig. 15 Fig. 16
FSC
FL1-viable cells
SSC
FSC
Identification and counting of Lactobacillus can easily be done in the FSC SSC plot (Fig. 15).
A simple DNA stain allows discrimination of living and dead cells (Fig. 16, living cells in Q2, dead cells in Q4).
11
APPLICATIONS
MAIN BENEFITS
_ Quality control of expensive wine products _ Fast detection and quantification of contaminants _ New developed, economic detection method
Suitable instruments for this application: _ CyFlow SL _ CyFlow space _ CyFlow ML For details please see section 14.
Count
Viable Brettanomyces
Cell siz
Viable Brettanomyces
12
APPLICATIONS
Industrial Applications
MAIN BENEFITS
_ Particle counting over a wide size range within minutes _ Optimization of large scale industrial processes _ Enormous time and cost saving
Suitable instruments for this application: _ CyFlow SL _ CyFlow space _ CyFlow ML For details please see section 14.
Particle count
FSC
Particle count
FSC
Fig. 19
Total particles
Particle size
Fig.19: The red and green curves show the overlay of wood pulp samples measurements before and after treatment with a crosslinker, resp. The results demonstrate the substantial removal of unwanted particles.
13
OVERVIEW
Instruments | Technology
Unique features of Partec flow cytometers which determine our instruments for low intensity applications _ Strongest available laser output power for maximum excitation _ Large selection of excitation light sources 375nm, 405nm, 488nm, 532nm, 561nm, 638nm and others _ Highest sensitivity Scatter 0,1 m Fluorescence < 100 MESF (FITC), < 50 MESF (PE) _ Different beam stop specifications and forward scatter angles available _ Portability (CyFlow SL) _ High stability, robustness and precision
14
PRODUCTS
Instruments | Reagents
Partec instruments
Article No. CY-S-1035 Item Partec CyFlow SL 1 Laser, 3 fluorescence colours
CY-S-3001
CY-S-2001
Instrument accessories
Article No. 12-01-1000 Item Partec Particle and Cell Sorter PPCS For CyFlow space
16-02-1000
Multi Well Plate Autolaoder Robby Well For CyFlow SL, CyFlow space and CyFlow ML
Reagents
Article No. 05-6000-01 Item YeastControl - Cell Cycle Reagent kit for biotechnological fermentation control Packaging Unit 50 Tests
05-6000-02
100 Tests
05-6000-03
50 Tests
05-6000-04
50 Tests
05-6000-05
50 Tests
on request
on request
on request
Cell staining reagents Reagents for labeling of cells and cell counting
on request
05-6001
50 Tests
15
ANALYZE
The Windows FloMax software integrates instrument control including acquisition, on- and offline data analysis, onand offline compensation into a complete software package.
Predefined and freely adaptable instrument settings and panels facilitate switching between different applications. FloMax is optimized for immunophenotyping, microbiology analysis, cell cycle, DNA ploidy, and other scientific flow cytometric analysis. Data are stored in FCS flow cytometry standard file format for easy exchange with other analysis software. One of the unique features is
the digital on- and offline color crosstalk compensation of the spectral overlap of fluorescence from simultaneously analysed dyes. The N-color compensation algorithm allows a correction of the crosstalk between any parameter without the need to rerun a sample. FloMax optimally supports the True Volumetric Absolute Counting feature of the Partec FCM instruments, displaying
particle concentrations for any subset of cells, even if defined by a gate at a later time after the acquisition.
The True Volumetric Absolute Counting (TVAC) is a unique feature of all Partec Flow Cytometers, offering highest absolute counting precision and accuracy.
The CyFlow instruments analyse concentrations of any particle or cell subpopulation of interest using True Volumetric Absolute Counting. This unique method is solely based on the fundamental definition of absolute counting respectively the particle concentration (c) equals the counted number (N) of particles (e.g. cells) in a given volume (V), c = N / V. In the CyFlow instruments, the volume is measured directly by mechanical means (rather than by calibration with expensive beads with asometimes doubtful given nominal concentration). Thus, the precision of volume measurement is defined by a fixed mechanical design, eliminating any errors related to varying bead concentrations or bead aggregation. The CyFlow instruments allow the analysis of a fixed volume as defined by the distance between two platinum electrodes reaching into the sample tube with a given diameter. Alternatively, a well defined volume of free choice involving the digital sample speed control can be used. Benefits of True Volumetric Absolute Counting:
_ digital volumetric precision by mechanical design: CV< 2 % _ no errors related to calibration _ no additional time and preparation steps for reference beads or haematology reference count _ no expenses for calibration beads _ no separate cell counter required
16
COMPANY
New sophisticated applications and increasing requirements for reliable results in research and routine within shortest possible time - The challenge for flow cytometry instrumentation, automation and software.
A well-established network of subsidiaries and distributors in more than 60 countries worldwide characterizes Partecs commitment to the increasing focus and need for global access to Flow Cytometry instrumentation and application support: www.partec.de/partec/distributors.html
17
LITERATURE
Karl-Josef Hutter, Michaela Miedl, Britta Kuhmann, Frank Nitzsche, James H. Bryce and Graham G. Stewart. Detection of Proteinases in Saccharomyces cerevisiae by Flow Cytometry. J. Inst. Brew. 111(1), 2632, 2005 Frederik A. Hammes and Thomas Egli. New Method for Assimilable Organic Carbon Determination Using Flow-Cytometric Enumeration and a Natural Microbiological Consortium as Inoculum. Environ Sci. Technol. 2005, 39, 3289 3294
J. P. Day, D. B. Kell and G. W. Griffith. Differentiation of Phytophtora infestans sporangia from other airborne biological particles by flow cytometry. Applied and Environmental Microbiology 2002, 68:1, 37-45
Paul H. Chlup, Dominic Bernard and Graham G. Stewart. The Disc Stack Centrifuge and its Impact on Yeast and Beer Quality. J. Am. Soc. Brew. Chem. 2007, 65(1): 29-37 L Chau T, A. Guilln, E. Roca, M.J. Nez and
G. Nebe-Von Caron, P. Stephens and A. R. Badley. Bacterial detection and differentiation by cytometry and fluorescent probes. Proceedings RMS 1999, 34/1, 321-327
J.M. Lema. Population dynamics of a continuous fermentation of recombinant Saccharomyces cerevisiae using flow cytometry. Biotechnol Prog. 2001, 17(5):951-7. J.C. Bouchez, M. Cornu, M. Danzart, J.Y. Leveau,
Jan Kolberg, Audun Aase, Simone Bergmann, Paul H. Bessette and Patrick S. Daugherty. Flow Cytometric Screening of cDNA Expression Libraries for Fluorescent Proteins. Biotechnol Prog. 2004 May-Jun;20(3):963-7 Claus Holm and Lene Jespersen. A Flow-Cytometric Gram-Staining Technique for Milk-Associated Bacteria. Appl. Envir. Microbiol., May 2003; 69: 2857 - 2863 E. Marza, N. Camougrand and S. Manon. Bax expression protects yeast plasma membrane against ethanol-induced permeabilization. FEBS Letters 2002, 521:1-3, 47-52 Paul H. Chlup, James Conery and Graham G. Stewart. Detection of Mannan from Saccharomyces cerevisiae by flow cytometry. J. Am. Soc. Brew. Chem. 2007, 65(3): 151-156 Michael Berney, Hans-Ulrich Weilenmann and Thomas Egli. Flow-cytometric study of vital cellular functions in Escheria coli during solar disinfection (SODIS). Microbiology (2006), 152, 1719-1729 Tove K. Herstad, Gunnhild Rodal, Ronald Frank, Manfred Rohde and Sven Hammerschmidt. Streptococcus pneumoniae enolase is important for plasminogen binding despite low abundance of enolase protein on the bacterial cell surface. Microbiology 152 (2006), 1307 1317
F. Duchiron and, M. Bouix. Physiological significance of the cytometric distribution of fluorescent yeasts after viability staining. Biotechnology and Bioengineering. 2004 Volume 86, Issue 5 , Pages 520 530 H. Hohenblum, N. Borth and D. Mattanovich. Assessing viability and cell-associated product of recombinant protein producing Pichia pastoris with flow cytometry. J Biotechnol. 2003, 102(3):281-90.
www.partec.com
OEM
Partec is your reliable Original Equipment Manufacturer (OEM) partner for a wide range of applications in the fields of healthcare, microbiology, agrosciences, and industrial applications, offering all benefits and advantages due to Partec's highest-depth in-house hardware and software development.
CONTACT
Headquarters North America Partec North America, Inc. 603 Heron Dr. Unit 9 Swedesboro, NJ 08085 USA Fon 856 467 0018 Fax 856 467 0188 partecna@partec.com Japan Partec Japan, Inc 3628-46 Kandatsu Tsuchiura City 300-0013 Japan Fon +81 (0) 29 834 7788 Fax +81 (0) 29 834 7772 partecjapan@partec.com
Partec GmbH Otto-Hahn-Strae 32 D-48161 Mnster Germany Fon +49 (0) 2534 8008-0 Fax +49 (0) 2534 8008-90 info@partec.com
France Partec S.A.R.L. 14/16 rue Gallieni 91700 Sainte Genevive des Bois France Fon +33 (0) 1 69 04 87 12 Fax +33 (0) 1 69 04 90 38 partecfrance@partec.com
United Kingdom Partec UK Ltd Suite BG10, Canterbury Enterprise Hub University of Kent Giles Lane Canterbury, Kent CT2 7NJ UK Fon +44 (0) 1227 823744 Fax +44 (0) 1227 824038 partecuk@partec.com
Italia Partec Italia S.r.l. Via G. Mascherpa 14 20048 Carate Brianza (MB) Italia
Fon +39 0362 909 143 Fax +39 0362 909 157 partecitalia@partec.com
Microbiology
Industrial Applications
Healthcare
Agrosciences
12.08
www.cyclos-design.de