Regards Gerhard If you attend the ISAC meeting in Montpellier you might want to attend the tutorial on microbiology. Below a rough guide on how to set up any cytometer for the measurement of bacteria. First of all buy some approx. 500nm reference beads (for example yellow-green from Polysciences calibration grade). Trigger instrument on green fluorescence and adjust the voltages to have the beads in the top right quadrant of a side scatter versus green fluorescence display and optimise calibration (if applicable). There should be a lot of nothing between the beads and the origin of the plot. Then set your instrument to trigger simultaneously side scatter and determine your noise background. Bugs will give you usually less scatter than the beads, so again, your threshold should be quite a bit / more than one decade below the bead cluster. On single channel triggered instruments you just have to go for side scatter only. Most instruments are unable to resolve bugs by their normal forward scatter signal. If you can not separate your bugs from the noise you have either got a dirty flow cell of lots of other bits in the sample or more likely sheath line (contact me for advice on cleaning) . This is how you usually find out about the first law of microbial cytometry: "sterile is not particle free". Best use filtered liquids in disposable labware. We actually put some disposable Millipore filter cartridges into the sheath line next to the flow cell. Extremely useful for sterile sorting! You can than mix beads with bugs to see them simultaneously. Ideally you start with heatfixed bugs stained with PI as you do not have to worry about dye extrusion pumps. However, you can also use green fluorescent bacteria from the Phagotest (Becton Dickinson / Orpegen) or make your own green bugs using carboxy-fluorescein-diacetate-succinimidylester. This combination should also allow you to set your red vs green compensation for this fitc/pi. Supravital stains are tricky as most bugs pump like mad. The highly permeable dyes sold by molecular probes work, but only if added fresh at the suggested concentration. Otherwise try to start with the neat cultures, stationary/starved e.coli (washed 4-5 times in PBS) to minimise the effect of extrusion pumps. E.coli from culture give quite some scatter signal. Listeria is more difficult. To classify the functional state of your bugs see Nebe-von Caron, G., Stephens, P., and Badley, R.A. Assessment Of Bacterial Viability Status By Flow Cytometry And Single Cell Sorting . J.Appl.Microbiol. 84:988-998, 1998. and the revised table added below. FUNCTIONAL - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - INTACT CELLS - - - - - - - - - - - - - - - - - - - - - - - - - - PERMEABILISED CELL - - - - - METABOLICALLY ACTIVE CELLS - - - - - - (DEAD) STATUS REPRODUCTIVE GROWING CELLS CELLS TEST CRITERIA CELL DIVISION METABOLIC ACTIVITY MEM. INTEGRITY MEMBRANE PERMEABILITY DETECTION METHOD CELL COUNTING FIXED VOLUME COUNT TIME INTEGRATION RATIOMETRIC COUNTING CELL TRACKING COVALENT LABELLING (INTRACELLULAR OR MEMBRANE) ENERGY DEPENDENT BIOSYNTHESIS (DNA / PROTEIN SYNTHESIS, CELL ELONGATION UNDER ANTIBIOTIC PRESSURE; Direct Viable Count, DVC) PUMP ACTIVITY (pH CONTROL, DYE EFFLUX) MEMBRANE POTENTIAL (ACCUMULATION OF CATIONIC DYES, DISSIPATION OF ANIONIC DYES) ENERGY INDEPENDENT ENZYME ACTIVITY SUBSTRATE CONVERSION BY ESTERASE or DEHYDROGENASE SELECTIVE MEMBRANE PERMEABILITY EXCLUSION OF MEMBRANE IMPERMEABLE DNA STAINS (PI) BUT UPTAKE OF SUPRAVITAL DNA STAINS (HOECHST 33342, DAPI, SYTO 9/13 ToPro-3, EB) INDISCRIMINATE UPTAKE OF CYTOPLASMIC STAINS - - - - - - - - - - - - - - - - - CELLS WITH NUCLEIC ACID DAMAGE (non-growing)- - - - - - - - - - - - - -- - - - - - - Viability measurements in their functional context: Reproductive growth as the most stringent proof for viability requires metabolic activity and in turn membrane integrity. In a lot of cases this function can not be measured due to irreversible DNA damage, fastidious growth conditions, lack of symbiotic partners or extremely slow growth. Detection of metabolic activity is less stringent but suggests the absence of cell death. Whilst it does not warrant reproductive growth, this function might be sufficient to generate unwanted effects such as food spoilage or accumulation of toxins or transfer of genes. In cases of injury, dormancy or extreme starvation metabolic functions might be below detection limit. Membrane integrity demonstrates the protection of cell constituents and its potential to generate gradients thus it’s capability of metabolic activity / repair and potentially reproductive growth unless the DNA is damaged beyond repair. It also separates bacteria from other organic matter and debris. Cells without an intact membrane can not maintain any electrochemical gradient and can be classified as dead cells. As their structures are freely exposed to the environment they will eventually decompose. -----Original Message----- From: Craig Shadbolt [SMTP:craig_ts@postoffice.utas.edu.au] Sent: Saturday, March 18, 2000 4:15 AM To: Cytometry Mailing List Subject: Basic setup for E. coli with Coulter EPICS Elite Dear Flow-Experts Being the rank flow cytometry novice that I am, I thought it best to ask others for some advice. I am very interested in using flow cytometry for my work involving bacterial heterogeneity during food processing of goods such as femented meats. Unfortunately there is little expertise here in dealing with bacteria in flow cytometers. The machine we have (Coulter EPICS Elite) is used only for clinical studies. I am hoping to change that, but I am currently having trouble breaking my cells out of the background clutter. I believe this may be due to the stain I was employing, propidium iodide (PI). The cell cultures I was using were a salt-stressed and a starved population of <italic>E. coli</italic>. I suspect that most of them were still reasonably healthy, so they would not have taken up the PI. Can anybody suggest a simple protocol using some common stains which would enable me to visualise my cultures in terms of viable/non-viable-injured/dead cells and get me started on this topic? Regards Craig Shadbolt ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ Craig Shadbolt School of Agricultural Science and Tasmanian Institute of Agricultural Research University of Tasmania GPO Box 252-54 Hobart, 7001 Australia Phone Intl. +61 (03) 6226 2735 Fax Intl. +61 (03) 6226 2642 email craig_ts@utas.edu.au
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