Microbiological Analysis With the Attune® Cytometer

The Attune® Acoustic Focusing Cytometer offers many advantages over traditional hydrodynamic focusing cytometers, including precise alignment of particles at increased collection rates (up to 1,000 μL/minute). As shown in Figure 1, consistent fluorescence emission is detected in samples of fluorescently labeled Staphylococcus aureus (S. aureus) analyzed at all collection rates using the Attune® cytometer. In addition, the Attune® cytometer is a valuable tool for cell vitality assessment (Figures 2 and 3), membrane potential measurement (Figure 4), and cell viability assays (Figure 5). To see a protocol for each assay used in this application note, go to lifetechnologies.com and search by catalog number.

		Figure 2. Analysis of relative cell viability within a bacterial culture using flow cytometry. Escherichia coli (E. coli) cells were stained with the LIVE/DEAD® BacLight™ Viability Kit before analysis using the Attune® Acoustic Focusing Cytometer equipped with 488 nm laser for SYTO® 9 and propidium iodide excitation. Samples were run at a collection rate of Standard 25 μL/min, and fluorescence emission was detected using a 530/30 bandpass filter for SYTO® 9 fluorescence and 640 longpass filter for propidium iodide fluorescence. Both live (L) and dead (D) cells fluoresce green (SYTO® 9) but only dead cells fluoresce red.
		Figure 3. Analysis of relative cell vitality within a bacterial culture using flow cytometry. Untreated E. coli cells and cells treated with an electron transport chain uncoupler (sodium azide) were stained with the BacLight™ RedoxSensor™ Green Vitality Kit before analysis using the Attune® Acoustic Focusing Cytometer equipped with 488 nm laser. Samples were run at a collection rate of Standard 25 μL/min, and fluorescence emission was detected using a 530/30 bandpass filter for BacLight™ RedoxSensor™ Green fluorescence. The histogram overlay indicates untreated cells have a brighter green fluorescence and greater redox potential than those treated with sodium azide.
		Figure 4. Analysis of relative membrane potential in an S. aureus culture before and after disruption with a proton ionophore. S. aureus cells were diluted to ~1 x 106 CFU/mL in PBS prior to staining with the BacLight™ Bacterial Membrane Potential Kit and 20 μM SYTOX® Blue. Samples stained with 30 μM 3,3’-diethyloxacarbocyanine iodide (DiOC2) alone, and samples stained with DiOC2 and treated with 5 μM carbonylcyanide 3-chlorophenylhydrazone (CCCP, for disruption of membrane potential), were analyzed on the Attune® Acoustic Focusing Cytometer equipped with 488 nm laser for DiOC2 fluorescence excitation. At increased membrane potential, DiOC2 molecules self-associate in the cytosol and shift DiOC2 fluorescence emission from green (detected in the BL1 channel using a 530/30 bandpass filter) to red (detected in the BL3 channel using a 640 longpass filter). In this example, dead cells have been removed from analysis by excluding SYTOX® Blue–positive cells from analysis. The dot plot overlay indicates increased red-shifted DiOC2 fluorescence in the untreated sample (-CCCP, green) as compared to the CCCP-treated sample (+CCCP, red).

Figure 5. Staining of bacteria using BacLight™ Green. Untreated and alcoholfixed E. coli (A) and S. aureus (B) cells were stained with BacLight™ Green before analysis using the Attune® Acoustic Focusing Cytometer equipped with 488 nm laser. Samples were run at a collection rate of Standard 25 μL/min, and fluorescence emission was detected using a 530/30 bandpass filter for BacLight™ Green fluorescence. The histogram overlays indicate that both untreated (L) and alcohol-fixed (F) gram-negative (E. coli) or gram-positive (S. aureus) cells have increased fluorescence over unstained (U) cells when stained with BacLight™ Green. Fluorescence staining of fixed cells is greater than staining in both unfixed and unstained cells.

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