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From simple to complex, coupled with the innovative technology of the Attune® Acoustic Focusing Cytometer, Life Technologies offers a complete solution for cytometric analysis of microbiology.

Flow cytometry has been widely used in microbiology research, including detection and quantification of viable and nonculturable organisms [1], analysis of host-microbe interactions [2], analysis of microbial cell cycle [3], and detailed spatial and temporal analysis of microbial metabolism in different environments [4].

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Consistent Fluorescent Detection

Consistent fluorescent detection at flow rates from 25 μL/min to 1,000 μL/min

Figure 1. Consistent fluorescent detection at flow rates from 25 μL/min to 1,000 μL/min.
S. aureus cells were stained with SYTO® 9 and analyzed on the Attune® Acoustic Focusing Cytometer using 488 nm excitation and the 530/30 bandpass filter (BL1) to collect SYTO® 9 fluorescence emission. (A) Typical scatter observed using a BL1 fluorescence threshold. S. aureus cells are shown in green and have a greater forward scatter signal than electronic noise/debris. (B) Fluorescence histogram overlay indicating SYTO® 9 fluorescence of the S. aureus population identified in (A), collected at Sensitive 25 μL/min (red), Sensitive 100 μL/min (blue), Standard 25 μL/min (green), Standard 100 μL/min (black), Standard 200 μL/min (purple), Standard 500 μL/min (burgundy), and Standard 1,000 μL/min (orange) collection rates. Unstained cells are shown in grey, collected at Standard 25 μL/min. Little variation is observed across all collection rates.

Sensitive Analysis for Many Routine Microbiology Applications

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.

 

Analysis of relative cell viability within a bacterial culture using flow cytometry 
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.

3. Analysis of relative cell vitality within a bacterial culture using flow cytometry
 
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.

Analysis of relative membrane potential in an S. aureus culture
 
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).

Staining of bacteria using BacLight™ Green

Figure 5. Staining of bacteria using BacLight™ Green. Untreated and alcohol-fixed 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.

Ordering Information

References

  1. Sachidanandham R, Gin KY, Poh CL (2005) Monitoring of active but non-culturable bacterial cells by flow cytometry. Biotechnol Bioeng 89:24–31.
  2. Hara-Kaonga B, Pistole TG (2007) A dual fluorescence flow cytometric analysis of bacterial adherence to mammalian host cells. J Microbiol Methods 69:37– 43.
  3. Marie D, Partensky F, Jacquet S et al. (1997) Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the nucleic acid stain SYBR Green I. Appl Environ Microbiol 63:186–193.
  4. Sachidanandham R, Gin KY (2009) Flow cytometric analysis of prolonged stress-dependent heterogeneity in bacterial cells. FEMS Microbiol Lett 290:143–148.
The Attune® Acoustic Focusing Cytometer is for research use only. It is not intended for animal or human therapeutic or diagnostic use.