MycoLight™ Live Bacteria Fluorescence Imaging Kit

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Fluorescence images of <em>E.coli</em> stained with CFDA or MycoLight™ Live Bacteria Fluorescence Imaging Kit. CFDA requires washing steps before imaging to minimize background, while no washing is needed using this kit (Cat#22409). The staining efficiency of MycoLight™ 520 is much higher than CFDA as more bacteria show green fluorescence. The signal of MycoLight™ 520 remains in cells after 1 hour of staining while CFDA leaks out readily. Same amount of bacteria were presented in each sample and fluorescence images were taken under the same exposure time.
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Unit Size: Cat No: Price (USD): Qty:
100 Tests 22409 $295


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Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
International: See distributors





Overview

Ex/Em (nm)496/524
Storage F/D/L
InstrumentsFluorescence microscope
Category Cell Biology
Labeling Cells
Related Fluorescence Imaging
The MycoLight™ Live Bacteria Fluorescence Imaging Kit provides an easy and convenient way for visualizing live bacteria through fluorescent microscope. MycoLight™ 520 is non-fluorescent esterase substrate that diffuse into both Gram positive and Gram-negative bacteria. Upon hydrolysis by bacterial intracellular non-specific esterase, a green fluorescent product is produced and accumulated within bacteria. Compare to the commonly used esterase substrate CFDA and CFDA-AM, the kit provides brighter and more stable signal with lower background and easier staining protocol.




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Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Protocol summary

  1. Prepare 100X dye stock solution.
  2. Prepare bacteria samples.
  3. Add MycoLight™ 520 and Signal Enhancer.
  4. Incubate bacteria samples with MycoLight™ 520 and Signal Enhancer at 37°C for 5-10 minutes or room temperature for 60 minutes in dark.
  5. Analyze sample by fluorescence microscope with FITC filter sets.

Important
Thaw one of each kit component at room temperature before starting the experiment.

Key parameters
Instrument:Fluorescence microscope
Excitation:488 nm
Emission:530 nm
Recommended plate:Black wall/clear bottom
Instrument specification(s):FITC filter
Preparation of stock solution
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. MycoLight™ 520 stock solution (100X):
Add 100 mL of DMSO (Component D) into the vial of MycoLight™ 520 (Component A) to make 100X stock solution.

Sample experimental protocol
  1. Prepare bacteria sample with concentration in range of 106 to 108 cells/ml. Grow bacteria into late log phase in appropriate medium. Remove medium by centrifugation at 10,000 x g for 10 minutes and re-suspend the pellet in Assay Buffer (Component B). Note: Measure the optical density of the bacterial culture at wavelength = 600 nm (OD600) to determine the cell number. For E. coli culture, OD600 = 1.0 equals 8 x 108 cells/ml.                                                                                                                                                                                                  
  2. Treat cells with test compounds as desired. Remove treatments by centrifugation at 10,000 x g for 10 minutes and re-suspend the pellet in appropriate amount of Assay buffer (Component B) so the concentration of bacteria in the treated sample is the same as the live.  Note:  Determine the concentration of the bacterial culture before starting the treatment. Note: Dead bacteria can serve as negative control, it is recommended to kill bacteria with 70% ethanol for 30 min followed by 60 min of boiling.  

  3. Add 1 µL of the 100X MycoLight™ 520 stock solution and 10 µL of 10X Signal Enhancer (Component B) to 90 µL of the bacterial sample in Assay Buffer.

  4. Mix well and incubate in dark for 5-10 min at 37°C or 60 min at RT for optimum staining results.

  5. Monitor fluorescence of bacteria with a fluorescent microscope through FITC (Ex/Em = 488/530 nm) channel. Note: Same protocol can also be used for microplate reader assays.
Example data analysis and figures

Figure 1. Fluorescence images of E.coli stained with CFDA or MycoLight™ Live Bacteria Fluorescence Imaging Kit. CFDA requires washing steps before imaging to minimize background, while no washing is needed using this kit Cat#22409. The staining efficiency of Mycolight™ 520 is much higher than CFDA as more bacteria show green fluorescence. The signal of MycoLight™ 520 remains in cells after 1 hour of staining while CFDA leaks out readily. Same amount of bacteria were presented in each sample and fluorescence images were taken under the same exposure time.
Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





References & Citations

Raman spectroscopic analysis of Lactobacillus rhamnosus GG in response to dehydration reveals DNA conformation changes
Authors: Myintzu Hlaing, M.; Wood, B.; McNaughton, D.; Ying, D.; Augustin, M. A.
Journal: J Biophotonics (2017): 589-597

Antibacterial and antigelatinolytic effects of Satureja hortensis L. essential oil on epithelial cells exposed to Fusobacterium nucleatum
Authors: Zeidan-Chulia, F.; Keskin, M.; Kononen, E.; Uitto, V. J.; Soderling, E.; Moreira, J. C.; Gursoy, U. K.
Journal: J Med Food (2015): 503-6

Inactivation of Cronobacter sakazakii in reconstituted infant formula by combination of thymoquinone and mild heat
Authors: Shi, C.; Jia, Z.; Chen, Y.; Yang, M.; Liu, X.; Sun, Y.; Zheng, Z.; Zhang, X.; Song, K.; Cui, L.; Baloch, A. B.; Xia, X.
Journal: J Appl Microbiol (2015): 1700-6

Fourier transform infra-red spectroscopy and flow cytometric assessment of the antibacterial mechanism of action of aqueous extract of garlic (Allium sativum) against selected probiotic Bifidobacterium strains
Authors: Booyens, J.; Thantsha, M. S.
Journal: BMC Complement Altern Med (2014): 289

Deposition and survival of Escherichia coli O157:H7 on clay minerals in a parallel plate flow system
Authors: Cai, P.; Huang, Q.; Walker, S. L.
Journal: Environ Sci Technol (2013): 1896-903

Observation of injured E. coli population resulting from the application of high-pressure throttling treatments
Authors: De Lamo-Castellvi, S.; Toledo, R.; Frank, J. F.
Journal: J Food Sci (2013): M582-6

Effect of air drying on bacterial viability: A multiparameter viability assessment
Authors: Nocker, A.; Fernandez, P. S.; Montijn, R.; Schuren, F.
Journal: J Microbiol Methods (2012): 86-95

patients and environment
Authors: Lindback, T.; Rottenberg, M. E.; Roche, S. M.; Rorvik, L. M., The ability to enter into an avirulent viable but non-culturable (VBNC) form is widespread among Listeria monocytogenes isolates from salmon
Journal: Vet Res (2010): 8

Behaviors of physiologically active bacteria in water environment and chlorine disinfection
Authors: Sawaya, K.; Kaneko, N.; Fukushi, K.; Yaguchi, J.
Journal: Water Sci Technol (2008): 1343-8

Long-term survival of Legionella pneumophila in the viable but nonculturable state after monochloramine treatment
Authors: Alleron, L.; Merlet, N.; Lacombe, C.; Frere, J.
Journal: Curr Microbiol (2008): 497-502


View More Citations




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