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MycoLight™ Live Bacteria Fluorescence Imaging Kit

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.
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.
A mixed population of Live/Dead <em>Bacillus subtilis </em>were stained with Cat# 22409 (MycoLight™ 520) and propidium iodide (Cat# 17517).
Ordering information
Price ()
Catalog Number22409
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Spectral properties
Correction Factor (260 nm)0.31
Correction Factor (280 nm)0.12
Extinction coefficient (cm -1 M -1)76000
Excitation (nm)498
Emission (nm)526
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Correction Factor (260 nm)
0.31
Correction Factor (280 nm)
0.12
Extinction coefficient (cm -1 M -1)
76000
Excitation (nm)
498
Emission (nm)
526
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.

Platform


Fluorescence microscope

Excitation488 nm
Emission530 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)FITC filter

Components


Component A: MycoLight™ 5201 vial
Component B: 10X Signal Enhancer1 vial (1 mL)
Component C: Assay Buffer1 bottle (10 mL)
Component D: DMSO1 vial (100 μL)

Example protocol


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.

PREPARATION OF STOCK SOLUTIONS

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.

MycoLight™ 520 stock solution (100X)
Add 100 µL 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 C).
    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 C) 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. 

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (260 nm)0.31
Correction Factor (280 nm)0.12
Extinction coefficient (cm -1 M -1)76000
Excitation (nm)498
Emission (nm)526

References


View all 16 references: Citation Explorer
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
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
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
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.; Fern and ez, 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
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
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