MycoLight™ Live Bacteria Fluorescence Imaging Kit

A mixed population of Live/Dead <em>Bacillus subtilis </em>were stained with Cat# 22409 (MycoLight™ 520) and propidium iodide (Cat# 17517).

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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
International	See distributors
Shipping	Standard 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-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12352200

Overview SDS Protocol

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

Excitation	488 nm
Emission	530 nm
Recommended plate	Black wall/clear bottom
Instrument specification(s)	FITC filter

Components

Example protocol

AT A GLANCE

Protocol Summary

Prepare 100X dye stock solution
Prepare bacteria samples.
Add MycoLight™ 520 and Signal Enhancer.
Incubate bacteria samples with MycoLight™ 520 and Signal Enhancer at 37°C for 5-10 minutes or room temperature for 60 minutes in dark.
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

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

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

Images

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.

Figure 2. A mixed population of Live/Dead Bacillus subtilis were stained with Cat# 22409 (MycoLight™ 520) and propidium iodide (Cat# 17517).

Citations

View all 3 citations: Citation Explorer

A novel bacteriocin against multiple foodborne pathogens from Lacticaseibacillus rhamnosus isolated from juice ferments: ATF perfusion-based preparation of viable cells, characterization, antibacterial and antibiofilm activity
Authors: Chen, Shi-Yu and Yang, Rui-Si and Ci, Bai-Quan and Xin, Wei-Gang and Zhang, Qi-Lin and Lin, Lian-Bing and Wang, Feng
Journal: Current Research in Food Science (2023): 100484

Antibacterial activity and action target of phenyllactic acid against Staphylococcus aureus and its application in skim milk and cheese
Authors: Jiang, Yu-Hang and Ying, Jian-Ping and Xin, Wei-Gang and Yang, Lin-Yu and Li, Xiu-Zhang and Zhang, Qi-Lin
Journal: Journal of Dairy Science (2022): 9463--9475

A novel bacteriocin against Staphylococcus aureus from Lactobacillus paracasei isolated from Yunnan traditional fermented yogurt: Purification, antibacterial characterization, and antibiofilm activity
Authors: Jiang, Yu-Hang and Xin, Wei-Gang and Yang, Lin-Yu and Ying, Jian-Ping and Zhao, Zi-Shun and Lin, Lian-Bing and Li, Xiu-Zhang and Zhang, Qi-Lin
Journal: Journal of Dairy Science (2022): 2094--2107

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