MycoLight™ Live Bacteria Fluorescence Imaging Kit

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.

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

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Citations

View all 6 citations: Citation Explorer

Lacticaseibacillus zeae PW3 combats multiple foodborne pathogens: Antibacterial activity and metabolomics analysis
Authors: Peng, Jia-Ni and He, Hong-Ru and Lu, Yao and Lin, Lian-Bing and Wang, Feng
Journal: LWT (2025): 117666

Antibacterial mechanism of action of plantaricin LP21-2 against Escherichia coli and its potential application in yak milk
Authors: Jiang, Yu-Hang and Zhao, Yu-Ting and Liu, Chang and Tan, Han and Bu, Lin-Li and Suo, Hua-Yi
Journal: LWT (2024): 116266

Antimicrobial effects and metabolomics analysis of cell-free supernatant produced by Pediococcus acidilactici LWX 401 isolated from Yunnan traditional pickles
Authors: Liu, Wei-Xin and Wang, Jia-Jia and Xiao, Xian-Kang and Chen, Cai-Rui and Lu, Xiao and Zhang, Xin-Yi and Lin, Lian-Bing and Wang, Feng
Journal: LWT (2023): 115626

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

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

Page updated on April 15, 2025

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