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AAT Bioquest

MycoLight™ Rapid Fluorescence Gram-Positive Bacteria Staining Kit

<em>Bacillus subtilis (Gram-positive) </em>(A) and <em>Escherichia coli</em> <em>(Gram-negative)</em> (B) was stained with MycoLight&trade; Rapid Fluorescence Gram-Positive Bacteria Staining Kit.
<em>Bacillus subtilis (Gram-positive) </em>(A) and <em>Escherichia coli</em> <em>(Gram-negative)</em> (B) was stained with MycoLight&trade; Rapid Fluorescence Gram-Positive Bacteria Staining Kit.
<em>Bacillus subtilis (Gram-positive) </em>(A) and <em>Escherichia coli</em> <em>(Gram-negative)</em> (B) was stained with MycoLight&trade; Rapid Fluorescence Gram-Positive Bacteria Staining Kit.
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Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
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Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


AAT Bioquest's MycoLight™ Rapid Fluorescence Gram-Positive Bacteria Staining Kit provides a novel one-step fluorescence assay for the determination of gram sign in living bacteria. The gram stain is an important and widely used method for the taxonomic classification of bacteria in clinical and research settings. The original method involves quite a few steps like heat fixation, two-steps staining protocol, alcohol extraction and counterstaining. These steps can create inconsistent staining. AAT Bioquest's one-step kit overcomes the existing problems by eliminating the labor-intensive steps. The kit uses a fluorescently labeled Concanavalin A (ConA), which is a lectin that selectively binds to N-acetyl glucosamine exposed on the surface of gram-positive bacteria. When gram-negative and gram-positive bacteria are stained with the fluorescently labeled ConA conjugate, only gram-positive bacteria fluoresce red. Stained bacteria can be monitored fluorimeterically. Our kit is robust and convenient since the fluorescently labeled ConA conjugate used in our kit demonstrates higher brightness and photo stability over other existing dyes.

Platform


Fluorescence microscope

Excitation650 nm
Emission669 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)Cy5 filter

Components


Example protocol


AT A GLANCE

Protocol Summary
  1. Prepare bacteria samples
  2. Prepare and add IF647-ConA stock solution
  3. Incubate bacteria samples with IF647-ConA for 5-15 minutes at room temperature in the dark
  4. Remove IF647-ConA staining solution and resuspend in Assay Buffer
  5. Analyze sample by fluorescence microscope with Cy5 filter set 
Important      Thaw all the kit components at room temperature before use.

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.

IF647-ConA stock solution (100X)
Add 50 µL of Assay Buffer (Component B) into one vial of IF647-ConA (Component A) and mix them well.
Note     Store stock solution at -20 °C, avoid light and store in smaller aliquots to avoid repeated freeze-thaw cycles.

SAMPLE EXPERIMENTAL PROTOCOL

Preparation of Bacterial Samples
  1. Grow bacteria into late log phase in appropriate medium. Prepare bacteria sample with concentration in range of 106 to 108 cells/mL.
    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. Remove medium by centrifugation at 10,000 x g for 5 minutes and re-suspend the pellet in Assay Buffer (Component B). 

Staining Protocol
  1. Add 1 µL of the IF647-ConA stock solution (100X) to 100 µL of the bacterial sample.
  2. Mix well and incubate in dark for 5-15 minutes at room temperature.
  3. Centrifuge at 10,000 x g for 5 minutes and remove the IF647-ConA staining solution.
  4. Resuspend in 100 µL of Assay Buffer (Component B).
  5. Monitor fluorescence of bacteria with a fluorescent microscope through Cy5 (Ex/Em = 650/669 nm) channel.
    Note     The protocol only provides a guideline, should be optimized with different bacterial strain or other specific needs. An optional washing step with Assay buffer (Component B) can be added before imaging if higher background is observed.
     

Images


Citations


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Authors: Wang, Zhihao and Guo, Long and Yuan, Changning and Zhu, Chengcheng and Li, Jun and Zhong, Haoran and Mao, Peng and Li, Jianji and Cui, Luying and Dong, Junsheng and others,
Journal: Virulence (2024): 2333271
Highly-branched poly(N-isopropyl acrylamide) functionalised with pendant Nile red and chain end vancomycin for the detection of Gram-positive bacteria
Authors: Swift, T., Katsikogianni, M., Hoskins, R., Teratarantorn, P., Douglas, I., MacNeil, S., Rimmer, S.
Journal: Acta Biomater (2019): se name="22415.enl" path="C:\Website\Referenc
Design and synthesis of biaryloxazolidinone derivatives containing a rhodanine or thiohydantoin moiety as novel antibacterial agents against Gram-positive bacteria
Authors: Wu, Y., Ding, X., Xu, S., Yang, Y., Zhang, X., Wang, C., Lei, H., Zhao, Y.
Journal: Bioorg Med Chem Lett (2019): 496-502
Rapid and Selective Discrimination of Gram-Positive and Gram-Negative Bacteria by Boronic Acid-Modified Poly(amidoamine) Dendrimer
Authors: Tsuchido, Y., Horiuchi, R., Hashimoto, T., Ishihara, K., Kanzawa, N., Hayashita, T.
Journal: Anal Chem (2019): se name="22415.enl" path="C:\Website\Referenc
Butenolide, a Marine-Derived Broad-Spectrum Antibiofilm Agent Against Both Gram-Positive and Gram-Negative Pathogenic Bacteria
Authors: Yin, Q., Liang, J., Zhang, W., Zhang, L., Hu, Z. L., Zhang, Y., Xu, Y.
Journal: Mar Biotechnol (NY) (2019): 88-98
Role of gram-positive bacteria in chronic pelvic pain syndrome (CPPS)
Authors: Murphy, S. F., Anker, J. F., Mazur, D. J., Hall, C., Schaeffer, A. J., Thumbikat, P.
Journal: Prostate (2019): 160-167
Synthesis of ZnO nanoparticles with chitosan as stabilizing agent and their antibacterial properties against Gram-positive and Gram-negative bacteria
Authors: Yusof, N. A. A., Zain, N. M., Pauzi, N.
Journal: Int J Biol Macromol (2019): 1132-1136
In Vitro Activity of Tebipenem (SPR859) Against Penicillin-Binding Proteins of Gram-negative and Gram-positive Bacteria
Authors: Lacasse, E., Brouillette, E., Larose, A., Parr, T. R., Jr., Rubio, A., Malouin, F.
Journal: Antimicrob Agents Chemother (2019): se name="22415.enl" path="C:\Website\Referenc
Antibacterial Potential of Aloe weloensis (Aloeacea) Leaf Latex against Gram-Positive and Gram-Negative Bacteria Strains
Authors: Emiru, Y. K., Siraj, E. A., Teklehaimanot, T. T., Amare, G. G.
Journal: Int J Microbiol (2019): 5328238
Reliability of the Verigene system for the identification for Gram-positive Bacteria and detection of antimicrobial resistance markers from children with bacteremia
Authors: Beckman, M., Washam, M. C., DeBurger, B., Haslam, D. B., Courter, J. D., Andersen, H., Schaffzin, J. K., Tchou, M. J., Ankrum, A., Mortensen, J.
Journal: Diagn Microbiol Infect Dis (2019): 191-195