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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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ShippingStandard overnight for United States, inquire for international
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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


View all 86 citations: Citation Explorer
Staphylococcus pseudintermedius induces pyroptosis of canine corneal epithelial cells by activating the ROS--NLRP3 signaling pathway
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
Redox regulation by reversible protein S-thiolation in Gram-positive bacteria
Authors: Imber, M., Pietrzyk-Brzezinska, A. J., Antelmann, H.
Journal: Redox Biol (2019): 130-145
Coriander essential oil and linalool - interactions with antibiotics against Gram-positive and Gram-negative bacteria
Authors: Aelenei, P., Rimbu, C. M., Guguianu, E., Dimitriu, G., Aprotosoaie, A. C., Brebu, M., Horhogea, C. E., Miron, A.
Journal: Lett Appl Microbiol (2019): 156-164
Antibacterial activity of synthetic 1,3-bis(aryloxy)propan-2-amines against Gram-positive bacteria
Authors: Serafim, M. S. M., Lavorato, S. N., Kronenberger, T., Sousa, Y. V., Oliveira, G. P., Dos Santos, S. G., Kroon, E. G., Maltarollo, V. G., Alves, R. J., Mota, B. E. F.
Journal: Microbiologyopen (2019): e814
In vitro antimicrobial efficacy of laser exposed chlorpromazine against Gram-positive bacteria in planktonic and biofilm growth state
Authors: Tozar, T., Nastasa, V., Stoicu, A., Chifiriuc, M. C., Popa, M., Kamerzan, C., Pascu, M. L.
Journal: Microb Pathog (2019): se name="22415.enl" path="C:\Website\Referenc
Bacteriocins of Gram-positive bacteria having activity spectra extending beyond closely-related species
Authors: Todorov, S. D., de Melo Franco, B. D. G., Tagg, J. R.
Journal: Benef Microbes (2019): 1-14
Depletion of Gram-Positive Bacteria Impacts Hepatic Biological Functions During the Light Phase
Authors: Oh, H. Y. P., Ellero-Simatos, S., Manickam, R., Tan, N. S., Guillou, H., Wahli, W.
Journal: Int J Mol Sci (2019): se name="22415.enl" path="C:\Website\Referenc
Discovery of small molecule antibiotics against a unique tRNA-mediated regulation of transcription in Gram-positive bacteria
Authors: Frohlich, K. M., Weintraub, S., Bell, J. T., Todd, G. C., Vare, V. Y., Schneider, R., Kloos, Z. A., Tabe, E. S., Cantara, W. A., Stark, C., Onwuanaibe, U., Duffy, B. C., Basanta-Sanchez, M., Kitchen, D. B., McDonough, K., Agris, P. F.
Journal: ChemMedChem (2019): se name="22415.enl" path="C:\Website\Referenc
Design, Synthesis and Biological Evaluation of Novel DNA Gyrase-Inhibiting Spiropyrimidinetriones as Potent Antibiotics for Treatment of Infections Caused by Multidrug-Resistant Gram-Positive Bacteria
Authors: Shi, C., Zhang, Y., Wang, T., Lu, W., Zhang, S., Guo, B., Chen, Q., Luo, C., Zhou, X. L., Yang, Y.
Journal: J Med Chem (2019): se name="22415.enl" path="C:\Website\Referenc
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