MycoLight™ Rapid Fluorescence Gram-Positive Bacteria Staining Kit

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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
International	See distributors
Bulk request	Inquire
Custom size	Inquire
Shipping	Standard overnight for United States, inquire for international

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

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

Excitation	650 nm
Emission	669 nm
Recommended plate	Black wall/clear bottom
Instrument specification(s)	Cy5 filter

Components

Example protocol

AT A GLANCE

Protocol Summary

Prepare bacteria samples
Prepare and add IF647-ConA stock solution
Incubate bacteria samples with IF647-ConA for 5-15 minutes at room temperature in the dark
Remove IF647-ConA staining solution and resuspend in Assay Buffer
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

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.
Remove medium by centrifugation at 10,000 x g for 5 minutes and re-suspend the pellet in Assay Buffer (Component B).

Staining Protocol

Add 1 µL of the IF647-ConA stock solution (100X) to 100 µL of the bacterial sample.
Mix well and incubate in dark for 5-15 minutes at room temperature.
Centrifuge at 10,000 x g for 5 minutes and remove the IF647-ConA staining solution.
Resuspend in 100 µL of Assay Buffer (Component B).
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

Figure 1. Bacillus subtilis (Gram-positive) (A) and Escherichia coli (Gram-negative) (B) was stained with MycoLight™ Rapid Fluorescence Gram-Positive Bacteria Staining Kit.

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