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MycoLight™ Rapid Fluorescence Gram-Positive Bacteria Staining Kit
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.
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
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.
Note Store stock solution at -20 °C, avoid light and store in smaller aliquots to 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.
Citations
View all 87 citations: Citation Explorer
Fish scale-inspired biomimetic nanocoatings on magnesium implants for vascularized bone regeneration in infected bone defects
Authors: Li, Dan and Dai, Danni and Wang, Jianrong and Ai, Zhen and Zhang, Chao
Journal: Journal of Magnesium and Alloys (2024)
Authors: Li, Dan and Dai, Danni and Wang, Jianrong and Ai, Zhen and Zhang, Chao
Journal: Journal of Magnesium and Alloys (2024)
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
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
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
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
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
Page updated on December 17, 2024
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