AAT Bioquest

MycoLight™ Rapid Fluorescence Bacterial Gram Stain Kit

Mixture of <em>Escherichia coli </em>and <em>Bacillus subtilis </em>were stained with MycoLight&trade; Rapid Fluorescence Bacterial Gram Stain Kit. Red &amp; Orange: Gram positive <em>Bacillus subtilis</em> cells; Green: gram-negative <em>Escherichia coli</em> cells.
Mixture of <em>Escherichia coli </em>and <em>Bacillus subtilis </em>were stained with MycoLight&trade; Rapid Fluorescence Bacterial Gram Stain Kit. Red &amp; Orange: Gram positive <em>Bacillus subtilis</em> cells; Green: gram-negative <em>Escherichia coli</em> cells.
Mixture of <em>Escherichia coli </em>and <em>Bacillus subtilis </em>were stained with MycoLight&trade; Rapid Fluorescence Bacterial Gram Stain Kit. Red &amp; Orange: Gram positive <em>Bacillus subtilis</em> cells; Green: gram-negative <em>Escherichia coli</em> cells.
Various proportions of&nbsp;<em>E.coli</em>&nbsp;and&nbsp;<em>B.sub</em>&nbsp;were prepared. 1 mL of bacteria suspension was stained with 4 &mu;L of dye working solution. The fluorescence emission spectrum (excitation 470 nm, emission 480-700 nm) were measured with Cary Eclipse Fluorescence Spectrophotometer (A).The integrated intensities of the green (490-510 nm) and red (590-610) were acquired and the green/red ratio were calculated for each bacteria suspension. A least-square fit of the relationship between % gram negative bacteria (<em>E.coli</em>) and Log<sub>10</sub>&nbsp;green/red ratio was plotted (B).&nbsp;
Ordering information
Catalog Number
Unit Size
Add to cart
Additional ordering information
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
ShippingStandard overnight for United States, inquire for international
Request quotation
Spectral properties
Excitation (nm)482
Emission (nm)512
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


Excitation (nm)
Emission (nm)
The MycoLight™ Rapid Fluorescence Bacterial Gram Stain Kit provides an easy and convenient way for determination of gram sign in live bacteria. Gram staining is a commonly used method in both clinical and research settings to taxonomically classify bacterial species into two large groups. Unfortunately, the traditional gram staining method is tedious and involves bacterial fixation which can be a significant drawback if the bacteria are to be characterized further. The MycoLight™ Rapid Fluorescence Bacterial Gram Stain Kit provides a one-step gram staining assay for live bacteria that overcomes the problems inherent in the traditional gram staining assays. The MycoLight™ Rapid Fluorescence Bacterial Gram Stain Kit utilizes two DNA dyes MycoLight™ Green and MycoLight™ Red with differential ability to stain gram positive and negative bacteria. MycoLight™ Green stains both gram positive and negative bacteria while MycoLight™ Red preferentially labels gram positive bacteria. The excitation/emission maxima for these two dyes are about 484/504 nm for MycoLight™ Green and 650/669 nm for MycoLight™ Red. Thus, when a mixture of gram positive and gram negative bacteria is stained with the dyes, gram positive bacteria will fluoresce red and gram negative bacteria will fluoresce green. The gram positive and negative staining can be monitored fluorimeterically with Cy5 and FITC filter set respectively.


Fluorescence microscope

Excitation488/650 nm
Emission530/669 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)FITC/Cy5 filters


Example protocol


Protocol Summary
  1. Prepare bactrerial samples
  2. Prepare and add MycoLight™ dye working solution to bacteria samples
  3. Incubate with MycoLight™ dye working solution at room temperature in dark for 15 minutes
  4. Analyze sample by fluorescence microscope or fluorescence spectroscopy with FITC and TRITC filter sets
Important Note

Thaw all the kit components at room temperature before use.


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™ Red stock solution

Add 100 µL of ddH2O into one vial of MycoLight™ Red (Component B) and mix them well.

Note: Store stock solution at -20 °C, avoid light and store in smaller aliquots to avoid repeated freeze-thaw cycles.


MycoLight™ dye working solution

Mix equal volume of MycoLight™ Green (Component A) and MycoLight™ Red stock solution in a tube and mix them well.


Preparation of Bacterial Samples
  1. Prepare bacteria sample with concentration around 107cells/ml. Grow bacteria into late log phase in appropriate medium.

    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 10 minutes and re-suspend the pellet in ddH2O, adjust bacteria concentration to ~ 107 cells/ml.
Staining Protocol
  1. Add 2 µL MycoLight™ dye working solution to 100 µL of the bacterial suspension.
  2. Mix well and incubate in dark for 15 min at room temperature.

  3. Remove the working solution by centrifugation at 10000 g for 10 minutes.

  4. Resuspend the bacteria pallate in ddH2O.
  5. Monitor fluorescence of bacteria with a fluorescent microscope through FITC (Ex/Em = 488/530 nm) channel for gram-negative bacteria and Cy5 (Ex/Em = 650/669 nm) channel for gram-positive bacteria.

    Note: The protocol only provides a guideline, should be optimized with different bacterial strains or other specific needs.

    Note: Relative ratio of gram positive and gram negative bacteria in a population can also be estimated with fluorescence spectroscopy with this kit. A sample analysis is included with the figures.


Open in Advanced Spectrum Viewer

Spectral properties

Excitation (nm)482
Emission (nm)512



View all 45 citations: Citation Explorer
In vitro efficacy of Er: YAG laser-activated irrigation versus passive ultrasonic irrigation and sonic-powered irrigation for treating multispecies biofilms in artificial grooves and dentinal tubules: an SEM and CLSM study
Authors: Bao, Pingping and Liu, He and Yang, Lan and Zhang, Lulu and Yang, Liwei and Xiao, Nannan and Shen, Jing and Deng, Jiayin and Shen, Ya
Journal: BMC Oral Health (2024): 1--14
TiO2-DNA Nanosensor In Situ for Quick Detection of Nasal Flora in Allergic Rhinitis Patients
Authors: Chen, Weihua and Zhang, Kaiyang and Zhong, Zewei
Journal: Computational and Mathematical Methods in Medicine (2022)
Gram Stain and Molecular Method for the Diagnosis of Bacterial Pneumonia
Authors: Guo, X. G., Liu, Q. F.
Journal: Chin Med J (Engl) (2016): 1884
Combination of Gram Stain, Sputum Culture, and Molecular Method for Diagnosis and Guiding Target Therapies of Bacterial Pneumonia
Authors: Wang, F., Gao, Z. C.
Journal: Chin Med J (Engl) (2016): 1885
Diagnosis of common bacterial causes of urethritis in men by Gram stain, culture and multiplex PCR
Authors: Jahan, F., Shamsuzzaman, S. M., Akter, S.
Journal: Malays J Pathol (2014): 175-80
Prognostic risk score for pleocytosis with a negative gram stain: valid but of limited utility in bacterial meningitis patients
Authors: Bijlsma, M. W., Brouwer, M. C., van de Beek, D.
Journal: Mayo Clin Proc (2013): 421
Comparison of clinical and gram stain diagnosis methods of bacterial vaginosis among pregnant women in ethiopia
Authors: Mengistie, Z., Woldeamanuel, Y., Asrat, D., Yigeremu, M.
Journal: J Clin Diagn Res (2013): 2701-3
A magnetic Gram stain for bacterial detection
Authors: Budin, G., Chung, H. J., Lee, H., Weissleder, R.
Journal: Angew Chem Int Ed Engl (2012): 7752-5
Diagnostic accuracy of cerebrospinal fluid gram stain in children with suspected bacterial meningitis
Authors: Brizzi, K., Hines, E. M., McGowan, K. L., Shah, S. S.
Journal: Pediatr Infect Dis J (2012): 195-7
The natural history of bacterial vaginosis diagnosed by gram stain among women in Rakai, Uganda
Authors: Thoma, M. E., Gray, R. H., Kiwanuka, N., Wang, M. C., Sewankambo, N., Wawer, M. J.
Journal: Sex Transm Dis (2011): 1040-5