AAT Bioquest

Gram Staining

MycoLight™ Rapid Fluorescence Bacterial Gram Stain Kit
Mycolight Rapid Fluorescence Bacterial Gram Stain Kit
Gram staining, named for its originator in 1884, remains the central method in microbiology for bacterial taxonomy by an initial separation into differently-colored Gram-positive or Gram-negative categories. The different colors are due to a polymer called peptidoglycan that is an important part of the bacterial cell wall. Peptidoglycan forms an alternating, tightly-bound crystal lattice that protects the bacteria from osmotic pressures and improves structural strength.

Gram-positive bacteria have a majority (50-90% of dry weight) of peptidoglycan present, and Gram-negative bacteria have a much smaller proportion (as little as 10%). This affects the amount of violet dye that is retained during the process. Since the Gram-negative bacteria have so little dye to start with, it is quickly removed during the procedure, and the final safranin counter-stain adds the pink color to aid visibility.

The possible applications of Gram staining have expanded beyond the initial categorization of unknown bacteria. The variable structure of the bacterial peptidoglycan layer has become a potential target for antimicrobial research, particularly in the past few years as new treatment-resistant bacterial strains are becoming an accelerating problem. The peptidoglycan lattice has gained attention in recent years due to its minimally-understood role in bacterial attachment and signaling. Gram staining has also begun to be included in various diagnostic methodologies with even greater frequency as part of customized medical therapies.



Traditional Fixed Cell Gram Stain vs Live Cell Gram Stain

The traditional five-step process results in two differently-colored fixed bacterial cells, depending on their cell wall composition. Gram-positive will appear dark purple (from the initial crystal violet stain) and gram-negative will be a pink to red color from the final counter-stain of safranin. Aside from a wider catalog of possible dyes available, the most definitive difference between traditional and modernized Gram staining is that the traditional methodology can only be done on fixed cells, eliminating the possibility of further characterization. Updated Gram staining techniques can often be done on live cells, allowing researchers more autonomy with experimental design and goals.

Cell Media Formulations

To assist in keeping optimal bacterial colony health, consult AAT Bioquests' interactive Cell Media Formulations Tool


Gram Staining Kits

MycoLight™ Live Bacteria Fluorescence Imaging Kit
Fluorescence images of E.coli stained with CFDA or MycoLight™ Live Bacteria Fluorescence Imaging Kit. CFDA requires washing steps before imaging to minimize background, while no washing is needed using this kit (Cat#22409). The staining efficiency of MycoLight™ 520 is much higher than CFDA as more bacteria show green fluorescence. The signal of MycoLight™ 520 remains in cells after 1 hour of staining while CFDA leaks out readily. Same amount of bacteria were presented in each sample and fluorescence images were taken under the same exposure time.
Traditional Gram staining is an inexpensive but labor-intensive 5-step staining process. Modern techniques and all-inclusive kits are more efficient and may be suited to time-sensitive experiments or procedures requiring minimization of possible human error. The MycoLight™ Kit series covers a wide range of frequent experimental needs, optimized for fluorescence microscope, microplate assays, Flow Cytometry or all of the above instrumental platforms. The series displays excellent background/signal ratio and brightness as well as minimal cytoxicity, allowing for normal bacterial growth and proliferation of target colonies.



Gram Staining Dyes

MycoLight™ Red JJ94
Rhodococcus qingshengii was stained with 2.5 µM of MycoLight™ Red JJ94 for 20 minutes. Image was taken by Keyence florescent microscope with Cy5 filter set.
With the development of improved dyes, a wide spectrum is available, allowing for Gram-staining to be included as part of multiparameter analysis of bacterial cells. Inexpensive selections like Hexidium Iodide and others are popular choices. For experiments requiring specificity as well as sensitivity, the MycoLight™ dye series are designed for low initial fluorescence, providing exceptional brightness only after nucleic acid binding. MycoLight™ Green JJ98 and JJ99 stain both Gram-positive and Gram-negative bacteria, and MycoLight™ Red JJ94 preferentially labels Gram-positive bacteria.

Table 2. MycoLight™ Dyes For Gram Staining Dyes

Product Name
Unit Size
23998MycoLight™ Green JJ98100 ug482512
23999MycoLight™ Green JJ981 mg482512
24000MycoLight™ Green JJ98 *5 mM in DMSO*100 uL482512
24001MycoLight™ Green JJ99 *5 mM in DMSO*100 uL482512
24006MycoLight™ Red JJ94 *2.5 mM in DMSO*100 uL630660