1. Fluorescent staining protocol for fungi (e.g., Candida albicans, Aspergillus niger):
- Sample preparation: Fungal cultures were grown on Sabouraud dextrose agar for 48–72 hours at 30°C. Fungal cells were harvested and suspended in sterile physiological saline to a concentration of 1×10⁶ cells/mL.
- Staining solution preparation: Brilliant Blue FCF was dissolved in sterile distilled water to prepare a 0.1% (w/v) stock solution, which was filtered through a 0.22 μm membrane for sterilization.
- Staining procedure: 100 μL of fungal cell suspension was mixed with 10 μL of 0.1% Brilliant Blue FCF stock solution (final concentration 0.01% w/v), and incubated at room temperature in the dark for 10–15 minutes.
- Observation: After incubation, 10 μL of the stained sample was placed on a glass slide, covered with a coverslip, and observed under a fluorescence microscope with an excitation wavelength of 450–490 nm and an emission wavelength of 515–565 nm. Fungal cells showed bright blue fluorescence, with clear visualization of cell walls and hyphal structures [1]
2. Fluorescent staining protocol for bacteria (e.g., Staphylococcus aureus, Escherichia coli):
- Sample preparation: Bacterial cultures were grown in Luria-Bertani broth for 18–24 hours at 37°C, then centrifuged at 3000×g for 5 minutes to collect bacterial pellets. Pellets were resuspended in sterile physiological saline to 1×10⁷ CFU/mL.
- Staining solution preparation: Same as fungal staining (0.1% w/v Brilliant Blue FCF stock solution, sterile-filtered).
- Staining procedure: 100 μL of bacterial suspension was mixed with 10 μL of 0.1% Brilliant Blue FCF stock solution (final concentration 0.01% w/v), incubated at room temperature in the dark for 8–12 minutes.
- Observation: Stained bacterial samples were observed under a fluorescence microscope (excitation 450–490 nm, emission 515–565 nm). Bacterial cells exhibited intense blue fluorescence, enabling clear distinction of individual cells and bacterial clusters [1]

Absorption, Distribution and Excretion
This study investigated the absorption, metabolism, and excretion of 14C-labeled Green S and Brilliant Blue FCF in rats, mice, and guinea pigs. Following a single oral administration of 100 μg/kg or 10 mg/kg of Green S, or 30 μg/kg or 3 mg/kg of Brilliant Blue FCF to male or female rats, most of the dose was excreted unchanged in feces within 72 hours. Pre-addition of unlabeled Green S or Brilliant Blue FCF (100 or 30 mg/kg/day, respectively) to the diet of male rats 21 days prior to administration of the 14C-labeled dye had no effect on the excretion pathway or the time required for complete clearance of the label. Similarly, in male mice and guinea pigs, all single oral doses of Green S or Brilliant Blue FCF were excreted in feces. Studies using isolated small intestinal loops confirmed that the gastrointestinal tracts of all three animal groups did not absorb or metabolize the labeled dyes. Results showed that no radioactive material was absorbed by the fetuses of pregnant rats after administration of 14C-labeled Green S or Brilliant Blue FCF. Female Sprague-Dawley rats were administered a single dose (0.27 mg; 1.74 μCi) of 14C-labeled dye via gavage. In bile duct ligated rats, 96 hours after oral administration, the intestinal absorption rate of FD&C Blue No. 1 was estimated at 2.05% of the administered dose, based on urinary 14C excretion, exhaled 14CO₂, and residual radioactivity in visceral organs and tissues. The mean fecal excretion rate was 97.28%, and the total radioactivity recovery rate was 99.38%. In intact rats, the average intestinal absorption rate of 14C-FD&C Blue No.1 was only 0.27% (with a recovery rate of 91%), while the average bile excretion rate in cannulated animals was 1.32% of the administered dose. Thin-layer chromatography analysis of urine and bile samples showed that approximately 95% of the excreted radioactivity was unaltered FD&C Blue No.1, and approximately 5% was unknown metabolites or degradation products of FD&C Blue No.1. These results indicate that FD&C Blue No.1 has a very low gastrointestinal absorption rate and is rapidly and completely excreted via bile after absorption. …The labeled dose (3 mg/kg body weight) excreted in rat bile within 5 hours was less than 0.05%. In pregnant rat fetuses orally administered 14C-labeled Brilliant Blue FCF on day 8 of gestation, extremely low radioactivity (0.004%–0.006% of the dose) was detected on day 11. Brilliant Blue FCF has a central methane ring labeled with ¹⁴C, exhibiting a radioactivity purity greater than 95%. …
After male or female rats were administered ¹⁴C-labeled Brilliant Blue FCF by gavage at doses of 30 μg/kg body weight or 3 mg/kg body weight, the majority of the dose was excreted unchanged in feces within 72 hours (99.9% for the low-dose group and 95.4% for the high-dose group). No radioactivity was detected in exhaled breath, and less than 0.5% was detected in urine. …
For more complete data on the absorption, distribution, and excretion of Brilliant Blue (6 items), please visit the HSDB record page.

Interactions
Although the content of food colorings is controlled according to the Acceptable Daily Intake (ADI), specific information is currently lacking regarding how these additive combinations affect food safety. This study investigated the effects of single and combined use of colorings on neural progenitor cell (NPC) toxicity (a developmental biomarker) and neurogenesis (reflecting adult central nervous system (CNS) function). In a developing CNS model, Allura Red AC and Amaranth Red reduced the proliferation and activity of pluripotent NPCs in mice. Among several combinations tested in mouse models, the combination of Tartrazine and Brilliant Blue FCF (at a dose 1000 times the average daily intake in Korea) significantly reduced the number of newly generated cells in the hippocampus of adult mice, indicating a significant adverse effect on hippocampal neurogenesis. However, other combinations, such as Allura Red AC and Amaranth Red, did not affect neurogenesis in the dentate gyrus of the adult hippocampus. Evidence suggests that the single and combined use of most tar-based food colorings may pose safety risks regarding developmental neural progenitor cells and adult hippocampal neurogenesis. However, the response to excessively high doses of the tartrazine and brilliant blue FCF combination suggests a possible synergistic effect, inhibiting the proliferation of adult hippocampal neural progenitor cells. Data indicate that combinations of tar-based pigments may have adverse effects on both developmental and adult hippocampal neurogenesis…

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Non-human toxicity values
Oral LD50 in rats >2.0 g/kg body weight
Subcutaneous LD50 in mice 4.6 g/kg body weight

[1]. An innovative brilliant blue FCF method for fluorescent staining of fungi and bacteria. Biotech Histochem. 2011 Aug;86(4):280-7.

Brilliant Blue is an organic molecular entity.
Erioglaucine A disodium salt is used as a food additive [EAFUS] (“EAFUS: United States Food Additives Directory. [http://www.eafus.com/]”).

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1. Background of Brilliant Blue FCF: Brilliant Blue FCF (CAS No. 3844-45-9) is a synthetic, water-soluble triarylmethane dye commonly used as a food additive (E133) for coloring beverages, confectionery, and baked goods. This study innovatively applied it to microbial fluorescent staining [1]
2. Advantages of the staining method: Compared with traditional staining methods (such as Gram staining and calcium fluorescent white staining), Brilliant Blue FCF staining has two main advantages: (1) It is simple and quick, without complicated washing steps, and staining can be completed within 8-15 minutes; (2) It has high specificity for microbial cells and will not stain background substances (such as culture medium residues) non-specifically, thus ensuring clear observation [1]
3. Fluorescent staining mechanism: Brilliant Blue FCF binds to carbohydrate components on the surface of microbial cells (such as chitin in fungal cell walls and peptidoglycan in bacterial cell walls) through hydrogen bonds and hydrophobic interactions. Under a specific excitation wavelength, the bound dye emits blue fluorescence, thereby realizing the visualization of microbial cells [1]

C37H34N2NA2O9S3

792.8484

792.122

3844-45-9

25305-78-6 (Parent)

19700

Pale purple to purple solid powder

283 °C (dec.)(lit.)

8.269

0

10

9

53

1510

0

SGHZXLIDFTYFHQ-UHFFFAOYSA-L

InChI=1S/C37H36N2O9S3.2Na/c1-3-38(25-27-9-7-11-33(23-27)49(40,41)42)31-19-15-29(16-20-31)37(35-13-5-6-14-36(35)51(46,47)48)30-17-21-32(22-18-30)39(4-2)26-28-10-8-12-34(24-28)50(43,44)45;;/h5-24H,3-4,25-26H2,1-2H3,(H2-,40,41,42,43,44,45,46,47,48);;/q;2*+1/p-2

disodium;2-[[4-[ethyl-[(3-sulfonatophenyl)methyl]amino]phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]cyclohexa-2,5-dien-1-ylidene]methyl]benzenesulfonate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~33.33 mg/mL (~42.04 mM)

Solubility in Formulation 1: 25 mg/mL (31.53 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)