Effect of phosphates on the disintegration of red blood cells by brilliant cresyl blue in correlation to age

The author describes age-related changes in the disintegration of rat red blood cells (RBC) caused by brilliant cresyl blue (BCB) in isotonic saline containing NaCl and phosphates. Blood was taken from rats aged 21, 45, 90-105, 340-360 and 690-720 days. The RBC were incubated four hours in the given solution. The incubation of RBC from different age groups in isotonic saline with phosphates showed no significant differences between the various age groups as far as disintegration of the RBC was concerned. When BCB was added to the solution, however, there was a marked increase in the disintegration of RBC from 21-, 42- and 690- to 720-day-old rats. The differences found in RBC disintegration can be attributed to age-determined qualitative changes in the structure of the RBC membrane--in the first place, changes in cross-linkages between the chains of the structural proteins of the RBC membrane.     

Selection of developmentally competent buffalo oocytes by brilliant cresyl blue staining before IVM

The brilliant cresyl blue (BCB) test determines the activity of glucose-6-phosphate dehydrogenase (G6PDH); the activity of this enzyme is greatest in growing oocytes, but it declines as oocytes mature. The objective was to develop and evaluate this test for assessing development of buffalo oocytes (to select developmentally competent oocytes for increased in vitro embryo production). Oocytes were exposed to BCB stain diluted in mDPBS (DPBS with 0.4% BSA) for 90 min at 38.5 °C in a humidified air atmosphere; those with or without blue coloration of the cytoplasm were designated as BCB+ and BCB−, respectively. In Experiment 1, oocytes were exposed to 13, 26, or 39 μM BCB. There were fewer BCB+ oocytes after exposure to 13 μM BCB (10%) than after exposure to 26 or 39 μM BCB (57.2 and 61.8%; P < 0.05), but there was no significant difference among treatments for blastocyst production rate. In Experiment 2, the diameter of BCB+ oocytes (144.4 ± 4.2 μm; mean ± S.E.M.) was higher (P < 0.05) than that of BCB− oocytes (136.8 ± 4.6 μm). In Experiment 3, oocytes were allocated into three groups: control (immediately cultured); holding-control (kept in mDPBS for 90 min before cultured); and treatment-incubation (incubated with 26 μM BCB). After IVM, oocytes were fertilized in vitro and cultured on an oviductal monolayer. The nuclear maturation rate was higher (P < 0.05) in BCB+ (86.2%), control (83.4%) and holding-control (82.6%) oocytes than BCB− (59.2%) oocytes. The BCB+ oocytes yielded more blastocysts than control or holding-control oocytes (33.4, 20.2, and 21.0%, P < 0.05); blastocyst development was lowest in BCB− oocytes (5.2%). In conclusion, staining of buffalo oocytes with BCB before IVM may be used to select developmentally competent oocytes for increased in vitro embryo production.     

Acid alcoholic brilliant cresyl blue stain for gastric and other mucins

Summary Some but not all samples of brilliant cresyl blue (6-methyl-7-dimethylamino-2-phenoxazin chloride) under C. I. No. 51010 in Conn's Biological Stains when dissolved at 1% level in 50–70% alcohol containing 1% concentrated (12 N) hydrochloric acid, stain (in 30 min) a wide variety of human and laboratory animal mucins blue black on an almost unstained background. The mucoprotein of the gastric surface epithelium and of the peptic gland neck cells of several species reacts strongly. A 16 hr 60° C methylation in 0.1 M methyl-sulfuric acid in methanol is required to block the staining of these gastric and some intestinal mucins, while 1–2 hr intervals suffice to prevent the staining of mast cells, cartilage and metachromatic sulfomucins generally. Saponification (1% KOH/70% alcohol, 20min) does not restore staining in either location group, indicating that sulfate mucins are probably reacting in both.Most other basic dyes fail to stain mucins from acid alcohol solutions: azure A, toluidine blue, resorcin blue, orcein, resorufin, azoresorufin brown, azolitmin, lacmoid, gallocyanin, Nile blue, methylene green, pararosanilin, crystal violet, Victoria blue R. Some staining occurred with one of three lots of Victoria blue B, with two lots of Victoria blue 4 R and with one lot each of Bernthsen's methylene violet, elastin violet PR and elastin purple PP.The stain may be preceded by the Feulgen reaction to give red nuclei, or followed by a brief collagen stain in an alcoholic acid fuchsin (0.05–0.1%), picric acid (1.5%) solution.Presented before the Symposium of the Histochemische Gesellschaft in Hamburg, 28. September 1968.Supported by National Cancer Institute Grant No. C-4816, National Institutes of Health.     

Selection of developmentally competent buffalo oocytes by brilliant cresyl blue staining before IVM

The brilliant cresyl blue (BCB) test determines the activity of glucose-6-phosphate dehydrogenase (G6PDH); the activity of this enzyme is greatest in growing oocytes, but it declines as oocytes mature. The objective was to develop and evaluate this test for assessing development of buffalo oocytes (to select developmentally competent oocytes for increased in vitro embryo production). Oocytes were exposed to BCB stain diluted in mDPBS (DPBS with 0.4% BSA) for 90 min at 38.5 degrees C in a humidified air atmosphere; those with or without blue coloration of the cytoplasm were designated as BCB+ and BCB-, respectively. In Experiment 1, oocytes were exposed to 13, 26, or 39 microM BCB. There were fewer BCB+ oocytes after exposure to 13 microM BCB (10%) than after exposure to 26 or 39 microM BCB (57.2 and 61.8%; P<0.05), but there was no significant difference among treatments for blastocyst production rate. In Experiment 2, the diameter of BCB+ oocytes (144.4+/-4.2 microm; mean+/-S.E.M.) was higher (P<0.05) than that of BCB- oocytes (136.8+/-4.6 microm). In Experiment 3, oocytes were allocated into three groups: control (immediately cultured); holding-control (kept in mDPBS for 90 min before cultured); and treatment-incubation (incubated with 26 microM BCB). After IVM, oocytes were fertilized in vitro and cultured on an oviductal monolayer. The nuclear maturation rate was higher (P<0.05) in BCB+ (86.2%), control (83.4%) and holding-control (82.6%) oocytes than BCB- (59.2%) oocytes. The BCB+ oocytes yielded more blastocysts than control or holding-control oocytes (33.4, 20.2, and 21.0%, P<0.05); blastocyst development was lowest in BCB- oocytes (5.2%). In conclusion, staining of buffalo oocytes with BCB before IVM may be used to select developmentally competent oocytes for increased in vitro embryo production.     

Selection of prepubertal goat oocytes using the brilliant cresyl blue test

Brilliant cresyl blue stain allows us to determine the activity of glucose-6-phosphate dehydrogenase (G6PD), an enzyme synthesized in growing oocytes but with decreased activity in oocytes that have finished their growth phase. The objective of this study was to evaluate the utility of the brilliant cresyl blue (BCB) test as an indirect measure of oocyte growth, in order to select competent prepubertal goat oocytes for in vitro embryo production. Oocytes were exposed to BCB diluted in PBS and were classified according to their cytoplasm coloration: oocytes with a blue cytoplasm or grown oocytes (BCB+) and oocytes without a blue cytoplasm or growing oocytes (BCB-). After exposure to different BCB concentrations, we evaluated in vitro maturation (IVM), in vitro fertilization (IVF) and embryo development parameters. We defined matured oocytes as those oocytes that reached the metaphase II (MII) stage after being cultured for 27 h. Oocytes showing two pronuclei at 20 h post-insemination were classified as normally fertilized oocytes. We assessed embryo development 8 days post-insemination and recorded the percentage of total embryos, morale and blastocysts. The mean percentage of BCB+ oocytes was 29.4%. Mean diameter of BCB+ oocytes (136.6+/-6.3 microm) was higher (P < 0.001) than that of BCB- oocytes (125.5+/-10.2 microm). The percentage of BCB+ oocytes reaching the MII stage (81.4%) was higher (P < 0.05) than that of BCB- (52.5%) and control oocytes (72.4%). Normal fertilization rate of BCB+ oocytes was also higher (23.5%) than that of BCB- (8.2%; P < 0.0001) and control oocytes (11.9%; P < 0.05). The percentages of total embryos undergoing development to >8-cell and the morula plus blastocyst stages were higher (P < 0.05) in the group of BCB+ (41.3 and 12.0%, respectively) than in BCB- oocytes (21.3 and 3.6%, respectively). In conclusion, the BCB test is a useful way to select more competent prepubertal goat oocytes for in vitro embryo production.     

Influence of acute hypoxia upon brilliant cresyl blue-induced disintegration of erythrocytes in rabbits and cats

The disintegration of erythrocytes by brilliant cresyl blue was studied in rabbits and cats during two hypoxic periods resulting in apnoea which was followed by artificial ventilation and recovery. The rate of erythrocyte disintegration was measured after 2 hours' incubation in isotonic NaCl or Krebs-Ringer solution plus brilliant cresyl blue (0.5 mmol.l-1). Significant increases of disintegration rates were found in rabbits during recovery in both incubation solutions. Erythrocytes in cats seemed to be more resistant to acute hypoxia, as their disintegration rate rose only in isotonic NaCl solution, and that only transiently. Brilliant cresyl blue--induced erythrocyte disintegration in cats did not differ from the control values during 10 min of spontaneous breathing after the first and the second period of hypoxia in the isotonic NaCl solution, but it was significantly lower in Krebs-Ringer solution. The possible factors influencing the erythrocyte disintegration rate in acute hypoxia are discussed.     

Developmental competence of porcine oocytes selected by brilliant cresyl blue and matured individually in a chemically defined culture medium

The objective of this study was to investigate the effects of oocyte selection using brilliant cresyl blue (BCB) and culture density during individual in vitro maturation (IVM) on porcine oocyte maturity and subsequent embryo development using a chemically defined medium. Cumulus-oocyte complexes (COCs) were classified as BCB-positive or BCB-negative after exposure to a BCB solution for 90 min. The classified COCs were matured in a group (15 COCs per 100-μL droplet) or individually (1 COC per 1-, 2.5-, 5-, or 10-μL droplet). Meiotic competence, intraoocyte glutathione concentration, and developmental competence after intracytoplasmic sperm injection were monitored. The BCB selected oocytes competent for nuclear and cytoplasmic maturation. Furthermore, meiotic competence for oocytes matured individually in a 5-μL droplet was superior (P < 0.05) to that of oocytes matured in a 1-μL droplet. Also, the culture density in a 5-μL droplet during IVM resulted in a higher (P < 0.05) rate of cleaved embryos than that in a 1-μL droplet and produced a similar rate of blastocysts compared with that of a group culture system. Conversely, BCB selection did not improve cleavage and blastocyst formation. In conclusion, it was possible to predict porcine oocytes competent for maturation using oocyte selection with BCB. Moreover, a 5-μL droplet during the individual IVM culture was most suitable for oocyte maturation and subsequent embryo development, although every culture density used in this study supported development up to the blastocyst stage.     

Developmental competence of prepubertal goat oocytes selected with brilliant cresyl blue and matured with cysteamine supplementation

The aim of this study was to assess the effect of oocyte selection using the brilliant cresyl blue (BCB) test plus the addition of cysteamine to the in vitro maturation (IVM) medium to improve the in vitro embryo development of prepubertal goat oocytes. The oocytes were exposed to 26 microM BCB and classified according to their cytoplasm coloration: BCB+ (oocytes with blue cytoplasm) and BCB- (unstained oocytes). The oocytes were matured in a conventional IVM medium supplemented with cysteamine 100 microM. The control group consisted of oocytes not exposed to BCB and matured without cysteamine. The IVM-oocytes were inseminated and cultured in synthetic oviductal fluid (SOF) for 7 days. The normal fertilisation rate (oocytes showing 2 pronuclei and 1 sperm tail) of BCB+ oocytes (40%) was higher than those of BCB- (21%) and control oocytes (22%). The percentage of morulae plus blastocysts was higher (P < 0.05) in the BCB+ group than in the BCB- group (23.8 vs. 5.1%, respectively). In conclusion, the integration of the BCB test and the addition of cysteamine in the protocol of in vitro embryo production from prepubertal goat oocytes has improved the developmental rates of embryo development.     

Selection of oocytes for in vitro maturation by brilliant cresyl blue staining： a study using the mouse model

Selecting oocytes that are most likely to develop is crucial for in vitro fertilization and animal cloning. Brilliant cresyl blue （BCB） staining has been used for oocyte selection in large animals, but its wider utility needs further evaluation. Mouse oocytes were divided into those stained （BCB＋） and those unstained （BCB-） according to their ooplasm BCB coloration. Chromatin configurations, cumulus cell apoptosis, cytoplasmic maturity and developmental competence were compared between the BCB＋ and BCB- oocytes. The effects of oocyte diameter, sexual maturity and gonadotropin stimulation on the competence of BCB＋ oocytes were also analyzed. In the large- and medium-size groups, BCB＋ oocytes were larger and showed more surrounded nucleoli （SN） chromatin configurations and higher frequencies of early atresia, and they also gained better cytoplasmic maturity （determined as the intracellular GSH level and pattern of mitochondrial distribution） and higher developmental potential after in vitro maturation （IVM） than the BCB-oocytes. Adult mice produced more BCB＋ oocytes with higher competence than the prepubertal mice when not primed with PMSG. PMSG priming increased both proportion and developmental potency of BCB＋ oocytes. The BCB＋ oocytes in the large-size group showed more SN chromatin configurations, better cytoplasmic maturity and higher developmental potential than their counterparts in the medium-size group. It is concluded that BCB staining can be used as an efficient method for oocyte selection, but that the competence of the BCB＋ oocytes may vary with oocyte diameter, animal sexual maturity and gonadotropin stimulation. Taken together, the series of criteria described here would allow for better choices in selecting oocytes for better development.     

Developmental competence of heifer oocytes selected using the brilliant cresyl blue (BCB) test

The aim of this study was to evaluate the usefulness of the brilliant cresyl blue (BCB) test in the selection of more competent heifer oocytes for in vitro embryo production (IVEP). IVEP from selected BCB heifer oocytes was compared to IVEP from morphologically selected heifer (control group) and cow oocytes. BCB staining determines the activity of glucose-6-phosphate dehydrogenase (G6PD), an enzyme synthesized in growing oocytes but with less activity in grown oocytes. Six hundred and fifty seven heifer cumulus-oocyte complexes (COC) were classified morphologically as Grade 1-3 and exposed to 26 microM of BCB and classified as: blue (or grown) oocytes (BCB+) or unstained oocytes or growing oocytes (BCB-). Grade 1-3 heifer oocytes showed significantly different percentages of BCB+ oocytes (78.6, 66.2, and 51.1%, respectively; P<0.05). The diameter of BCB+ oocytes was significantly higher than BCB- oocytes (152.6+/-5.8 microm and 147+/-5.9 microm, respectively; P<0.001). The percentage of BCB+ oocytes reaching the blastocyst stage was significantly higher than those of BCB- and control heifer oocytes (12.3, 1.6, and 5.2%, respectively; P<0.05), but lower than those of cow oocytes (30.0%; P<0.05). In conclusion, heifer oocytes selected by the BCB test (BCB+) are larger and more competent for IVEP than control heifer oocytes. However, fewer heifer oocytes selected using the BCB test develop to blastocyst stage compared to cow oocytes.     

Combination of oocyte and zygote selection by brilliant cresyl blue (BCB) test enhanced prediction of developmental potential to the blastocyst in cattle

The cumulus oocyte complexes (COCs) were obtained from local abattoir. After aspiration, the COCs were allotted into four treatments to evaluation of brilliant cresyl blue (BCB) test. Control treatment (C): oocytes were cultured directly (without exposure to BCB) after recovery in in vitro production (IVP) process. Oocyte treatment (OBCB): immediately after aspiration, COCs were incubated in modified Dulbecco's phosphate-buffered saline (mDPBS) supplemented with 26 μM of BCB for 90 min and classified into two classes: oocytes with blue cytoplasm coloration (OBCB+: more competent oocytes) and oocytes without blue cytoplasm coloration (OBCB−: low competent oocytes). Directly after classification, the oocytes were maintained undisrupted in the IVP process. Zygote treatment (ZBCB): After oocyte collection, maturation and fertilization, zygotes were stained with BCB for 10 min and categorized into three ways, according to whether they were highly stained (ZBCB++: low competent zygotes), moderately stained (ZBCB+: moderate competent zygotes) and unstained (ZBCB−: more competent zygotes). Directly after classification, the zygotes were maintained undisrupted in the culture process. Oocyte and zygote treatments (OBCB/ZBCB): COCs were stained with BCB after recovery and classified into two classes (OBCB+ and OBCB−). After fertilization, the zygotes produced from OBCB+ and OBCB− oocytes were further stained with BCB for 10 min and categorized six ways (OBCB+/ZBCB++, OBCB+/ZBCB+, OBCB+/ZBCB−, OBCB−/ZBCB++, OBCB−/ZBCB+ and OBCB−/ZBCB−). Directly after classification, the zygotes were maintained undisrupted in the culture process. The selection rate produced from OBCB treatment (OBCB+; 54.3%) was greater (P < 0.05) than ZBCB treatment (ZBCB−; 44.3%). In addition, the selection rate produced from double application (combination of oocyte and zygote selection) of BCB test (OBCB+/ZBCB−: 28.8%) was less (P < 0.01) than single application of BCB test (ZBCB−: 44.3%or OBCB+: 54.3%). The percentage of blastocyst production from OBCB+ oocytes (35.7%) and ZBCB− zygotes (36.6%) were greater (P < 0.05) than that from C oocytes (25.7%), OBCB− oocytes (16.5%), ZBCB++ (13.5%) and ZBCB+ zygotes (21.3%). However, there were no significant differences (P > 0.05) in the percentages of blastocyst production between OBCB+ oocytes (35.7%) and ZBCB− zygotes (36.6%). The proportion of blastocyst production from double application of BCB test (OBCB+/ZBCB−: 48.0%) was greater (P < 0.05) than that from single application of BCB test (OBCB+: 35.7% or ZBCB−: 36.6%). In conclusion, current results confirmed that combination of oocyte and zygote selection by BCB test enhanced the efficiency of selecting for high quality embryos, compared to the single BCB test.     

Effects of single and double exposure to brilliant cresyl blue on the selection of porcine oocytes for in vitro production of embryos

The aim of this study was to evaluate the effectiveness and toxicity of single and double application of the brilliant cresyl blue (BCB) test on the selection of porcine oocytes as an indirect measure of oocyte growth for in vitro fertilization (IVF) and nuclear transfer. In the first experiment, oocytes were exposed to BCB before and after maturation culture and classified according to their cytoplasmic coloration: blue coloration and colorless. The classified oocytes were fertilized with spermatozoa and then cultured for 7 days. The percentages of maturation to metaphase II in blue oocytes at the start of maturation culture were higher than those of colorless oocytes (68.7-70.1% versus 0.8-3.6%, P < 0.05). However, double application of BCB test before and after maturation culture had a toxic effect on fertilization and embryonic development. No significant differences in the blastocyst formation were found between blue oocytes without double application of BCB test and control oocytes without any application of BCB test, whereas the total cell number per blastocyst from the blue oocytes was higher than that from the control oocytes (48.0 versus 34.2, P < 0.05). In the second experiment, oocytes were exposed to the BCB test before or after maturation culture, and then the matured oocytes were activated to evaluate the ability of parthenogenetic development. The proportion of blastocyst formation of blue oocytes classified after maturation culture was lower than that of blue oocytes classified before maturation culture (10.0% versus 27.0%, P < 0.05). Therefore, double application of the BCB test before and after maturation culture impaired fertilization and embryonic development, whereas a single application before maturation culture was efficient to select oocytes for IVF and nuclear transfer.     

Selection of developmentally competent oocytes through brilliant cresyl blue stain enhances blastocyst development rate after bovine nuclear transfer

The aim of the present investigation was to study the effect of oocyte selection on the efficiency of bovine nuclear transfer in terms of increased blastocyst production. For this purpose, prior to in vitro maturation (IVM), oocytes were selected for their developmental competence on the basis of glucose-6-phosphate dehydrogenase (G6PDH) activity indicated by brilliant cresyl blue (BCB) staining. It has been hypothesized that growing oocytes have a higher level of active G6PDH in comparison to the mature oocytes. Compact cumulus oocyte complexes (COCs) were recovered from slaughterhouse-collected bovine ovaries and classified either as control group, which were placed immediately into culture without exposure to BCB stain, or treatment group, which were stained with BCB for 90min before culture. Treated oocytes were then divided into BCB- (colourless cytoplasm, increased G6PDH) and BCB+ (coloured cytoplasm, low G6PDH) based on their ability to metabolize the stain. After IVM, oocytes were subjected to nuclear transfer procedure for the production of cloned embryos which were then cultured for a period of 8 days to determine the blastocyst rate. The BCB+ oocytes yielded a significantly higher blastocyst rate (39%) than the control (21%) or BCB- oocytes (4%). These results show that the staining of bovine cumulus-oocyte complexes with BCB before in vitro maturation could be used to select developmentally competent oocytes for nuclear transfer. In addition, G6PDH activity could prove to be a useful marker for determining the oocyte quality in future.     

Gene expression pattern of adiponectin and adiponectin receptors in dominant and atretic follicles and oocytes screened based on brilliant cresyl blue staining

Adiponectin and its receptors (AdipoR1 and AdipoR2) are novel endocrine systems that act at various levels to control male and female fertility. The aim of this study was to determine whether adiponectin and its receptors gene expression levels differ between dominant follicle (DF) and atretic follicle (AF) and also between oocytes which were stained positively and negatively with brilliant cresyl blue (BCB(+) and BCB(-)). Based on estradiol/progesterone ratio, follicles from ovaries were classified as AFs and DFs. The stages of estrous cycle (follicular or luteal phases) were defined by macroscopic observation of the ovaries and the uterus. Oocytes were stained with BCB for 90 min. The relative expression of adiponectin, AdipoR1 and AdipoR2 mRNA in theca and cumulus cells and oocytes of different follicles were determined by quantitative real time PCR. Adiponectin and its receptors genes were clearly expressed higher (P<0.05) in theca and cumulus cells and oocytes of DFs than those of AFs during the follicular and luteal phases. BCB(+) oocytes showed a higher (P<0.05) expression of adiponectin and its receptors compared with their BCB(-) counterparts. Positive correlation (r>0.725, P<0.001) was observed between adiponectin mRNA level in ovarian cells of DFs and follicular fluid E2 concentration in follicular phase. Adiponectin mRNA abundance in ovarian cells of AFs showed a significant negative correlation with follicular fluid progesterone concentration in follicular and luteal phases (r<-0.731, P<0.001). This work has revealed the novel association of adiponectin and its receptors genes with follicular dominance and oocyte competence, thereby opening several new avenues of research into the mechanisms of dominance and competence in animal and human.     

Supplementation with cysteamine during maturation and embryo culture on embryo development of prepubertal goat oocytes selected by the brilliant cresyl blue test

Our previous studies have shown that the addition of 100 mircroM cysteamine to the in vitro maturation (IVM) medium increased the embryo development of prepubertal goat oocytes. The aim of the present study was to evaluate the effect of adding different concentrations of cysteamine to the IVM medium and to the in vitro embryo culture medium (IVC) on the embryo development of prepubertal goat oocytes selected by the brilliant cresyl blue (BCB) test. Oocytes were exposed to BCB and classified as: oocytes with a blue cytoplasm or grown oocytes (BCB+) or oocytes without blue cytoplasm or growing oocytes (BCB-). In Experiment 1, oocytes were matured in a conventional IVM medium supplemented with 100 microM, 200 microM or 400 microM cysteamine. In Experiment 2, oocytes were matured with 400 microM cysteamine and following in vitro fertilization (IVF) were cultured in SOF medium supplemented with 50 microM and 100 microM cysteamine. In Experiment 1, BCB+ oocytes matured with 100 microM and 200 microM cysteamine showed higher normal fertilization and embryo development rates than BCB- oocytes. Oocytes matured with 400 microM cysteamine did not present these differences between BCB+ and BCB- oocytes. In Experiment 2, the addition of 50 microM and 100 microM cysteamine to culture medium did not affect the proportion of total embryos obtained from BCB+ oocytes (35.89% and 38.29%, respectively) but was significantly different in BCB- oocytes (34.23% and 29.04%, respectively, P < 0.05). In conclusion, the addition of 400 microM cysteamine to the IVM improved normal fertilization and embryo development of BCB- oocytes at the same rates as those obtained from BCB+ oocytes. The proportions of morulae plus blastocyst development were not affected by the treatments.     

Preparative binding of Coomassie brilliant blue to bovine serum

Laboratory scale preparation of bovine serum albumin (BSA) stained with Coomassie brilliant blue (CBB) at alkaline pH is first described. Physical-chemical analyses of CBB-BSA showed that the unprotonated (anion) CBB dye binds tightly to BSA in buffered media of pH 8.2. Characteristic differences in spectra lambda(max) and molar absorptivities were found for the free anion CBB dye versus the CBB-BSA complex. Binding studies with low versus high dye/protein concentration ratios at alkaline pH gave values for n, binding site numbers, and K, intrinsic binding coefficient, consistent with those reported in analytical studies under acidic pH, but higher than values for neutral pH. Comparative analyses of Beer's law plots for the alkaline CBB-BSA complex under different experimental conditions showed its high stability toward various interferences, such as pH, strong detergents, temperature, light, prolonged storage, as well as high affinity for tannins. The hydrophobic nature of the CBB-BSA association at alkaline pH was tested.