Dye Exchange in Bacterial Cells, and the Theory of Staining

Bacterial cells were stained in sequence, and at various pH's, by 22 different basic dyes. It was found that any dye could replace another already present in the bacterial cell. This replacement was shown to act according to mass action laws for reversible reactions, and hence was influenced by concentration of reagent and time of application. Since basic dyes are also known to react at carboxyl group sites, the phenomena of staining of bacterial cells by ordinary basic dyes must be a chemical adsorption exchange reaction.     

The Nature of Mycobacterial Acid-Fastness

Phenol is not essential to acid-fast staining, for it will occur in the absence of phenol where such lipoid-soluble basic dyes as night blue, Victoria blue B or Victoria R are used; it is essential for acid-fast staining with water soluble basic dyes such as basic fuchsin. When phenol is added to the staining solution, such water soluble basic dyes behave in effect like their lipid-soluble counterparts. The loss of mycobacterial acid-fastness with carbolfuchsin after bromination or chromation indicates that this phenomenon is related to the presence of unsaturated lipids in the bacterial cells. Within the cells these acid-fast lipids are bound in such a way that they are easily removed from all mycobacteria by hot dilute HCl; from leprosy bacilli alone they are easily removed with hot pyridine. From the results of various blocking reactions it appears that carboxyl and especially hydroxyl groups of these cellular lipids are essential to the acid-fast reaction of mycobacteria.     

The nature of mycobacterial acid-fastness.

Phenol is not essential to acid-fast staining, for it will occur in the absence of phenol where such lipoid-soluble basic dyes as night blue, Victoria blue B or Victoria R are used; it is essential for acid-fast staining with water soluble basic dyes such as basic fuchsin. When phenol is added to the staining solution, such water soluble basic dyes behave in effect like their lipid-soluble counterparts. The loss of mycobacterial acid-fastness with carbol-fuchsin after bromination or chromation indicates that this phenomenon is related to the presence of unsaturated lipids in the bacterial cells. Within the cells these acid-fast lipids are bound in such a way that they are easily removed from all mycobacteria by hot dilute HCl; from leprosy bacilli alone they are easily removed with hot pyridine. From the results of various blocking reactions it appears that carboxyl and especially hydroxyl groups of these cellular lipids are essential to the acid-fast reaction of mycobacteria.     

Effect of alternating current exposure on the resistivity of resting Escherichia coli B cells to crystal violet and other basic dyes

Phosphate buffer suspensions of resting Escherichia coli B cells at pH 70 were anaerobically exposed to alternating current (a.c.) of 50 Hz at a current density of 600 60 mA/cm² and 34 3C. The minimum inhibitory concentrations of eight basic dyes: crystal violet, malachite green, brilliant green, fuchsin, methylene blue, toluidine blue, safranin and acriflavine for exposed cells were decreased to about the half values of those for unexposed ones when both cells were grown in the minimal medium including one of the dyes. The integrated viabilities of exposed cells tended to decline with increasing concentration of the dyes markedly more than those of unexposed ones, whereas the exposed cells took up the dyes less readily than the unexposed cells. These results suggested that a.c. exposure may serve as an agent which renders E. coli cells susceptible to the basic dyes.     

Effect of alternating current exposure on the resistivity of resting Escherichia coli B cells to crystal violet and other basic dyes

Phosphate buffer suspensions of resting Escherichia coli B cells at pH 7.0 were anaerobically exposed to alternating current (a.c.) of 50 Hz at a current density of 600 +/- 60 mA/cm2 and 34 degrees +/- 3 degrees C. The minimum inhibitory concentrations of eight basic dyes: crystal violet, malachite green, brilliant green, fuchsin, methylene blue, toluidine blue, safranin and acriflavine for exposed cells were decreased to about the half values of those for unexposed ones when both cells were grown in the minimal medium including one of the dyes. The integrated viabilities of exposed cells tended to decline with increasing concentration of the dyes markedly more than those of unexposed ones, whereas the exposed cells took up the dyes less readily than the unexposed cells. These results suggested that a.c. exposure may serve as an agent which renders E. coli cells susceptible to the basic dyes.     

Cell permeability as a rate limiting factor in the microbial reduction of sulfonated azo dyes

Summary Permeabilization of cells of B. cereus and other bacterial strains by toluene treatment significantly increased the passage of sulfonated and carboxylated azo dyes from the external medium into the cells with a concomittant increase of the reduction rate of the dyes. Dyes which are not reduced at all by intact cells were readily decolorized. The reduction rate of sulfonated compounds was consistently larger than of their carboxylated analogues, once the dyes had entered the cells.     

Isolation, identification and application of novel bacterial consortium TJ-1 for the decolourization of structurally different azo dyes

A novel bacterial consortium (TJ-1), which could decolorize Acid Orange 7 (AO7) and manyother azo dyes, was developed. In TJ-1 three bacterial strains were identified as Aeromonas caviae, Proteus mirabilis and Rhodococcus globerulus by 16S rRNA gene sequence analysis. AO7 decolorization was significantly higher with the use of consortium as compared to the use of individual strains, indicating complementary interactions among these strains. AO7 decolorization was observed under microaerophilic condition in the presence of organic carbon source. Either yeast extract (YE) alone or a combination of YE and glucose resulted in much higher decolorization of AO7 as compared to glucose alone, peptone or starch. Kinetic studies with different initial AO7 concentrations showed that more than 90% decolorization could be achieved even at 200mg/l within 16h. Fed-batch studies showed that AO7 decolorization required 10h during the first cycle and 5h in the second and third cycles, showing that bacterial cells could be used for multiple cycles. The consortium also decolorized fifteen other azo dyes individually as well as a simulated wastewater containing a mixture of all the sixteen azo dyes, thus, conferring the possibility of application of TJ-1 for the treatment of industrial wastewaters.     

Combination of Certain Fluorane Derivative dyes with Bacterial cells at Different Hydrogen on Concentrations

While acid dyes have not been widely used for staining bacteria, several suggestions for their use have been advanced (Conn and Holmes, 1926; Maneval, 1941). In general these procedures call for adding an acid to the stain to intensify the combination of the dye with the bacterial cell. McCalla and Clark (1941) have demonstrated greater adsorption of acid dyes at lower pH levels. Conn and Holmes (1926) list a number of dyes derived from fluorane and compare their relative acidic tendencies, color intensities, and other properties. In a study of the influence of the acidic properties of dyes on their combination with bacterial cells at various hydrogen-ion concentrations, the data herein reported were obtained.     

Combination of Certain Fluorane Derivative dyes with Bacterial cells at Different Hydrogen on Concentrations

While acid dyes have not been widely used for staining bacteria, several suggestions for their use have been advanced (Conn and Holmes, 1926; Maneval, 1941). In general these procedures call for adding an acid to the stain to intensify the combination of the dye with the bacterial cell. McCalla and Clark (1941) have demonstrated greater adsorption of acid dyes at lower pH levels. Conn and Holmes (1926) list a number of dyes derived from fluorane and compare their relative acidic tendencies, color intensities, and other properties. In a study of the influence of the acidic properties of dyes on their combination with bacterial cells at various hydrogen-ion concentrations, the data herein reported were obtained.     

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4'',6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.     

Use of fluorescent dyes as molecular probes for the study of multidrug resistance

Fluorescence microscopy has shown that 18 different fluorescent dyes, staining various intracellular structures in transformed hamster fibroblasts (DM-15), did not stain or stained weakly multidrug-resistant cells selected from DM-15 by colchicine. Reduced staining by fluorescent dyes was characteristic also of five other tested multidrug-resistant cell lines of hamster and mouse origin, selected by actinomycin D, colcemid, rubomycin, and ruboxyl. The intensity of staining of two revertant cell lines was similar to that of parental sensitive cells. All tested inhibitors of multidrug resistance, including weak detergent, metabolic inhibitors, calcium channel blockers, calmodulin inhibitors, and reserpine, restored normal staining of multidrug-resistant cells. The dyes accumulated in resistant cells in presence of these inhibitors left the cells several minutes after the removal of the inhibitor from the incubation medium. Sensitive cells retained the dyes for several hours. The efflux of the dyes from resistant cells is an active process since it occurred even in the presence of the dyes in the incubation medium. The efflux could be blocked by all tested inhibitors of multidrug resistance and it is possibly a basic mechanism of the reduced staining of resistant cells. These data support the idea that multidrug resistance is based on active nonspecific efflux of the drugs and indicate that the simple procedure of cell staining can be used for the detection of resistant cells and further study of the phenomenon of multidrug resistance.     

细菌脱色酶TpmD对三苯基甲烷类染料脱色的酶学特性研究

从嗜水气单胞菌DN322中分离纯化出能够对三苯基甲烷类染料结晶紫、碱性品红、灿烂绿及孔雀绿进行有效脱色的脱色酶,命名为TpmD。该酶的亚基分子量为29.4kDa,等电点为5.6。该酶催化上述4种三苯基甲烷类染料脱色反应的适合温度为40~60℃,适合pH范围为5.5~9.0。动力学参数测定结果显示TpmD对结晶紫、碱性品红、灿烂绿及孔雀绿的Km值分别为24.3、40.65、4.2、68.5μmol-1.L-1,Vmax值分别为19.6、74.1、82.8、115.6μmol.L-1.s-1。结晶紫为该酶的最适反应底物。TpmD催化的脱色反应依懒于NADH/NADPH及分子氧的存在,显示该酶属于NADH/NADPH依赖型的氧化酶类。这是国内外首次关于细菌中三苯基甲烷类染料脱色酶酶学性质的描述。     

The mutant gene wellhaarig disturbs the differentiation of hair follicle cells in the mouse]

First generation (G1) hairs in mice homozygous for the wellhaarig (we) gene are wavy and shorter than in normal mice; basal regions of the hairs are deformed. Follicles of G1 hairs in mid-dorsal region of 8-, 12- and 16-days old we/we mice were examined. Huxley cells of the inner root sheath (IRS) in apical region of hair follicles appeared to be hypertrophied. Cytoplasm of these cells was not stained by basic dyes and showed no birefringence. Cytoplasm of the IRS Henle cells was not stained by basic dyes either. These data indicate that keratinization of the IRS cells is disturbed in mutant homozygotes. The layer of outer root sheath in the we/we mice was thinner than in normal mice; this is probably due to hypertrophy of the IRS cells. The structure of differentiating cells of the hair shaft in normal and mutant mice was similar. The data obtained suggest that abnormal G1 hairs in we/we mice result from disturbance in IRS cells differentiation.     

The Effect of the Chemical Nature of a Decolorizer on Its Functioning. II. the Apparent Isoelectric Point

The isoelectric point of a bacterial system is the hydrogen-ion concentration at which there is equal retention of anion and cation. Defining this point as that at which there is equal retention of acidic and basic stain when acetone is used as a decolorizer, it is shown that acidic decolorizers shift the experimentally determined point to a higher pH-value while basic decolorizers shift it to a lower value. Thus basic decolorizers show abnormally high decolorizing power toward smears stained with acid dyes, and acid decolorizers show the same abnormal behavior toward smears stained with basic dye. By basic decolorizer is meant, not one of high pH-value, but one which will form a salt with acids, as for example pyridin or anilin. This indicates an ionic chemical equilibrium as a factor in the mechanism of staining.     

Decolorization of basic, direct and reactive dyes by pre-treated narrow-leaved cattail (Typha angustifolia Linn.)

The efficiency of basic, direct and reactive dye removal from water by narrow-leaved cattail (NLC) powder treated with distilled water (DW-NLC), 37% formaldehyde+0.2 N sulfuric acid (FH-NLC), or 0.1 N sodium hydroxide (NaOH-NLC) at various pH levels (3, 5, 7, and 9) was tested. Desorption of the adsorbed dyes was also investigated. The type of NLC treatment and pH of the dye solution had little effect on removal of basic dyes, and efficiencies ranged from 97% to 99% over the range of pH used. Over a wide range of pH levels, all types of treated cattail powder had negative charges and probably attracted the basic dyes possessing positive charges. Efficiency of removal by the three NLC treatments ranged from 37% to 42% for direct dyes and from 22% to 54% for direct dyes at pH 7. The pH of the dye solution had substantial effects on the efficiency of removal in direct and reactive dyes. Dye removal was highest at pH 3, with 99% for a direct dye (Sirius Red Violet RL) and 96% for a reactive dye (Basilen Red M-5B). There was mutual attraction between negatively charged direct dye molecules and positively charged molecules on the surface of the FH-treated cattail. In tests of desorption of dyes from cattail in distilled water, the desorption percentage for FH-NLC after adsorbing basic, direct and reactive dyes was 6%, 10% and 35%, respectively, which indicated a chemisorption mechanism for basic and direct dyes and some physiosorption for reactive dyes.     

Evaluation of impact of exposure of Sudan azo dyes and their metabolites on human intestinal bacteria

Sudan azo dyes are banned for food usage in most countries, but they are illegally used to maintain or enhance the color of food products due to low cost, bright staining, and wide availability of the dyes. In this report, we examined the toxic effects of these azo dyes and their potential reduction metabolites on 11 prevalent human intestinal bacterial strains. Among the tested bacteria, cell growth of 2, 3, 5, 5, and 1 strains was inhibited by Sudan I, II, III, IV, and Para Red, respectively. At the tested concentration of 100 μM, Sudan I and II inhibited growth of Clostridium perfringens and Lactobacillus rhamnosus with decrease of growth rates from 14 to 47%. Sudan II also affected growth of Enterococcus faecalis. Growth of Bifidobacterium catenulatum, C. perfringens, E. faecalis, Escherichia coli, and Peptostreptococcus magnus was affected by Sudan III and IV with decrease in growth rates from 11 to 67%. C. perfringens was the only strain in which growth was affected by Para Red with 47 and 26% growth decreases at 6 and 10 h, respectively. 1-Amino-2-naphthol, a common metabolite of the dyes, was capable of inhibiting growth of most of the tested bacteria with inhibition rates from 8 to 46%. However, the other metabolites of the dyes had no effect on growth of the bacterial strains. The dyes and their metabolites had less effect on cell viability than on cell growth of the tested bacterial strains. Clostridium indolis and Clostridium ramosum were the only two strains with about a 10 % decrease in cell viability in the presence of Sudan azo dyes. The present results suggested that Sudan azo dyes and their metabolites potentially affect the human intestinal bacterial ecology by selectively inhibiting some bacterial species, which may have an adverse effect on human health.     

Biodegradation perspectives of azo dyes by yeasts

Azo dyes are the largest class of synthetic dyes, which are widely used in the textile industry. The amount of dyestuff does not bind to the fibers and is lost in wastewater during textile processing. The discharge of colored effluents into the environment is not only aesthetically unpleasing. Moreover, dyes and their break-down products cause toxic effects and they affect photosynthetic activity of aquatic systems by reducing light penetration. A number of microorganisms belonging to different taxonomic groups of bacteria, algae, fungi and yeast have been reported for their ability to decolorize azo dyes. In the literature the ability to decolorize azo dyes by yeasts, compared to bacterial and fungal species, has been studied in a few reports. Within this review, an attempt is made to elucidate some basic biological aspects associated with the azo dye degradation by yeasts and enzymes involved that are responsible for degradation process.     

Mechanism of chlorpromazine binding by Gram-positive and Gram-negative bacteria

Chlorpromazine forms charge-transfer complexes with xanthene dyes in bacteria. These complexes permit the differentiation of Gram-positive and Gram-negative bacteria in both light and polarization microscopy. The birefringence induced by the charge-transfer complex might explain the molecular basis of bacterial staining.The charge-transfer complexes formed between chorpromazine and xanthene dyes accumulate in the bacterial cell, mainly inside the bacterial cell wall. The complexes give the cells a color, which depends on the chemical composition of the staining structure, and in particular the polysaccharides of the cell wall in bacteria.Metachromatic granules were seen inside Gram-positive bacteria after chlorpromazine and rose bengal staining. Although the nature of these granules remains unclear, this type of binding may have a role in the inhibition of biochemical processes in the bacterial cells.     

The Mechanism of the Gram Reaction I. The Specificity of the Primary Dye

Dyes of all major types were tested for their suitability as the primary dye in the Gram stain. When a counterstain was not used, some dyes of all types were found to differentiate Gram-positive from Gram-negative organisms. When a counterstain was used, these dyes were found to vary greatly in their suitability. Those dyes found to be good substitutes for crystal violet were: Brilliant green, malachite green, basic fuchsin, ethyl violet, Hoffmann's violet, methyl violet B, and Victoria blue R. All are basic triphenylmethane dyes. Acid dyes were generally not suitable. Differences in the reaction of Gram-positive and Gram-negative cells to Gram staining without the use of iodine were observed and discussed but a practical differentiation could not be achieved in this manner. Certain broad aspects of the chemical mechanism of dyes in the gram stain are discussed.     

The mode of cell wall growth in selected archaea is similar to the general mode of cell wall growth in bacteria as revealed by fluorescent dye analysis

The surfaces of 8 bacterial and 23 archaeal species, including many hyperthermophilic Archaea, could be stained using succinimidyl esters of fluorescent dyes. This allowed us for the first time to analyze the mode of cell wall growth in Archaea by subculturing stained cells. The data obtained show that incorporation of new cell wall material in Archaea follows the pattern observed for Bacteria: in the coccoid species Pyrococcus furiosus incorporation was in the region of septum formation while for the rod-shaped species Methanopyrus kandleri and Methanothermus sociabilis, a diffuse incorporation of cell wall material over the cell length was observed. Cell surface appendages like fimbriae/pili, fibers, or flagella were detectable by fluorescence staining only in a very few cases although their presence was proven by electron microscopy. Our data in addition prove that Alexa Fluor dyes can be used for in situ analyses at temperatures up to 100°C.