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.     

RNA-binding activities of the different domains of a spinach chloroplast ribonucleoprotein.

An RNA-binding protein of 28 kD (28RNP) has been previously isolated from spinach chloroplasts and was found to be required for 3' end processing of chloroplast mRNAs. The amino acid sequence of 28RNP revealed two approximately 80 amino-acid RNA-binding domains, as well as an acidic and glycine-rich amino terminal domain. Each domain by itself, as well as in combination with other domains, was expressed in bacterial cells and the polypeptides were purified to homogeneity. We have investigated the RNA-binding properties of the different structural domains using UV-crosslinking, saturation binding and competition between the different domains on RNA-binding. It was found that the acidic domain does not bind RNA, but that each of the RNA-binding domains, expressed either individually or together, do bind RNA, although with differing affinities. When either the first or second RNA-binding domain was coupled to the acidic domain, the affinity for RNA was greatly reduced. However, the acidic domain has a positive effect on the binding of the full-length protein to RNA, because the mature protein binds RNA with a better affinity than the truncated protein which lacks the acidic domain. In addition, it was found that a stretch of two or three G residues is enough to mediate binding of the 28RNP, whereas four U residues were insufficient. The implications of the RNA-binding properties of 28RNP to its possible function in the processing of chloroplast RNA is discussed.     

Molecular dynamics study of biodegradation of azo dyes via their interactions with AzrC azoreductase

Azo dyes are one of the most important class of dyes, which have been widely used in industries. Because of the environmental pollution of azo dyes, many studies have been performed to study their biodegradation using bacterial systems. In present work, the AzrC of mesophilic gram-positive Bacillus sp. B29 has been considered to study its interaction with five common azo dyes (orange G, acid red 88, Sudan I, orange I, and methyl red). The molecular dynamics simulations have been employed to study the interaction between AzrC and azo dyes. The trajectory was confirmed using root mean square deviation and the root mean square fluctuation analyses. Then, the hydrogen bond and alanine scanning analyses were performed to reveal active site residues. Phe105 (A), Phe125 (B), Phe172 (B), and Pro132 (B) have been found as the most important hydrophobic residues whereas Asn104 (A), Tyr127 (B), and Asn187 (A) have key role in making hydrogen bond. The results of molecular mechanics Poisson–Boltzmann surface area and molecular mechanics generalized Born surface area calculations proved that the hydrophobic azo dyes like Acid red 88 binds more tightly to the AzrC protein. The calculated data suggested MR A 121 (B) I as a potential candidate for improving the AzrC–MR interactions.     

The antimicrobial properties of magnesium monoperoxyphthalate hexahydrate

The antimicrobial properties of magnesium monoperoxyphthalate hexahydrate (MMPP), which is a stable, water-soluble solid peroxyacid, have been investigated. A 2% (w/w) solution of MMPP rapidly kills yeasts and vegetative bacteria and slowly inactivates bacterial endospores at 22°C. Biocidal activity, which is greater under mildly acidic conditions, is retained in the presence of organic contamination or hard water. Sporicidal action is greatly increased by a moderate rise in temperature or by using MMPP in combination with propan-2-ol. Such mixtures appear to be suitable for use as liquid chemical sterilants.     

The antimicrobial properties of magnesium monoperoxyphthalate hexahydrate

The antimicrobial properties of magnesium monoperoxyphthalate hexahydrate (MMPP), which is a stable, water-soluble solid peroxyacid, have been investigated. A 2% (w/w) solution of MMPP rapidly kills yeasts and vegetative bacteria and slowly inactivates bacterial endospores at 22 degrees C. Biocidal activity, which is greater under mildly acidic conditions, is retained in the presence of organic contamination or hard water. Sporicidal action is greatly increased by a moderate rise in temperature or by using MMPP in combination with propan-2-ol. Such mixtures appear to be suitable for use as liquid chemical sterilants.     

Sulfidogenesis in low pH (3.8-4.2) media by a mixed population of acidophilic bacteria

A defined mixed bacterial culture was established which catalyzed dissimilatory sulfate reduction, using glycerol as electron donor, at pH 3.8-4.2. The bacterial consortium comprised a endospore-forming sulfate reducing bacterium (isolate M1) that had been isolated from acidic sediment in a geothermal area of Montserrat (West Indies) and which had 94% sequence identity (of its 16S rRNA gene) to the Gram-positive neutrophile Desulfosporosinus orientis, and a Gram-negative (non sulfate-reducing) acidophile (isolate PFBC) that shared 99% gene identity with Acidocella aromatica. Whilst M1 was an obligate anaerobe, isolate PFBC, as other Acidocella spp., only grew in pure culture in aerobic media. Analysis of microbial communities, using a combination of total bacterial counts and fluorescent in situ hybridization, confirmed that concurrent growth of both bacteria occurred during sulfidogenesis under strictly anoxic conditions in a pH-controlled fermenter. In pure culture, M1 oxidized glycerol incompletely, producing stoichiometric amounts of acetic acid. In mixed culture with PFBC, however, acetic acid was present only in small concentrations and its occurrence was transient. Since M1 did not oxidize acetic acid, it was inferred that this metabolite was catabolized by Acidocella PFBC which, unlike glycerol, was shown to support the growth of this acidophile under aerobic conditions. In fermenter cultures maintained at pH 3.8-4.2, sulfidogenesis resulted in the removal of soluble zinc (as solid phase ZnS) whilst ferrous iron remained in solution. Potential syntrophic interactions, involving hydrogen transfer between M1 and PFBC, are discussed, as is the potential of sulfidogenesis in acidic liquors for the selective recovery of heavy metals from wastewaters.     

Mechanism of connective tissue techniques I. The effect of dye concentration and staining time on anionic dye procedures

Summary Anionic dye connective tissue procedures were performed by staining for 5 min and 24 h with (a) 0.00018m and 0.0018m solutions of 28 dyes, and 0.018m solutions of 21 dyes in saturated picric acid (SPA), and (b) 0.0018m and 0.018m solutions of 20 dyes in 1% (w/v) phosphomolybdic acid (PMA). The staining obtained with dyes in SPA was classified as selective (no cytoplasmic staining), moderately selective (traces of cytoplasmic staining) and non-selective (all other staining patterns). The staining of collagen and cytoplasm with dyes in PMA was separately classified on a scale of 1–5 (1 = no staining, 5 = maximum staining). The selectivity of the staining obtained with SPA with solutions of dyes at concentrations of 0.00018m and 0.0018m, and both staining times, was correlated (p < 0.001) with an empirical sulphonic acid constant (SAC) defined as the (number of dye sulphonic acid groups/dye molecular weight) × 10³. Correlation with molecular weight was poor and was significant only when staining was performed with 0.00018m dye solutions for 24 h. The dyes were divisible into three groups: group 1 (selectivity independent, or almost independent of staining time), group 2 (selective to moderately selective when staining was performed for 5 min), and group 3 (non-selective). The SAC of the group 1 dyes differed significantly from those of the group 2 and 3 dyes. Selectivity was essentially lost at dye concentrations of 0.018m. The staining with acidic dyes (no amines or substituted amines) in PMA differed significantly (p < 0.001) from that obtained with amphoteric dyes (containing basic substituents). In general, acidic dyes stained cytoplasm. Amphoteric dyes with the exception of indigocarmine stained collagen. However, most of these dyes also stained cytoplasm. In contrast to the results obtained with dyes in SPA, selectivity correlated strongly with molecular weight and only poorly with the SAC. Staining time and dye concentration affected selectivity only when the acidic dyes were used for 5 min at concentrations of 0.0018m and 0.018m. The data obtained do not permit a clear distinction between the rate control and chemical affinity models for the mechanism of staining with anionic dyes. However, it seems possible that different groups of dyes stain by different mechanisms. Part of this work was performed by M.I., S.N., M.J. and L.M. in partial fulfilment of the requirements for the completion of Pathology 438. A partial account of this work was presented at the annual convention of the British Columbia Society of Medical Technology, Victoria, British Columbia, October 1991.     

Isolation and characterization of four major neutral glycosphingolipids from the liver of the English sole (Parophrys vetulus). Presence of a novel branched lacto-ganglio-iso-globo hybrid structure

We have previously reported on carbohydrate structures of the major acidic glycosphingolipids from the liver of the English sole, Parophrys vetulus (Ostrander, G. K., Levery, S. B., Hakomori, S., and Holmes, E. H. (1988) J. Biol. Chem. 263, 3103-3110). We have now isolated four major neutral glycosphingolipids from English sole liver. They have been characterized by 1H nuclear magnetic resonance spectroscopy, methylation analysis, and by fast atom bombardment-mass spectrometry. In addition to neutral glycosphingolipids with known structures (CMH, lactosylceramide, and Gg3), a major polar neutral glycosphingolipid was isolated and characterized as having the following novel structure: (formula; see text) The compound represents a novel hybrid of neolacto-, ganglio-, and iso-globo-series glycosphingolipid structures, a combination not previously encountered. Furthermore, the linkage Fuc alpha 1----3GalNac has not been previously reported for a glycosphingolipid. The relationship of these structural elements to known glycoconjugates is discussed.     

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.     

Bacterial decolorization and degradation of azo dyes

Azo compounds constitute the largest and the most diverse group of synthetic dyes and are widely used in a number of industries such as textile, food, cosmetics and paper printing. They are generally recalcitrant to biodegradation due to their xenobiotic nature. However microorganisms, being highly versatile, have developed enzyme systems for the decolorization and mineralization of azo dyes under certain environmental conditions. Several genera of Basidomycetes have been shown to mineralize azo dyes. Reductive cleavage of azo bond, leading to the formation of aromatic amines, is the initial reaction during the bacterial metabolism of azo dyes. Anaerobic/anoxic azo dye decolorization by several mixed and pure bacterial cultures have been reported. Under these conditions, this reaction is non-specific with respect to organisms as well as dyes. Various mechanisms, which include enzymatic as well as low molecular weight redox mediators, have been proposed for this non-specific reductive cleavage. Only few aerobic bacterial strains that can utilize azo dyes as growth substrates have been isolated. These organisms generally have a narrow substrate range. Degradation of aromatic amines depends on their chemical structure and the conditions. It is now known that simple aromatic amines can be mineralized under methanogenic conditions. Sulfonated aromatic amines, on the other hand, are resistant and require specialized aerobic microbial consortia for their mineralization. This review is focused on the bacterial decolorization of azo dyes and mineralization of aromatic amines, as well as the application of these processes for the treatment of azo-dye-containing wastewaters.     

Novel fluorescent ceramide derivatives for probing ceramidase substrate specificity

Ceramidases are key regulators of cell fate. The biochemistry of different ceramidases and of their substrate ceramide appears to be complex, mainly due to specific biophysical characteristics at the water-membrane interface. In the present study, we describe the design and synthesis of a set of fluorescently labeled ceramides as substrates for acid and neutral ceramidases. For the first time we have replaced the commonly used polar NBD-dye with the lipophilic Nile Red (NR) dye. Analysis of kinetic data reveal that although both the dyes do not have any noticeable preference for the substitution at acyl or sphingosine (Sph) part in ceramide towards hydrolysis by acid ceramidase, the ceramides with acyl-substituted NBD and Sph-substituted NR dyes have been found to be a better substrate for neutral ceramidase.     

In Search of Engineered Prokaryotic Chlorophyllases: A Bioinformatics Approach

Chlorophyllase (Chlase) is considered as the first and most important enzyme in chlorophyll degradation pathway. Although there is abundant information regarding plant Chlases and their biological functions, comparatively little is known about their prokaryotic counterparts. In the present study, we employed several bioinformatics tools to assess the phylogenetic relationships in bacterial and cyanobacterial Chlases as well as predicting their molecular and physicochemical properties. The phylogenetic tree analysis classified the bacterial and cyanobacterial chlorophyllases into three distinct clades. All bacterial and cyanobacterial chlorophyllases possessed at least one alpha/ beta hydrolase family domain (pfam12695). Cyanobacterial chlorophyllases pI analysis indicated that they generally have acidic pH, while the pI of bacterial chlorophyllases ranged from acidic (4.58) to highly basic (10.78). Cyanobacterial chlorophyllases generally contained 1 disulfide bond, while bacterial chlorophyllases averagely contained 3 disulfide bonds. Interestingly, while cyanobacterial chlorophyllases contained one or two N-glycosylation sites, bacterial chlorophyllases contained higher numbers of N-glycosylation sites (6 and 7). The findings of the present study would be useful in paving the road for sophisticated engineering of prokaryotic chlorophyllases for biotechnological applications. It was also exhibited that catalytic triad (serine, glutamate or aspartate and histidine) is a critical factor for chlorophyllase activity.     

The bactericidal, fungicidal and sporicidal properties of hydrogen peroxide and peracetic acid

The antimicrobial properties of aqueous solutions of peracetic acid and hydrogen peroxide have been compared. Peracetic acid exhibited excellent antimicrobial properties, especially under acidic conditions. Reductions by a factor of 10⁶ in the numbers of vegetative bacteria are obtained within 1 min at 25°C using a solution containing 1.3 mmol/l of peracetic acid. Rapid activity against bacterial spores and yeasts also occurs. Hydrogen peroxide is more effective as a sporicide than as a bactericide, with sporicidal action being obtained using a solution containing 0.88 mol/l. Bactericidal action is poor but hydrogen peroxide was bacteriostatic at concentrations above 0.15 mmol/l.     

A Fluorescent Gram Stain for Flow Cytometry and Epifluorescence Microscopy

The fluorescent nucleic acid binding dyes hexidium iodide (HI) and SYTO 13 were used in combination as a Gram stain for unfixed organisms in suspension. HI penetrated gram-positive but not gram-negative organisms, whereas SYTO 13 penetrated both. When the dyes were used together, gram-negative organisms were rendered green fluorescent by SYTO 13; conversely, gram-positive organisms were rendered red-orange fluorescent by HI, which simultaneously quenched SYTO 13 green fluorescence. The technique correctly predicted the Gram status of 45 strains of clinically relevant organisms, including several known to be gram variable. In addition, representative strains of gram-positive anaerobic organisms, normally decolorized during the traditional Gram stain procedure, were classified correctly by this method.Gram’s staining method is considered fundamental in bacterial taxonomy. The outcome of the Gram reaction reflects major differences in the chemical composition and ultrastructure of bacterial cell walls. The Gram stain involves staining a heat-fixed smear of cells with a rosaniline dye such as crystal or methyl violet in the presence of iodine, with subsequent exposure to alcohol or acetone. Organisms that are decolorized by the alcohol or acetone are designated gram negative.Alternative Gram staining techniques have recently been proposed. Sizemore et al. (19) reported on the use of fluorescently labeled wheat germ agglutinin. This lectin binds specifically to N-acetylglucosamine in the peptidoglycan layer of gram-positive bacteria, whereas gram-negative organisms contain an outer membrane that prevents lectin binding. Although simpler and faster than the traditional Gram stain, this method requires heat fixation of organisms.Other Gram stain techniques suitable for live bacteria in suspension have been described. Allman et al. (1) demonstrated that rhodamine 123 (a lipophilic cationic dye) rendered gram-positive bacteria fluorescent, but its uptake by gram-negative organisms was poor. This reduced uptake by gram-negative bacteria was attributed to their outer membranes. The outer membrane can be made more permeable to lipophilic cations by exposure to the chelator EDTA (4). Shapiro (18) took advantage of this fact to form the basis of another Gram stain, one which involved comparing the uptake of a carbocyanine dye before and after permeabilizing organisms with EDTA. All of these methods, however, rely on one-color fluorescence, making analysis of mixed bacterial populations difficult.An alternative to the use of stains is the potassium hydroxide (KOH) test. The method categorizes organisms on the basis of differences in KOH solubility. After exposure to KOH, gram-negative bacteria are more easily disrupted than gram-positive organisms. This technique has been used to classify both aerobic and facultatively anaerobic bacteria, including gram-variable organisms (8). In a study by Halebian et al. (9), however, this technique incorrectly classified several anaerobic strains, giving rise to the recommendation that the method should only be used in conjunction with the traditional Gram stain.In this study we demonstrate a Gram staining technique for unfixed organisms in suspension, by using clinically relevant bacterial strains and organisms notorious for their gram variability. The method uses two fluorescent nucleic acid binding dyes, hexidium iodide (HI) and SYTO 13. Sales literature (11) published by the manufacturers of HI (Molecular Probes, Inc., Eugene, Oreg.), which displays a red fluorescence, suggests that the dye selectively stains gram-positive bacteria. SYTO 13 is one of a group of cell-permeating nucleic acid stains and fluoresces green (11). These dyes have been found to stain DNA and RNA in live or dead eukaryotic cells (16). Both dyes are excited at 490 nm, permitting their use in fluorescence instruments equipped with the most commonly available light sources. We reasoned that a combination of these two dyes applied to mixed bacterial populations would result in all bacteria being labeled, with differential labeling of gram-positive bacteria (HI and SYTO 13) and gram-negative bacteria (SYTO 13 only). The different fluorescence emission wavelengths of the two dyes would ensure differentiation of gram-positive from gram-negative bacteria by either epifluorescence microscopy or flow cytometry when equipped with the appropriate excitation and emission filters. While a commercial Gram stain kit produced by Molecular Probes includes HI and an alternative SYTO dye, SYTO 9, we are unaware of any peer-reviewed publications regarding either its use or its effectiveness with traditionally gram-variable organisms.     

Fluorescent DNA probes: Study of mechanisms of changes in spectral properties and features of practical application

Spectral data and nucleic acid complex formation properties for more than 30 both newly synthesized and widely used fluorescent nucleotide-specific compounds of various classes have been analyzed. These include phenylbenzene, bisbenzimidazole, psoralen, angelicin, tetrahydrocarbazole, oxophenoxazine, and others. The main rules of a generalized model adequately explaining changes in fluorescent properties of synthetic, low molecular weight nucleotide-specific dyes depending on their chemical structure, mode of interaction with substrate, properties of assay medium, etc. are proposed. Fluorescent nucleotide-specific dyes have been originally used in newly developed methods for: express evaluation of "generalized microbial dissemination" of liquid media; evaluation of possible genotoxic effects of various foodstuffs, pharmaceutical drugs, hazardous environmental factors (including their combined effects on living organisms), etc.     

A gene required for the regulation of photosynthetic light harvesting in the cyanobacterium Synechocystis 6803

A gene required for the short-term regulation of photosynthetic light harvesting (the state transition) has been identified in the cyanobacterium Synechocystis sp. PCC6803. The open reading frame is designated sll1926 in the complete Synechocystis gene sequence. The deduced amino acid sequence has no homologues in current sequence databases and no recognizable sequence motifs. It encodes a putative integral membrane protein of 16 kDa, which we have designated RpaC (regulator of phycobilisome association C). Fluorescence measurements of an insertional inactivation mutant of rpaC (Deltasll1926) show that it is specifically unable to perform state transitions. Deltasll1926 has approximately wild-type levels of PS1, PS2 and phycobilisomes. Measurements of oxygen evolution and uptake show Deltasll1926 to have no deficiency in electron transport rates. In vitro [gamma-32P]-ATP labelling experiments suggest that RpaC is not the 15 kDa membrane phosphoprotein previously implicated in state transitions. Deltasll1926 grows more slowly than the wild type only at very low light intensities.     

Monitoring Changes in Membrane Polarity,Membrane Integrity,and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC₄(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca²⁺, K⁺, and H⁺, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.     

Sulfidogenesis in Low pH (3.8–4.2) Media by a Mixed Population of Acidophilic Bacteria

A defined mixed bacterial culture was established which catalyzed dissimilatory sulfate reduction, using glycerol as electron donor, at pH 3.8–4.2. The bacterial consortium comprised a endospore-forming sulfate reducing bacterium (isolate M1) that had been isolated from acidic sediment in a geothermal area of Montserrat (West Indies) and which had 94% sequence identity (of its 16S rRNA gene) to the Gram-positive neutrophile Desulfosporosinus orientis, and a Gram-negative (non sulfate-reducing) acidophile (isolate PFBC) that shared 99% gene identity with Acidocella aromatica. Whilst M1 was an obligate anaerobe, isolate PFBC, as other Acidocella spp., only grew in pure culture in aerobic media. Analysis of microbial communities, using a combination of total bacterial counts and fluorescent in situ hybridization, confirmed that concurrent growth of both bacteria occurred during sulfidogenesis under strictly anoxic conditions in a pH-controlled fermenter. In pure culture, M1 oxidized glycerol incompletely, producing stoichiometric amounts of acetic acid. In mixed culture with PFBC, however, acetic acid was present only in small concentrations and its occurrence was transient. Since M1 did not oxidize acetic acid, it was inferred that this metabolite was catabolized by Acidocella PFBC which, unlike glycerol, was shown to support the growth of this acidophile under aerobic conditions. In fermenter cultures maintained at pH 3.8–4.2, sulfidogenesis resulted in the removal of soluble zinc (as solid phase ZnS) whilst ferrous iron remained in solution. Potential syntrophic interactions, involving hydrogen transfer between M1 and PFBC, are discussed, as is the potential of sulfidogenesis in acidic liquors for the selective recovery of heavy metals from wastewaters.     

Biological control of crown gall, Agrobacterium tumefaciens

Biological control of crown gall caused by Agrobacteriurn turnefaciens (Smith & Townsend) Conn, pioneered by Dr A. Kerr in South Australia, is effected through the establishment of a high population of the related non-pathogen A. radiobacter (Beijerinck & van Delden) Conn, strain 84 in the rhizosphere of susceptible plants. Strain 84 produces a bacteriocin to which many strains of the pathogen in Australasia, North America and Britain are sensitive. The disease is present in Britain on a variety of hosts including cherry. At East Malling cherry leaf scars, invaluable as an avenue of infection for bacterial canker infectivity titrations, have been used successfully in crown gall studies. Live cells of strain 84, but neither an avirulent strain of the pathogen nor a soil bacterium highly antagonistic to A. tumefaciens in vitro, inhibited gall formation in cherry leaf scars. Heat-killed cells had no effect. In a field experiment at East Malling hardwood cuttings of the new rootstock Colt have been dipped in strain 84 and total inhibition of crown gall is expected to ensue. The results of other experiments where the disease is already established on cherry-rootstock layer-beds and in blackberry plantations are less predictable. In time we hope to solve this problem. Only time will show whether this method of biological control is long lasting or will eventually break down.     

A Brief History of the Biological Stain Commission: its Founders,its Mission and the First 75 Years

The need for batch-to-batch consistency in available dyes and stains used for biological purposes posed a considerable problem for United States scientists following World War I. Prior to that time, most of the acceptable stains in this country were of German origin. In an attempt to standardize the performance of biological stains and dyes, the Society of American Bacteriologists in 1922 appointed Dr. Harold Conn to form the Committee on the Standardization of Biological Stains. To assist him, Dr. Conn recruited scientists from several major professional scientific societies. Mr. Holland Will, a Rochester, NY, vendor of stains, was also instrumental in the Committee's success. This article traces the origin, mission and accomplishments of the product of that Committee, the Biological Stain Commission, through the past 75 years, and focuses on some of the major events that influenced and shaped its development.