Comparison of methods to detect biosurfactant production by diverse microorganisms

Three methods to detect biosurfactant production, drop collapse, oil spreading, and blood agar lysis, were compared for their ease of use and reliability in relation to the ability of the cultures to reduce surface tension. The three methods were used to test for biosurfactant production in 205 environmental strains with different phylogenetic affiliations. Surface tension of select strains that gave conflicting results with the above three methods was also measured. Sixteen percent of the strains that lysed blood agar tested negative for biosurfactant production with the other two methods and had little reduction in surface tension (values above 60 mN/m). Thirty eight percent of the strains that did not lyse blood agar tested positive for biosurfactant production with the other two methods and had surface tension values as low as 35 mN/m. There was a very strong, negative, linear correlation between the diameter of clear zone obtained with the oil spreading technique and surface tension (rs = -0.959) and a weaker negative correlation between drop collapse method and surface tension (rs = -0.82), suggesting that the oil spreading technique better predicted biosurfactant production than the drop collapse method. The use of the drop collapse method as a primary method to detect biosurfactant producers, followed by the determination of the biosurfactant concentration using the oil spreading technique, constitutes a quick and easy protocol to screen and quantify biosurfactant production. The large number of false negatives and positives obtained with the blood agar lysis method and its poor correlation to surface tension (rs = -0.15) demonstrated that it is not a reliable method to detect biosurfactant production.     

On the suitability of gelatin for plate cultures. II

Summary In continuation of our investigation into the factors which determine the suitability of gelatin for colony formation it was demonstrated that surface tension lowering substances added in small concentrations to plate media prepared with a poor gelatin have a decidedly favourable effect. Especially Tweens were examined. An addition of 0.01% Tween 60 or 80 to the gelatin media was sufficient to bring about growth of typical lobated colonies of bothBacterium coli andFlavobacterium aquatile. In none of the experiments made did the Tweens, in the low concentrations applied, exhibit any toxic effect. No direct method for the determination of the surface tension of gels being available, we resorted to the determination of the wettability of the various gelatin gels. With the aid of this method it was found that the contact angle of water droplets on 10% gelatin gels correlated satisfactory with colony appearance. It seems probable that gelatins of good quality contain some constituent which increases the wettability of the gel. Finally it was shown that addition of Tweens to agar gels produces analogous effects on colony growth.     

Effects of cations and polyamines on the aggregation and fusion of phosphatidylserine membranes

Effects of various metal cations and polyamines on aggregation and fusion of phosphatidylserine vesicles and their associated physicochemical properties (such as surface tension and vesicle electrophoretic mobility) have been studied. It was found that metal polycations and hydrogen ion caused an increase in the surface tension of a phosphatidylserine monolayer, whereas the polyamines and other monovalent cations did not increase the surface tension of the membrane appreciably. All cations used affected the vesicle mobility roughly in the order of the number of their valencies and linearly with respect to the logarithm of their concentrations of ions; vesicle surface charge densities are reduced by adsorption and screening of the counter ions depending on their valencies and concentrations. The degree of aggregation of lipid vesicles parallels somewhat that of the reduction of vesicle electrophoretic mobilities. However, the degree of membrane fusion induced by these cations parallels that of the increase in surface tension of the membranes induced by these cations.     

Detection of microbial proteolytic activity by a cultivation plate assay in which different proteins adsorbed to a hydrophobic surface are used as substrates.

A screening technique for microbial proteases, the thin-layer enzyme assay cultivation technique, was developed. The inner surface of a polystyrene petri dish was coated with protein and then covered with a culture agar medium. The enzymes, produced during growth of the microorganisms, reach the protein-coated surface by diffusion in the agar. Degradation of the protein was visualized by condensation of water vapor on the surface after removal of the agar medium. The wettability of the enzyme-affected protein-coated polystyrene surface was decreased compared with the unaffected protein surface. Enzyme substrates used were fibrinogen, immunoglobulin G, egg albumin, human serum albumin, bovine serum albumin, hemoglobin, mucin, and gelatin. It was possible to use a variety of culture agar media, nonselective as well as selective, in the assay. The technique provides a sensitive, convenient, and inexpensive method for screening various microbial proteases. In addition, the technique can be used for screening proteolytic enzyme activity of specific microbial species in a mixed microbial sample as well as for studies of factors that influence the cultivation conditions for protease production and activity.     

Detection of microbial proteolytic activity by a cultivation plate assay in which different proteins adsorbed to a hydrophobic surface are used as substrates

A screening technique for microbial proteases, the thin-layer enzyme assay cultivation technique, was developed. The inner surface of a polystyrene petri dish was coated with protein and then covered with a culture agar medium. The enzymes, produced during growth of the microorganisms, reach the protein-coated surface by diffusion in the agar. Degradation of the protein was visualized by condensation of water vapor on the surface after removal of the agar medium. The wettability of the enzyme-affected protein-coated polystyrene surface was decreased compared with the unaffected protein surface. Enzyme substrates used were fibrinogen, immunoglobulin G, egg albumin, human serum albumin, bovine serum albumin, hemoglobin, mucin, and gelatin. It was possible to use a variety of culture agar media, nonselective as well as selective, in the assay. The technique provides a sensitive, convenient, and inexpensive method for screening various microbial proteases. In addition, the technique can be used for screening proteolytic enzyme activity of specific microbial species in a mixed microbial sample as well as for studies of factors that influence the cultivation conditions for protease production and activity.     

Microencapsulation: a Strategy for Formulation of Inoculum

A non-toxic phase separation method was developed for microencapsulation of inoculum used in biological control. Aqueous sodium alginate or gelatin and agar was mixed with inocula of various biopesticides and emulsified in a mixture of corn oil, n-hexadecane, and lecithin. Gelatin and agar globules gelled in the emulsion; alginate globules gelled after settling into a lower phase of aqueous CaCl₂. A layer of gelatinous material thus surrounded the inoculum as 'capsules'. Mixing with n-hexadecane reduced the specific gravity and surface tension of the oil, allowing aqueous extraction of the capsules. Successful extraction of alginate capsules depended upon lecithin (>0.17%), n-hexadecane (>30%), and CaCl₂ (>0.01 M) concentrations. Alginate-encapsulated macroconidia of Fusarium avenaceum caused 23±3% leaf area damage to seedlings of marsh reed grass, versus 4±3% for unformulated controls. In green foxtail seedlings, gelatin and agar-encapsulated conidia of Bipolaris sorokiniana caused 21.3 vs. 7.9 lesions per plant for encapsulated versus unformulated conidia. Mortality of Douglas-fir tussock moth larvae caused by a nuclear polyhedrosis virus was delayed when 23 polyhedral inclusion bodies (PIB) were incorporated into alginate capsules, but it proceeded normally for 2.3 PIB/capsule, where efficacy was also higher versus positive controls. Microencapsulation enhances the activity of biological control agents and protects them from adverse conditions.     

Divalent cation-induced interaction of phospholipid vesicle and monolayer membranes

The effects of phospholipid vesicles and divalent cations in the subphase solution on the surface tension of phospholipid monolayer membranes were studied in order to elucidate the nature of the divalent cation-induced vesicle-membrane interaction. The monolayers were formed at the air/water interface. Various concentrations of unilamellar phospholipid (phosphatidylserine, phosphatidylcholine and their mixtures) vesicles and divalent cations (Mg²⁺, Ca²⁺, Mn²⁺, etc.) were introduced into the subphase solution of the monolayers. The changes of surface tension of monolayers were measured by the Wilhelmy plate (Teflon) method with respect to divalent ion concentrations and time.When a monolayer of phosphatidylserine and vesicles of phosphatidylserine/phosphatidylcholine (1 : 1) were used, there were critical concentrations of divalent cations to produce a large reduction in surface tension of the monolayer. These concentrations were 16 mM for Mg²⁺, 7 mM for Sr²⁺, 6 mM for Ca²⁺, 3.5 mM for Ba²⁺ and 1.8 mM for Mn²⁺. On the other hand, for a phosphatidylcholine monolayer and phosphatidylcholine vesicles, there was no change in surface tension of the monolayer up to 25 mM of any divalent ion used. When a phosphatidylserine monolayer and phosphatidylcholine vesicles were used, the order of divalent ions to effect the large reduction of surface tension was Mn²⁺ > Ca²⁺ > Mg²⁺ and their critical concentrations were in between the former two cases. The threshold concentrations also depended upon vesicle concentrations as well as the area/molecule of monolayers. For phosphatidylserine monolayers and phosphatidylserine/phosphatidylcholine (1 : 1) vesicles, above the critical concentrations of Mn²⁺ and Ca²⁺, the surface tension decreased to a value close to the equilibrium pressure of the monolayers within 0.5 h.This decrease in surface tension of the monolayers is interpreted partly as the consequence of fusion of the vesicles with the monolayer membranes. The     

Evaluation of surface tension and ion occupancy effects on gramicidin A channel lifetime.

The surface tension of glycerylmonooleate-hexadecane lipid bilayer membranes and the lifetime of gramicidin A channels were measured at various concentrations of the surrounding solutions. For HCl the surface tension is essentially constant at approximately 5 mN/m up to approximately 1 M, whereas the average lifetime increases approximately 40-fold. At higher concentrations the surface tension decreases markedly. For CsCl the surface tension is constant up to about 1 M then increases with salt level. The average lifetime in this case increases about sixfold. In both cases the lifetime levels off and even decreases at higher salt levels. The increase in lifetime observed with ion activity is therefore qualitatively different from, and not explained by, the established dependence of lifetime on membrane properties (Elliot, J.R., D. Needham, J.P. Dilger, and D.A. Haydon. 1983. Biochim. Biophys. Acta. 735:95-103). We have previously proposed that ion occupancy is a determinant of channel stability, and to test this hypothesis the voltage dependence of channel lifetime was measured in asymmetrical solutions. For the case of a potassium chloride solution on one side of the membrane and a hydrogen chloride solution, on the other, the voltage dependence of the lifetime is asymmetrical. The asymmetry is such that when the electrical field is applied in the direction of the chemical gradient for each of the ions, the channel lifetime approaches, at increasing field strengths, that of a symmetrical solution of the respective ion. The voltage dependence of the surface tension, on the other hand, is negligible for the range of voltages used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative characterization of antagonistic activity of lactobacilli

In this work the method of serial dilutions of lactobacilli in two-layer agar was used. On the agar surface bacterial or fungal cultures were applied at different time intervals. A special quantitative characteristic was introduced. L. plantarum strain 8P-A3 was shown to have the maximum antagonistic activity. In great amounts L. casei and L. reuteri are capable to suppress the growth of bacteria and fungi. All lactobacilli under study produced a pronounced bactericidal effect on Pseudomonas, had different influence on the viability of Escherichia and staphylococci and exhibited fungistatic and fungicidal action only when inoculated at high concentrations.     

New screening test to determine the acceptability of 0.45-micron membrane filters for analysis of water

During routine membrane filter (MF) quality control testing, irregularities such as partial or complete inhibition of microbial growth at grid lines, abnormal spreading of colonies, growth in or along the grid lines, nonwetting areas, poor colony sheen and metallic sheen on the MF surface with mEndo agar, brittleness, decreased recovery, and severe wrinkling were observed with several lots of filters. To study these effects and to develop a more sensitive screening test for MF quality, we compared five different MFs with various types and degrees of defects by using five stock coliform cultures and five different media. Results showed that the Enterobacter aerogenes-tryptic soy agar test system detected more MF defects than any other combination did and was superior to the Escherichia coli-mFC agar American Society for Testing and Materials method for grid line inhibition. Filtered natural samples grown on the same media showed the same effects as were observed with the pure cultures. Poor colony sheen and sheen on the MF surface were best detected with Enterobacter aerogenes on mEndo agar. The use of tryptic soy agar and mEndo agar with this organism permitted the maximum detection of MF irregularities. Of the 142 MF lots tested by this method, 30% were acceptable, 10% were marginally acceptable, and 61% were unacceptable. This method provides a valuable screening test for determining the acceptability of 0.45-microm-pore-size MFs used for coliform and heterotroph analysis and may also be useful in conjunction with other methods.     

New screening test to determine the acceptability of 0.45-micron membrane filters for analysis of water.

During routine membrane filter (MF) quality control testing, irregularities such as partial or complete inhibition of microbial growth at grid lines, abnormal spreading of colonies, growth in or along the grid lines, nonwetting areas, poor colony sheen and metallic sheen on the MF surface with mEndo agar, brittleness, decreased recovery, and severe wrinkling were observed with several lots of filters. To study these effects and to develop a more sensitive screening test for MF quality, we compared five different MFs with various types and degrees of defects by using five stock coliform cultures and five different media. Results showed that the Enterobacter aerogenes-tryptic soy agar test system detected more MF defects than any other combination did and was superior to the Escherichia coli-mFC agar American Society for Testing and Materials method for grid line inhibition. Filtered natural samples grown on the same media showed the same effects as were observed with the pure cultures. Poor colony sheen and sheen on the MF surface were best detected with Enterobacter aerogenes on mEndo agar. The use of tryptic soy agar and mEndo agar with this organism permitted the maximum detection of MF irregularities. Of the 142 MF lots tested by this method, 30% were acceptable, 10% were marginally acceptable, and 61% were unacceptable. This method provides a valuable screening test for determining the acceptability of 0.45-microm-pore-size MFs used for coliform and heterotroph analysis and may also be useful in conjunction with other methods.     

The role of palmitic acid in pulmonary surfactant: enhancement of surface activity and prevention of inhibition by blood proteins

The surface activity of two surfactant preparations, Lipid Extract Surfactant (LES) and Survanta, was examined during adsorption and dynamic compression using a pulsating bubble surfactometer. At low surfactant phospholipid concentrations (1-2.5 mg/ml), Survanta reduces surface tension at minimum bubble radius faster than LES: however, with continued pulsation LES obtains a lower surface tension. Addition of surfactant-associated protein A (SP-A) to LES significantly reduces the time required to reduce surface tension. Survanta is completely unresponsive to the addition of SP-A in that no further reduction of surface tension is observed. Addition of various blood components has been previously shown to inactivate surfactants in vitro. Addition of fibrinogen to Survanta causes an increase in surface tension when measured in the absence of calcium. When assayed in the presence of calcium, inhibition by fibrinogen is not observed possibly due to aggregation of this protein. Albumin and alpha-globulin strongly inhibit Survanta at physiological serum concentrations both in the presence and absence of calcium. The surface activity of Survanta is also inhibited by lysophosphatidylcholine (lyso-PC). The role of palmitic acid in the surface activity of pulmonary surfactant was examined by adding palmitic acid to LES. At low phospholipid concentrations addition of palmitic acid (10% w/w of the surfactant phospholipid) greatly enhances the surface activity of LES. Maximal enhancement of surface activity and adsorption was observed at or above 7.5% added palmitic acid (w/w of surfactant lipid). LES supplemented with palmitic acid is more resistant to inhibition by fibrinogen, albumin, alpha-globulin and lyso-PC than LES alone, however, the counteraction of blood protein inhibition is not as pronounced as that observed with SP-A.     

Effect of different grades of agar-agar on the nature of the test microbe growth inhibition zones in controlling antibiotic activity by means of agar diffusion]

The effect of various agar grades on the size and margin character of the inhibition growth zones in assay of antibiotic activity by the agar diffusion method was studied. It was shown that not all the agar grades could be used in antibiotic activity assay. Depending on the agar type the size of the inhibition growth zones produced by the same antibiotic concentration significantly varied. The variations in the size of the inhibition growth zones depended on the agar ability to bind antibiotics and were mainly defined by the agar purity. The agars with low content of nitrogen admixtures bound the antibiotics to a low extent. The commerical grades of the agars of the South-Sea and Korsakov Plants, the experimental grade of the TINRO agar with additional purification, as well as the agars imported from Argentina and France proved to be most useful for determination of the antibiotic activity by the agar diffusion method.     

Patterns of growth and gliding motility inSimonsiella

Forty-six strains ofSimonsiella—large, Gram-negative, aerobic, multicellular filamentous, gliding bacteria from the oral cavities of cats, dogs, sheep, and humans—were grown under various environmental conditions to elucidate features of gliding motility in the genus. Under standard growth conditions on bovine serum-tryptic soy-yeast extract (BSTSY) agar at 37°C, few strains glided. Nongliding strains displayed edges of microscopic colonies ranging from entire to rhizoid (filamentous outgrowth). Gliding strains displayed motility on agar in individual, often well-separated filaments, forming etched tracks in the agar. In some strains, gliding on agar led to the formation of satellite colonies, suggesting that motility is a possible mechanism for sustaining growth. Gliding was often pronounced in regions of heavy growth bordering on unoccupied agar surfaces, suggesting that motility might be triggered by growth metabolite accumulations, but, also, might require certain levels of fresh nutrients. Motility rates of 4- to 12-h-old cultures of selected strains in BSTSY broth or on BSTSY plus 0.5% agar (measured in sealed slide preparations held at approximately 37°C) ranged from 5 to 23.8 μm/min. Rate variations, obtained for the same as well as different trials, would be expected due to variations in oxygen tension and in metabolite and nutrient concentrations on agar sealed under glass.     

Image analysis method for evaluation of specific and non-specific hand contamination

AIMS: To evaluate a quantifying image analysis method for assessing the degree of hand contamination and efficacy of hand washing procedures. METHODS AND RESULTS: Two types of experimental design were used. In one, different concentrations of pure cultures of Escherichia coli, Listeria innocua and Pseudomonas flourescens were applied to hands. In the other, hands were contaminated by handling various raw foods. Imprints of the contaminated palms were made on 24.5 x 24.5 cm agar plates using appropriate agars. After incubation, digital photographs of the plates were analysed using image analysis. In pure culture studies with selective agars, levels from 1 to 10(6) CFU cm(-2) palm could be monitored. For aerobic, mesophilic organisms from raw chicken, levels from 10(3) to 10(6) CFU cm(-2) palm were correlated linearly to image analysis data. CONCLUSIONS: The image analysis of palm imprints made on agar plates was suitable for assessing the degree of contamination from foods on the palms. Sensitivity and specificity depended on the agar used and the type of contamination encountered. SIGNIFICANCE AND IMPACT OF THE STUDY: Data capture by the image analysis method is simple and can be partly automated. Sampling time is short for the person to be tested, which makes it an attractive method for assessing hand hygiene status in larger field trials.     

Direct quantitative method for evaluating the effect of biocides on Pseudomonas fluorescens in different culture media

A method for quantitative evaluation of the effects of biocides is presented. The method was tested in experiments with Pseudomonas fluorescens grown on various agar nutrient media. The effective concentrations of biocides that decreased the maximum specific rate of the colony biomass growth (mu'm) were called S (suppressing) concentrations, and concentrations that decreased the number of colony-forming units (CFU) were taken as L (sublethal) concentrations. The efficiency of the reported approach was demonstrated in experiments with three biocides tested in four nutrient media. It was found that the biocide sensitivity of Pseudomonas fluorescens varied by a factor of 30, depending on the amount and the type of the nutrient substrate.     

TEMPERATURE-DEPENDENCE OF THE TENSION AT THE SURFACE OF SEA-URCHIN EGGS*

Tension at the surface of unfertilized sea-urchin eggs was measured at various temperatures by compression method. It was confirmed that the tension definitely decreases with a rise in temperature. This indicates that (1) the tension at the surface as determined with the compression method is due solely to the plasma membrane, and (2) the membrane is fluid in nature.     

An Agar Diffusion Method for the Determination Of Antibodies Against Staphylococcus Aureus Deoxyribonuclease

On the addition of small concentrations of deoxyribonuclease, produced by Staphylococcus aureus, to Toluidine Blue DNA agar, a medium is produced on which antibodies against S. aureus deoxyribonuclease may be detected. When samples of milk, or blood serum, containing antibodies against S. aureus are applied into wells in the agar, the deoxyribonuclease activity is inhibited by the antibodies diffusing into the agar. As a result of this inhibition, blue zones are produced around the wells in the otherwise bluish-red agar. The diameters of the zones correspond to the concentrations of antibodies, and the method may consequently be used for qualitative and quantitative examinations of antibodies against S. aureus deoxyribonuclease in milk and serum. The procedure and certain limitations of the method are described.     

THE ADAPTATION OF FUNGI TO FUNGICIDES: ADAPTATION TO THIRAM, ZIRAM, FERBAM, NABAM AND ZINEB

The concentrations of thiram preventing germination of spores of Botrytis cinerea in drops of a 1% solution of sucrose, and on the surface of a sucrose-nitrate agar have been determined. Thiram had much less effect on germination in the agar medium, even when a purified agar was used. There was no growth on sucrose-nitrate agar if the concentration of thiram exceeded 31 p.p.m. Attempts to obtain strains able to grow at higher concentrations were unsuccessful.
Similar results were obtained with ziram, nabam and zineb.
Ferbam also was more effective in preventing spore germination in spore drops than on agar media; this effect was obtained with ordinary and with purified agar.
On a sucrose-nitrate agar generally there was no growth if the concentration of ferbam exceeded 125 p.p.m., but in one of forty-eight plates containing 250 p.p.m. ferbam, five slowly growing colonies were produced, and from these colonies arose mycelium which grew and sporulated rapidly on 500 p.p.m. ferbam agar. Agar disk inocula were transferred from these cultures to agar containing higher concentrations of ferbam and in this way, and by repeating the process, a strain was obtained which grew slowly but continuously, and sporulated on agar containing 5000 p.p.m. ferbam. However, the poor solubility of this fungicide made it difficult to assess quantitatively the degree of adaptation.
A proportion of the spores from this strain germinated in drops containing about twice the concentration of ferbam which prevented germination of parent spores.
The resistance of the mycelium of the resistant strain was not lost after repeated subculture on fungicide-free agar. The resistant strain was as susceptible as the parent strain to thiram, ziram, nabam and zineb.
Attempts to obtain strains of Venturia inaequalis resistant to thiram, ferbam, ziram and zineb were unsuccessful.