Showing papers on "Agar plate published in 1993"
TL;DR: It was concluded that the cells in these 15 dilution cultures had adapted to growth under laboratory conditions after several months of nutrient deprivation prior to isolation and were potentially a very important part of picoplankton biomass in the areas investigated.
Abstract: Marine bacteria in Resurrection Bay near Seward, Alaska, and in the central North Sea off the Dutch coast were cultured in filtered autoclaved seawater following dilution to extinction. The populations present before dilution varied from 0.11 × 109 to 1.07 × 109 cells per liter. The mean cell volume varied between 0.042 and 0.074 μm3, and the mean apparent DNA content of the cells ranged from 2.5 to 4.7 fg of DNA per cell. All three parameters were determined by high-resolution flow cytometry. All 37 strains that were obtained from very high dilutions of Resurrection Bay and North Sea samples represented facultatively oligotrophic bacteria. However, 15 of these isolates were eventually obtained from dilution cultures that could initially be cultured only on very low-nutrient media and that could initially not form visible colonies on any of the agar media tested, indicating that these cultures contained obligately oligotrophic bacteria. It was concluded that the cells in these 15 dilution cultures had adapted to growth under laboratory conditions after several months of nutrient deprivation prior to isolation. From the North Sea experiment, it was concluded that the contribution of facultative oligotrophs and eutrophs to the total population was less than 1% and that while more than half of the population behaved as obligately oligotrophic bacteria upon first cultivation in the dilution culture media, around 50% could not be cultured at all. During one of the Resurrection Bay experiments, 53% of the dilution cultures obtained from samples diluted more than 2.5 × 105 times consisted of such obligate oligotrophs. These cultures invariably harbored a small rod-shaped bacterium with a mean cell volume of 0.05 to 0.06 μm3 and an apparent DNA content of 1 to 1.5 fg per cell. This cell type had the dimensions of ultramicrobacteria. Isolates of these ultramicrobacterial cultures that were eventually obtained on relatively high-nutrient agar plates were, with respect to cell volume and apparent DNA content, identical to the cells in the initially obligately oligotrophic bacterial dilution culture. Determination of kinetic parameters from one of these small rod-shaped strains revealed a high specific affinity for the uptake of mixed amino acids (a°A, 1,860 liters/g of cells per h), but not for glucose or alanine as the sole source of carbon and energy (a°A, ± 200 liters/g of cells per h). The ultramicrobial strains obtained are potentially a very important part of picoplankton biomass in the areas investigated.
305 citations
TL;DR: The results showed that EO (oregano, thyme and clove) were most effective as antimicrobial agents when the bacterial load was low and minimum inhibitory concentrations were found to vary as a function of the emulsifying agent used.
Abstract: The influence of the number of bacterial cells inoculated and the emulsifying agent used to disperse essential oils (EO) into the culture media on the measurement of the antibacterial activity of EO in an agar medium was determined. The results showed that EO (oregano, thyme and clove) were most effective as antimicrobial agents when the bacterial load was low. Minimum inhibitory concentrations (MICs) were found to vary as a function of the emulsifying agent used. However, MICs obtained by dispersing EOs into 0.2% agar solution without the use of solvents and detergents were greatly reduced compared to when they were used. This demonstrates the fact that solvents and detergents often used in antimicrobial studies significantly decrease the antibacterial activity of EO.
220 citations
TL;DR: This article corrects the article on p. 3534 in vol.
Abstract: A new membrane filter agar medium (MI agar) containing a chromogen, indoxyl-beta-D-glucuronide, and a fluorogen, 4-methylumbelliferyl-beta-D-galactopyranoside, was developed to simultaneously detect and enumerate Escherichia coli and total coliforms (TC) in water samples on the basis of their enzyme activities. TC produced beta-galactosidase, which cleaved 4-methylumbelliferyl-beta-D-galactopyranoside to form 4-methylumbelliferone, a compound that fluoresced under longwave UV light (366 nm), while E. coli produced beta-glucuronidase, which cleaved indoxyl-beta-D-glucuronide to form a blue color. The new medium TC and E. coli recoveries were compared with those of mEndo agar and two E. coli media, mTEC agar and nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide, using natural water samples and spiked drinking water samples. On average, the new medium recovered 1.8 times as many TC as mEndo agar, with greatly reduced background counts (< or = 7%). These differences were statistically significant (significance level, 0.05). Although the overall analysis revealed no statistically significant difference between the E. coli recoveries on MI agar and mTEC agar, the new medium recovered more E. coli in 16 of 23 samples (69.6%). Both MI agar and mTEC agar recovered significantly more E. coli than nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide. Specificities for E. coli, TC, and noncoliforms on MI agar were 95.7% (66 of 69 samples), 93.1% (161 of 173 samples), and 93.8% (61 of 65 samples), respectively. The E. coli false-positive and false-negative rates were both 4.3%. This selective and specific medium, which employs familiar membrane filter technology [corrected] to analyze several types of water samples, is less expensive than the liquid chromogen and fluorogen media and may be useful for compliance monitoring of drinking water.
178 citations
TL;DR: Compared for cost and efficiency in recovering Strongyloides stercoralis larvae in a hospital setting in Honduras, the modified Baermann technique was 3.6 times more efficient than the direct smear, and the agar plate culture increased the modifiedBaermann efficiency by 0.8 times.
Abstract: The direct smear, a modified Baermann technique, and the agar plate culture method were compared for cost and efficiency in recovering Strongyloides stercoralis larvae in a hospital setting in Honduras. Of 427 stool samples, 9 were positive by the direct smear and 33 additional ones were detected by the Baermann technique; the agar plate culture method disclosed 28 more cases for a total of 70 S. stercoralis infections. The modified Baermann method was 3.6 times more efficient than the direct smear, and the agar plate culture increased the modified Baermann efficiency by 0.8 times. Cost for materials alone was cheapest for the direct smear; it increased 4 times for the modified Baermann method and 15 times for the agar plate culture method. This last technique required a better equipped laboratory, more time to perform, and the best trained, most skillful laboratory personnel. In field studies, prevalence of infection should determine the cost-effectiveness of the agar plate culture method; in a clinical setting, when S. stercoralis infection must be ruled out, the agar plate culture method should be made available to the medical community.
113 citations
TL;DR: The potential use of yeasts to control grain-storage molds was evaluated by coculturing the yeast Pichia anomala with Penicillium roqueforti and Aspergillus candidus on agar plates.
Abstract: The potential use of yeasts to control grain-storage molds was evaluated by coculturing the yeast Pichia anomala with Penicillium roqueforti and Aspergillus candidus on agar plates, using different temperatures, water activities (aw), and nutrient concentrations. Addition of 10 ppm cycloheximide to malt-extract agar inhibited Pichia anomala completely without affecting mold growth, making it possible to quantify the inhibition as a reduction in colony-forming units (cfu). For A. candidus, numbers of cfu and hyphal lengths were reduced at an initial yeast concentration of 104 cells/plate and reduced below detection limit at 108 cells/plate. A clear reduction in growth of Penicillium roqueforti was only observed at 108 yeast cells/plate. The antagonistic effect was generally more pronounced at low (6, 15 °C) and high (30, 37 °C) temperatures than at ambient ones. Pichia anomala inhibited growth of both molds more strongly in a substrate-rich medium than in a medium with a low substrate content. In water aga...
76 citations
TL;DR: There is need for new and improved media for selectively isolating various groups, genera, species and strains of yeasts capable of growing only under specific environmental conditions in specific types of foods and beverages.
62 citations
TL;DR: A wide range of moulds representing several genera and species, was recorded in this study from 5 seed samples of each almond, cashew nut, chestnut, hazelnut, pistachio nut and walnut collected from different markets in Ar' Ar, Saudi Arabia.
Abstract: A wide range of moulds representing several genera and species, was recorded in this study from 5 seed samples of each almond, cashew nut, chestnut, hazelnut, pistachio nut and walnut collected from different markets in Ar' Ar, Saudi Arabia. The total counts of fungi were widely fluctuated between 1960–7704 and 1948–7434 colonies/g dry seeds on glucose-Czapek's and glycerol agar media at 28°C, respectively, and represented twenty genera, 53 species and 2 varieties of fungi. The prevalent fungi on the 2 agar media wereAspergillus flavus, A. niger andPenicillium chrysogenum. On glucose-Czapek's agar,Rhizopus stolonifer andAspergillus flavus var.columnaris were isolated from all 6 kinds of nut,A. parasiticus from 5 kinds andA. fumigatus from 4 kinds with high frequencies.Eurotium species were completely absent on glucose-Czapek's agar but they were isolated in high frequency from all kinds of nut on glycerol agar medium. The different nut samples were analyzed by thin layer chromatography for the presence of aflatoxins B1, B2, G1 & G2, citrinin, ochratoxins, patulin, sterigmatocystin, diacetoxyscirpenol, T-2 toxin and zearalenone. Aflatoxins B1 & G1 were detected in 3 out of the 5 samples tested of chestnut at concentrations ranging between 20 to 60 µg/kg. All other samples of almond, cashew nut, hazelnut, pistachio nut, and walnut that were analyzed were mycotoxin free.
60 citations
TL;DR: Results indicate that some cells exposed to BAC were not detected by the plate count procedure but were still viable, and increased resistance of listeriae grown in diluted TSB was associated with dilution of the salt/buffer components of the medium.
56 citations
Journal Article•
TL;DR: Of all the compounds tested, Gluma cleanser and Gluma etchant showed the strongest growth inhibition for all bacterial strains, and the antibacterial effect of XR-Bond on S sanguis and A viscosus was not affected by light curing.
Abstract: The susceptibility of five bacterial species to seven dentinal bonding agents was examined in vitro. Agar diffusion tests using filterpaper disks containing 10 microL each of conditioner, primer, or resin were performed on blood agar and mitis salivarius bacitracin agar. Chlorhexidine (0.2%) was used as a positive control. After incubation, zones of inhibited bacterial growth were measured. Of all the compounds tested, Gluma cleanser and Gluma etchant showed the strongest growth inhibition for all bacterial strains. No antibacterial effect was noted for Prisma Universal Bond 2 and Superlux Universal Bond 2 systems. The primers of Gluma, Denthesive, and Scotchbond 2 displayed antibacterial activity that, in some cases, was comparable to that of 0.2% chlorhexidine. Zones of inhibition were seen for the resin materials of Scotchbond 2 and Tripton with Streptococcus mutans, Streptococcus sanguis and Actinomyces viscosus. No inhibition was seen after these resins were cured, whereas the antibacterial effect of XR-Bond on S sanguis and A viscosus was not affected by light curing.
47 citations
TL;DR: An agar medium, designated X-Glu agar, was developed for the selective enumeration of Lactobacillus acidophilus in yogurt-related milk products containing a mixed microflora of lactobacilli, streptococci and bifidobacteria as mentioned in this paper.
44 citations
TL;DR: The results indicate the possibility of using S. marcescens as a biocontrol agent for Botrytis spp.
Abstract: Serratia marcescens (isolate B2 from tomato phylloplane) inhibited markedly the growth of Botrytis allii, B. byssoidea, B. cinerea, B. fabae and B. tulipae on LB agar medium containing colloidal chitin. The conidial germination and hyphal growth of B. cinerea and B. fabae were also suppressed by culture filtrate of the bacterium. The inhibitory effect of the bacterium correlated with its chitinolytic activity. On leaf disks of broad bean, the bacterium could control chocolate spot disease caused by B. fabae. These results indicate the possibility of using S. marcescens as a biocontrol agent for Botrytis spp.
TL;DR: It is suggested that a major factor influencing the ability of a pseudomonad isolate to act as a biocontrol agent against P. ultimum, is their ability to metabolize the constituents of seed exudate in order to produce compounds inhibitory to P.Ultimum.
Abstract: There was a significant (P ⩽ 0.05) linear relationship between the ability of eight pseudomonad isolates to increase the emergence of sugarbeet seeds in a soil (in which sugarbeet seeds are subject to damping-off by Pythium ultimum) and their ability to inhibit P. ultimum on seed exudate agar derived from normal (non-surface sterilized) seeds. There was also a significant (P ⩽ 0.05) linear relationship between the ability of the eight pseudomonad isolates to increase the emergence of sugarbeet seeds in soil and their ability to inhibit P. ultimum on agar medium, containing the same concentration of the most abundant sugars (cellobiose + galactose + sorbose) found in seed exudate agar derived from normal seeds and on agar medium containing cellobiose and galactose alone. These results suggest that a major factor influencing the ability of a pseudomonad isolate to act as a biocontrol agent against P. ultimum on sugarbeet seeds in soil, is their ability to metabolize the constituents of seed exudate in order to produce compounds inhibitory to P. ultimum.
TL;DR: This study was undertaken to determine the antibacterial effects of various endodontic medicaments against six selected anaerobic bacteria by testing the vapors of six medicaments using agar plates streaked with bacteria.
TL;DR: Hemolytic plaque isolates appeared to be regulated by the concentration of environmental iron: exogenous hemin was found to inhibit hemolysin production, and the iron scavenging compound, 2,2'- dipyridyl, was finding to promote hemoly sin production by all of the strains except for the S. constellatus isolate.
Abstract: Three-quarters of the patients with periodontal diseases surveyed in this study had one or more distinct types of hemolytic bacteria in their subgingival plaque. Twelve different species of bacteria were identified, belonging to five genera (Actinomyces, Streptococcus, Staphylococcus, Prevotella, and Actinobacillus). Nine hemolytic isolates, consisting of four Prevotella denticola strains, two Actinomyces naeslundii genospecies 2 strains, and one each of P. melaninogenica, Streptococcus constellatus, and A. naeslundii genospecies 1 strains were characterized. Incorporation of pronase into blood agar medium inhibited hemolysis by all of the isolates, suggesting a proteinaceous component for each of their hemolysins. With one exception, hemolysin production appeared to be regulated by the concentration of environmental iron: exogenous hemin was found to inhibit hemolysin production, and the iron scavenging compound, 2,2'- dipyridyl, was found to promote hemolysin production by all of the strains except for the S. constellatus isolate. Genomic libraries of each of the hemolytic plaque isolates were prepared in Escherichia coli using pBR322. Hemolytic clones were isolated on blood agar medium containing ampicillin at frequencies ranging from 1-6.7 x 10(-4). Extensive restriction mapping revealed regions of homology in the case of clones derived from three P. denticola strains isolated from the same subjects. Two of the P. denticola-derived clones were virtually identical throughout the entrety of their > 5 Kb inserts. The clone derived from the third strain showed good homology to the other two within a 1.3 Kb region, but the flanking DNA showed no homology even though all three P. denticola isolates were shown to be clonally related by ribotyping.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: It was revealed that enforcement cloning was applicable in the new host-vector systems on normal nutrient agar medium, except for a combination of TH3α and pKF2, with the TH2 strain in combination with pK f2 or pKf3 seeming to be most suitable for enforcement cloning, even without Sm.
TL;DR: In this paper, the degradation of starch-plastic films in soil was monitored using scanning electron microscopy, where the films were buried in a forest soil where rapid colonization by fungal hyphae could be detected.
TL;DR: The findings indicate that anaerobic bacteria that produce these metabolic intermediates and anaerilic bacteria that can convert the intermediates to volatile fatty acids may be important components of probiotic cultures that can be provided to chicks to reduce colonization by salmonellae.
TL;DR: The Centers for Disease Control, Atlanta, Georgia, confirmed the disease to be acanthamebiasis and specifically identified the amebas as Acanthamoeba culbertsoni by fluorescent antibody testing on brain tissue from the dog.
Abstract: Acanthamoeba were isolated from a naturally occurring animal infection of granulomatous amebic encephalitis. The amebas were grown from lung lesions from a 1-year-old greyhound puppy, which was 1 of several dogs in a kennel that was affected by a progressive fatal neurologic and respiratory disease. The Centers for Disease Control, Atlanta, Georgia, confirmed the disease to be acanthamebiasis and specifically identified the amebas as Acanthamoeba culbertsoni by fluorescent antibody testing on brain tissue from the dog. The amebas were cultured initially on potato dextrose agar and on nonnutrient agar plates that were seeded with a lawn of nonpathogenic Escherichia coli. The isolate was then transferred to nonnutrient agar plates containing killed Enterobacter aerogenes and subsequently to axenic medium and cell cultures. The isolate was highly pathogenic by intranasal inoculation into 2-week-old mice.
TL;DR: Bioluminescent genes from Vibrio fischeri and V. harveyi were introduced into Bacillus subtilis on a plasmid vector and by chromosomal integration and the chromosomally marked strains emitted less light but were found to be stable without the requirement for antibiotic selection and following sporulation and germination.
Abstract: Bioluminescence (lux) genes from Vibrio fischeri and V. harveyi were introduced into Bacillus subtilis on a plasmid vector and by chromosomal integration. The plasmid-bearing strain was highly luminescent and stable under antibiotic selection, but luminescence was lost in the absence of selection and following sporulation and germination. The chromosomally marked strains emitted less light but were found to be stable without the requirement for antibiotic selection and following sporulation and germination. Individual luminescing colonies of both B. subtilis strains could be detected against a high background of non-bioluminescent indigenous soil microbial colonies on agar plates using a charge-coupled device camera. These bioluminescent Gram-positive strains could be of value in studies concerning the survival and spread of genetically-modified micro-organisms in soil environments.
TL;DR: The effect of long-term energy starvation (lack of electron acceptor in respiration) on the culturability of Pseudomonas aeruginosa PAO303 was studied by subsequent incubations for growth on aerobic medanaerobic media and resulted in a subpopulation of cells, which were detectable under denitrifying, but not under aerobic growth conditions.
TL;DR: Individual yeast cells can be successfully isolated and recultured on plates with a new isolation method making use of optical trapping with infrared laser light, and close to 100% of the isolated stationary‐phase cells formed colonies on agar plates, indicating a high recovery.
Abstract: Individual yeast cells can be successfully isolated and recultured on plates with a new isolation method making use of optical trapping with infrared laser light. The cells can be selected on morphological criteria by high resolution microscopy. The isolation device is constructed from two coverslips separated by spacers, in which selected cells are transferred to a plastic capillary, using the optical trap. To test the procedure, selection experiments were done with a mixture of two Saccharomyces cerevisiae strains, distinguishable both in fluorescence microscopy and on agar plates. These experiments showed that only selected cells were isolated, and close to 100% of the isolated stationary-phase cells formed colonies on agar plates, indicating a high recovery. A lower recovery was obtained with exponential-phase cells, possibly because of a higher sensitivity to laser irradiation. Applications for this method may include the isolation of mutants with altered morphology and the isolation of subpopulations of yeast cultures, for their separate investigation or for the initiation of pure cultures.
TL;DR: The results suggest that in soil, factors other than pH and inorganic nutrients are important or that a strain's genetic background is of such importance that it can mask the improved nodulation in an acid soil expected from selecting for tolerance on an agar medium.
Abstract: We report field experiments which tested the performance of ten strains of Rhizobium leguminosarum bv. trifolii in acid soils. The strains had been classified as either tolerant or sensitive to mineral imbalances associated with acid soils and to low pH, on an agar medium. The soils of the two sites were pH4.0 and 4.2 (10 mm CaCl 2 ) and all strains with one exception originated from acid soils at or near these sites. All strains colonized the rhizospheres of subterranean clover equally well but were more successful at the site where the naturalized population of rhizobia was smaller. Nodulation by the inoculant strains in the first year was poor; they formed only 8 and 2.9% of the nodules at Sites 1 and 2 respectively. The two strains originating from Site 1 formed a mean of only 2% of the 173 nodules tested from plots where it had been introduced at Site 1. The naturalized strains formed a disproportionate number of nodules relative to their number in the rhizosphere. Higher rates of inoculation in the second year, although not markedly affecting rhizosphere numbers, increased nodule initiation by the inoculant strains but not in proportion to their numbers. Unexpectedly, the most successful strains in each year were those classified as acid-sensitive; they formed a higher proportion of the nodules than those classified as acid-tolerant. The results suggest that in soil, factors other than pH and inorganic nutrients are important or that a strain's genetic background is of such importance that it can mask the improved nodulation in an acid soil expected from selecting for tolerance on an agar medium.
01 Jan 1993
TL;DR: Many alternatives to plate counts have been proposed in order to decrease the time required to determine numbers of viable cells.
Abstract: In microbiology it is often necessary to determine the number of viable cells in a sample or culture of interest. This is usually achieved by plating out the sample (diluted as required) on to an agar plate (Postgate 1969; Hattori 1988). There are several problems associated with this technique, the greatest of which is the length of time required to obtain the results. For some slowly growing organisms (e.g. Mycobacteria) it may take in excess of a week to determine how many cells were “viable” in the original sample, and even when the sample contains fast-growing organisms and the plates are incubated under optimal growth conditions a minimum of overnight growth is usually required before the resulting colonies can be counted. For some clinical specimens even an overnight incubation may be too long to be of use and consequently many alternatives to plate counts have been proposed in order to decrease the time required to determine numbers of viable cells (Harris and Kell 1985).
TL;DR: In well-aerated broth cultures, good growth of Renibacterium salmoninarum was obtained in a serum-free medium consisting of 1% peptone, 1% yeast extract, and 0.1% l-cysteine (PYC broth), which was essential for optimal growth.
Abstract: In well-aerated broth cultures, good growth of Renibacterium salmoninarum was obtained in a serum-free medium consisting of 1% peptone, 1% yeast extract, and 0.1% l-cysteine (PYC broth). In contrast, serum or charcoal is required for growth on agar medium. Charcoal treatment of broth media, either before bacterial inoculation or during growth, increased the growth of R. salmoninarum, whereas the surfactants Tween 20 and Tween 80 inhibited growth. l-Cysteine was essential for optimal growth. Other organic sulfur compounds, such as d-cysteine, l-methionine, homocysteine, homocysteine thiolactone, and reduced glutathione, supported only lower levels of growth, while cystine and dithiothreitol did not allow growth.
TL;DR: The medium facilitates the detection of strains having atypical fermentation patterns, such as the lactose- or sucrose-positive salmonellae, and detects Salmonella typhi after enrichment.
Abstract: An agar medium for the isolation of Salmonella spp. is described. The medium, lysine-mannitol-glycerol agar, has features of both xylose-lysine-deoxycholate agar and mannitol-lysine-crystal violet-brilliant green agar, but glycerol is added for the differentiation of Salmonella and Citrobacter spp. The medium facilitates the detection of strains having atypical fermentation patterns, such as the lactose- or sucrose-positive salmonellae. The medium also detects Salmonella typhi after enrichment.
TL;DR: Three strains of Listeria monocytogenes were heat-treated at three temperatures in physiological saline by a capillary tube method and better average recovery was obtained on blood agar than on TPA-FE.
Abstract: Three strains of Listeria monocytogenes were heat-treated at three temperatures in physiological saline by a capillary tube method. Recovery of heat-treated bacteria was performed on blood agar and on tryptose phosphate agar with ferric citrate and aesculin (TPA-FE). Both media were used in two ways: (1) incubation at 37 degrees C for 7 d, and (2) preincubation at 4 degrees C for 5 d in order to obtain repair of heat-injured bacteria, followed by incubation at 37 degrees C for 7 d. D and z values were determined. In both incubation procedures, better average recovery was obtained on blood agar than on TPA-FE. Thus, higher D values were recorded when blood agar was used. In most cases the differences were statistically significant. Repair at 4 degrees C of heat-injured bacteria occurred on both media but the proportions of repaired bacteria were higher on blood agar. The repair on this medium was generally reflected in higher D values for preincubated samples. Some significant differences in heat resistance were noted between the strains.
Journal Article•
TL;DR: A comparative study for the identification of 32 known strains of Candida species on the basis of morphology on glucose agar, rice extract agar and corn meal agar with and without Tween 80 revealed that when Tween80 is incorporated in the media identification is possible for 96.8% of the species within 48 hours on rice extract Agar and for 96% ofthe species on glucose Agar.
Abstract: A comparative study for the identification of 32 known strains of Candida species on the basis of morphology on glucose agar, rice extract agar and corn meal agar with and without Tween 80 revealed that when Tween 80 is incorporated in the media identification is possible for 96.8% of the species within 48 hours on rice extract agar and for 96.8% of the species within 48 hours on rice extract agar and for 90.6% of the species on glucose agar. The germ tubes and chlamydospores were also produced more on rice extract agar than on 0.1% glucose agar. Rice extract agar with Tween 80 can be used as single medium for morphologic identification of Candida species. The inoculated medium is first incubated at 37 degrees C for 3 hours and examined for germ tube formation and then incubated at 25 degrees C for 24 to 72 hours and examined for appearance of chlamydospores and mycelial morphology.
TL;DR: The latex agglutination test can be used as an alternative rapid procedure of the coagulase test, which needs several hours or even overnight incubation for completion, and all S. aureus isolates grown on Baird-Parker agar also could be correctly identified by the latex reagent.
TL;DR: AChemotactic responses of Acanthamoeba castellanii to bacteria, several representative bacterial products, and chemotactic peptides were studied by following migration of amebas under agar, showing a positive chemotaxis response to all bacterial species tested.
Abstract: Chemotactic responses of Acanthamoeba castellanii to bacteria, several representative bacterial products, and chemotactic peptides were studied by following migration of amebas under agar. Amebas showed a positive chemotactic response to all bacterial species tested, even to those which were not ingested by amebas because of toxic pigments. Lipopolysaccharide and lipoteichoic acid, components of the outer membrane and cell wall, respectively, of Gram-negative and Gram-positive bacteria, evoked a neutral response from the amebas indicating that they are not attractants. Cyclic adenosine monophosphate, either as a bacterial product or as a pure compound was not an attractant. The chemotactic peptide formyl-methionyl-leucyl-phenylalanine served as an attractant, but the antagonist peptides N-t-boc-norleucyl-leucyl-phenylalanine and N-t-boc-methionylleucyl-phenylalanine did not. Amebas respond to these chemical stimuli, probably by means of membrane receptors. Chemotaxis is an oriented response to a stimulus (Wilkinson, 1982). It may be an important factor in the location of bacteria by soil amebas, as it has been shown to be for mammalian phagocytes locating invading microbes (Hugli, 1989). Amebas such as Acanthamoeba, a free-living protozoon found in soil and water, share a number of similarities with phagocytes in that both types of cells ingest bacteria and probably possess some mechanism that enables them to locate these microbes. A large body of information is available about the chemotactic responses of mammalian phagocytes (Devreotes & Zigmond, 1988; Wilkinson, 1982), but less is known about mechanisms used by amebas in locating bacteria. Studies of Acanthamoeba and Hartmannella (see McIntyre & Jenkin, 1969; Tharavanij, 1965; Urquhart, 1984) have indicated that a chemotactic factor present in, or released from, bacteria serves as the signal in attracting amebas, and that both chemotaxis and chemokinesis are exhibited by Naegleria fowleri toward bacteria and chemotactic peptide (MarcianoCabral & Cline, 1987). Entamoeba histolytica was shown to migrate toward filtrates of Escherichia coli, as well as toward complement component C5a, and lysed human erythrocytes (Urban et al., 1983). This study was undertaken to examine chemotaxis in Acanthamoeba, using migration under agar toward bacteria, bacterial components, and chemotactic peptides as a means of evaluating the ability of soil amebas to locate microbial I The authors thank Dr. Savanat Tharavanij, Mahidol University, Bangkok, Thailand, for making available sections of his doctoral thesis dealing with chemotaxis of amebas. We thank Professor David Raab, Brooklyn College, Psychology Department, for advice on statistical treatment of data. This research was supported in part by a City University of New York research award 669171 and by NIH-NIGMS Grant 5T34 GM08078 (NIH-MARC Program). Portions of this research were presented at meetings of the Society of Protozoologists. TRANS. AM. MICROSC. Soc., 112(1): 43-61. 1993. ? Copyright, 1993, by the American Microscopical Society, Inc. This content downloaded from 207.46.13.57 on Sat, 10 Sep 2016 05:40:07 UTC All use subject to http://about.jstor.org/terms TRANS. AM. MICROSC. SOC. food sources. Our results support a chemotactic response of amebas to bacteria, as well as to the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine, a chemoattractant for mammalian phagocytes (Schiffman et al., 1975). MATERIALS AND METHODS Acanthamoeba castellanii (Neff strain) was grown axenically in Oxoid proteose-peptone (2% w/v)/yeast extract (0.5% w/v)/glucose (0.5% w/v), pH 7.2 at room temperature (ca. 25?C), with a generation time of about 13 h. Cultures were maintained in Corning tissue culture flasks (75 cm2), and amebas for use in experiments were harvested while in logarithmic growth phase (2-3 days old). At time of harvest, flasks were chilled on ice and amebas dislodged from the growth surface by several sharp raps to the side of the growth vessel. Amebas were washed three times in dilute saline (Page, 1967) at 4?C in a clinical centrifugation (200 x g), and kept chilled until ready to use. The ameba suspension was counted in a Coulter Counter (model ZF) and cell numbers were adjusted for use in experiments. Preparation of chemotaxis plates. Five ml of sterile melted Difco Noble agar (1.5% w/v) prepared in dilute saline was pipetted into sterile 60 x 15 mm Nunclon? tissue culture Petri dishes (Nunc, Denmark) having a grid pattern of 2-mm squares. The agar was allowed to harden, at which time wells (6-mm diameter) were punched in the agar with a no. 3 cork borer, using a template. The agar plug was removed with a Pasteur pipette attached to a vacuum line. Agar plates were freshly prepared at the time of each experiment to minimize drying of the agar and, for the same reason, holes were punched in the agar at the time they were to be filled. The cork borer used for making wells was sterilized by immersion in 95% (v/v) ethanol, followed by flaming to burn off the alcohol. Agarose (Litex, type HSA; Accurate Chemical & Scientific Corp., Westbury, New York) was used instead of agar in several experiments at 1 and 1.5% w/v. Three wells were made in a line in the agar (Nelson & Herron, 1988). One well was located in the center of the plate and the other two wells along an axis with the center well (one well at 1200 hours and the other at 1800 hours), using the grid pattern for orientation. The center-to-center spacing between wells was 20 mm (or 10 squares). Amebas were added to the center well; the test substance (bacteria, bacterial products, etc.) in agar was added to the second well (the 1200 hours well). The third well (the 1800 hours well) served as the control well, receiving saline or solvent used in preparing the test material added to agar. Each well could hold 50 tl of fluid. Figure 1 is a schematic representation illustrating the experimental design as seen on the plate surface, with stippling to indicate the distribution of amebas around the center well. Preparation of test substance. When live bacteria were used as test substance, they were grown either in nutrient broth or brain heart-infusion media at 37?C. Cultures were transferred daily over several days. For experiments, overnight cultures grown with shaking were harvested and washed three times with dilute saline using a Sorvall RC-2 centrifuge (12,000 x g), and suspended in saline to 44 This content downloaded from 207.46.13.57 on Sat, 10 Sep 2016 05:40:07 UTC All use subject to http://about.jstor.org/terms VOL. 112, NO. 1, JANUARY 1993
TL;DR: The development of an easy in vitro method for culturing Populus sp.
Abstract: We report here the development of an easy in vitro method for culturing Populus sp. embryos. Half-capsules were cultured in an agar medium with Murashige and Skoog inorganic salts and sucrose. Embr...