Direct Microscopic Methods Research Articles

Recovery of hydrogen peroxide-sensitive culturable cells of Vibrio vulnificus gives the appearance of resuscitation from a viable but nonculturable state.

The viabilities of five strains of Vibrio vulnificus were evaluated during the storage of the organisms in sterile seawater at 5 degrees C. The number of CFU was measured by plate count methods on rich media. The total cell numbers were determined by direct microscopic count methods. The titer of CFU declined logarithmically to undetectable levels over a period of 2 to 3 weeks, while the total cell numbers were unchanged. Midway through each study, higher culturable cell counts began to be observed on plates containing catalase or sodium pyruvate; during the latter stages of the study, the plate counts on such media were up to 1,000-fold higher than those on unsupplemented plates. Because autoclaving is known to generate hydrogen peroxide in rich media, and because catalase and sodium pyruvate are known to eliminate hydrogen peroxide, it appears that the conditions of the experiments led to the selection of a hydrogen peroxide-sensitive culturable cell subpopulation. At the time of the final stage of the decline in viability of each culture, hydrogen peroxide-sensitive cells were the only culturable cells present. Warming samples of the cultures to room temperature led to the growth of these residual culturable cells, utilizing nutrients provided by the nonculturable cells. The cells that grew recovered hydrogen peroxide resistance. When mixtures of culturable and nonculturable cells were diluted to the point where only nonculturable cells were present, or when the hydrogen peroxide-sensitive culturable cells had declined to undetectable levels, warming had no effect; no culturable cells were recovered. Warming has been reported to "resuscitate" nonculturable cells. Recognition of the existence of hydrogen peroxide-sensitive culturable cell populations, as well as their ability to grow to high levels in the warmed seawater microcosms, leads instead to the conclusion that while warming permits culturable cells to grow, it has no effect on nonculturable cells.

Kinetic Lattice Model for Long-Time Chemical Phenomena: Introduction of Time-Scale into Monte Carlo Simulation

The kinetic lattice model (KLM) has been proposed, as a model of the slow dynamics in chemical systems, where the molecular diffusion can be treated and the time notion can be introduced within the Monte Carlo (MC) simulation scheme by combining Stokes-Einstein relation and Einstein's formula for the diffusion coefficient D of the solute molecule. Then, its mean square displacement calculated via MC simulations, brings about directly a time-scale per 1 MC step. For a model chemical system consisting of a spherical molecule with a radius of 10 Å in such a solvent with the coefficient of viscosity η of toluene, 1 MC step was found to correspond to the order of 100 ps for both 2-dimensional and 3-dimensional KLM. At the same time, the temperature dependence and the dimensionality were discussed within the present KLM. It was established that the KLM should be a plausible and unique tool to understand the long-time chemical phenomena which have been, for a long time, difficult problems that can not be dealt by any direct microscopic methods.

Dynamics of fungal pathogens in host plant tissues

To develop efficient control measures against fungal plant pathogens, the dynamics of host plant colonization during disease development and the interactions among fungi within host plant tissues need to be clarified. These studies require accurate quantitative estimation of specific fungal biomass in plant tissues. This has been approached by direct-microscopic methods, cultural methods, chemical determinations of fungal components, serological methods, and molecular methods. Among these methods, serological and molecular methods provide rapid, specific, and sensitive quantitative measures of fungal biomass in host plant tissues. Therefore, studies on fungal dynamics of host plant colonization using these two methods are presented. Some examples of species interactions among pathogenic fungi within host plants, such as synergism and competition, are reviewed and the usefulness of serological and molecular methods for studies on these interactions is presented. These quantitative methods will provide helpful information for understanding the ecology of plant pathogenic fungi, such as the dynamics of host plant colonization and species interactions. Key words: quantitative methods, fungal biomass, ELISA, PCR, fungal colonization, species interaction.

Sources of error in direct microscopic methods for estimation of fungal biomass in soil

Sources of error in direct microscopic measurement of fungal hyphae in soil were examined and fungal biomass estimates and associated variability obtained by the direct counting method and the ergosterol technique were compared. Nested random effects ANOVA indicated that the major source of variance in the direct microscopic counting method were the people examining the prepared microscope slides, accounting for 83% of the total variance. Sampling variability accounted for approximately 14% of the total variance. Fungal biomass values calculated from soil ergosterol concentrations were close to the range of values derived from hyphal length estimates but coefficients of variation were much lower for soil ergosterol determinations (6–13%) than for hyphal length estimates (16–32%). For one soil sample, we compared total hyphal length and fungal biomass estimates from our lab to those of another lab. Values obtained by the other laboratory were outside the range of values and 95% confidence intervals reached in our lab. Comparison of fungal hyphal length estimates from undisturbed prairie soil and an adjacent cultivated soil indicated that the undisturbed soil contained more than twice as much fungal biomass. Results of our study indicate: (1) extreme caution must be used when comparing hyphal length and fungal biomass estimates made by different laboratories using the direct counting method; and (2) soil ergosterol determinations can provide information on fungal biomass that may be useful in comparing direct count estimates by different labs.

Ecomethodology for organoosmotrophs: Prokaryotic unicellular versus eukaryotic mycelial

Although they are very unlikely to play large direct roles in water-column microbial loops, eukaryotic mycelial decomposers (the mycelial true fungi, eumycotes, and zoosporic "fungi," oomycotes) have the potential to be important secondary producers in decaying plant material in shallow aquatic systems. Their secondary productivity may lead to important exchanges of material with microbial loops: output of ascospores, conidia, zoosporic flagellates, leaked lysates, and particles of decayed plants containing mycelium; input of dissolved organics and inorganic nutrients. Development of methods for ecological study of the aquatic mycelial eukaryotic decomposers has not advanced as rapidly as that for the prokaryotes of microbial loops, probably because (1) there are fewer aquatic microbial ecologists with mycological training and inclination than with prokaryotic leanings; and (2) the mycelial decomposers are difficult to work with, because they produce their mycelial mass virtually entirely within opaque solid substrates. Direct microscopic methods have emerged as prime tools for the measurement of prokaryotic mass, whereas an index-chemical assay (ergosterol) is currently the most efficient way to measure the mass of eumycotes. For measuring productivity of prokaryotes of microbial loops, microbial ecologists may choose from several (>10) published and field-tested methods, involving direct microscopy or monitoring of radiotracers. Extensive reviews of distribution and dynamics of aquatic bacterial mass and productivity have appeared. For measuring productivity of eukaryotic mycelial decomposers, one has only two published methods from which to choose, a direct-microscopic and a radiotracer method, neither of which has had adequate field testing. We are, furthermore, much less well equipped to obtain mass and productivity information for the poorly known mycelial oomycotes than we are for the eumycotes. Application of productivity techniques and nucleic-acid technology, may within the next decade allow knowledge of ecology of aquatic eukaryotic mycelial decomposers to advance to levels approaching that for the prokaryotes of microbial loops.

Sulfate‐reducing and methanogenic bacteria from deep aquifers in montana

Thermophilic sulfate‐reducing and methanogenic bacteria were detected in deep (1200 to 1800 m) ground waters from geologic units that make up the Madison Formation, which underlies a large portion of the northern Great Plains. Some sulfate‐reducing bacteria were isolated and tentatively identified as Desulfotomaculum nigrificans. These organisms probably produce the hydrogen sulfide that is in the ground water. Direct microscopic counting methods demonstrated the presence of about 1000 bacteria/ml in the ground waters. Attempts to detect aerobic and other anaerobic bacteria were unsuccessful.

Comparisons between direct microscopic and cultural methods for recognition of Corynebacterium vaginale in women with vaginitis

The frequency with which clue cells could be detected in Gram-stained vaginal smears and/or cervical Papanicolaou (Pap) smears was compared with the frequency of Corynebacterium vaginale (Haemophilus vaginalis) isolation in a group of 236 female patients, of whom 221 had vaginitis. Vaginal clue cells were found most often in women from whom C. vaginale was isolated (P = 0.00006) whereas, conversely, clue cells in cervical Pap smears were reported more frequently in women with negative cultures for this organism (P = 0.006). C. vaginale isolations were made more frequently from women with both vaginal and cervical clue cells reported (P = 0.000088). However, the combined false positive-false negative vaginal clue cell rate in the patients studied was 36.5%. Neither the detection of vaginal clue cells nor the isolation of C. vaginale was significantly affected by whether or not patients had trichomoniasis (P = 0.25). Trichomonas vaginalis detection in cervical Pap smears and vaginal isolation were related (P = 0.00005), whereas the same relationship was not significant for fungi (P = greater than 0.05).

Localization and measurement of microvascular and microcirculatory responses to venous pressure elevation in the rat.

The effect of increase in regional draining venous pressure on the response of mesoappendix microvasculature and microcirculation was studied by direct microscopic methods <i>in vivo</i>. In 9 out of 21 experiments in pentobarbital anesthetized rats, elevation of ileo-colic vein pressure (13.0 ± 1.3 mm Hg; mean and SE) above resting levels (9.9 ± 2.2 mm Hg) resulted in persistent lumen expansion of small venular microvessels (18-68 <i>µ</i>m<i>, </i>i.d.) and evoked a vasoconstrictor response of consecutive segments of pre-capillary vessels and the sphincter. The vasoconstricor response was stimulus-dependent and reversible. The lumen reductions in percent for 9 arterioles (21.0 ± 4.37 <i>µ</i>m<i>, </i>i.d.) 9 metarterioles (9.3 ± 1.57 <i>µ</i>m<i>, </i>i.d.), and 5 precapillary sphincters (5.7 ± 1.23 <i>µ</i>m<i>, </i>i.d.) were: 17.9 ± 6.60, 43.6 ± 4.20, and 96.0 ± 3.81, respectively. In none of the experiments did the response propagate to larger arteriolar segments ( > 35–38 <i>µ</i>m, i.d.) in the tissue under observation. These microscopic studies confirmed the presence of a venous-pre-capillary and sphincter constrictor response in splanchnic vasculature in the rat.

Candidiasis: reflections on the problems of clinical assessment and treatment.

Over a period of eight years 256 cases of suspected candidacies were investigated in private dermatological office practice by direct microscopic and cultural methods. Laboratory investigation cannot define the limits of disease with precision and the significance of the laboratory findings can only be made by the clinician, who cannot delegate responsibility for the final diagnosis. The majority of cases of intertriginous candidacies appeared to be secondary to intertriginous (seborrhoeic) dermatitis, diabetes, pregnancy or neoplasia, and primary candidiasis without a predisposing factor was not common. Gandidal balanitis was the clinical state most frequently associated with previously undiagnosed diabetes. Because of the inadequate response of intertriginous candidiasis to topical polyene antibiotics and the frequency of a co-existing dermatitis, it was felt that hydroxyquinoline (e.g. vioform)—steroid preparations are the best available method of treatment.

Studies of the mineral phase of coral skeleton

Abstract Direct and electron microscopic and X-ray analytical methods were used to describe the microscopic and submicroscopic units of aragonite and their organization in the skeletons of one hermatypic species and one ahermatypic species of scleractinian coral. Weight and volume relationships of aragonite and other skeletal components are estimated in the different zones of the skeleton. The results are described in terms of coral growth and tissue differentiation.

Bacterial Populations in Sea Water as Determined by Different Methods of Enumeration1

Five different cultural and two direct microscopic methods were employed for estimating the abundance of bacteria in samples of sea water collected from both oceanic and neritic areas. The cultural methods included macrocolony counts on nutrient agar, silica gel, and membrane filters, microcolony counts on membrane filters, and the extinction dilution method. Direct microscopic counts were made of microbes on membrane filters and of microbes transferred from membrane filters to glass slides. Direct counts showed the presence of from 13 to 9,700 times as many bacteria as cultural methods. The extinction dilution method and the microcolony membrane filter method gave counts 20 and 35 times higher, respectively, than did any of the macrocolony methods. Direct microscopic counts on membrane filters were approximately 150 times higher than plate counts, and the numbers of microbes transferred from membrane filters to glass slides were approximately 2,000 times higher than plate counts. In all of the cultural methods, a peptone‐yeast extract medium was used.Differences in the abundance of microorganisms obtained by the various methods are attributed to a variety of factors: the presence of bacteria in aggregates, selective effects of the media, and the presence of inactive cells. A marked decrease in bacterial numbers was observed just below the thermocline as reflected in the macrocolony methods but not by the direct microscopic methods. A considerable population of spirilli‐like forms was noticed directly under the microscope but not after cultivation.

CHANGES IN THE TENTH EDITION OF STANDARD METHODS FOR THE EXAMINATION OF DAIRY PRODUCTS

Methods for extraneous matter and vitamin determinations have been omitted and new material added on growth inhibitors, detection of psychrophilic and thermoduric bacteria, improved sampling procedures for milk in tanks or vats, and for gassed or pressurized creams. Changes in microbiological methods include milk tree plating agars and use of metal 0.01 ml. transfer syringes and 3 alternative staining procedures in the direct microscopic methods. Numerous revisions or modifications made in other bacteriological and chemical procedures are reviewed.

The development of fundulus heteroclitus embryos in solutions of metrazol

Speidel1 has attempted to ascertain the specific effect of metrazol on the nervous system by immersing frog tadpoles in aqueous solutions of it and by studying, by direct microscopic and cine-photomicrographic methods, the outgrowth of living nerve fibers in the tadpoles'tails. Using a 2% solution for approximately 20 minutes, Speidel found that the growing nerve endings degenerated during the period of immersion, but recovered and subsequently regenerated when the tadpoles were returned to pond-water.The present communication reports the results of studying the development of certain aspects of gross behavior in whole embryos (Fundulus heteroclitus) which were immersed in solutions of metrazol at different stages of development and which remained in them for varying periods of time. The investigation was undertaken with the possibility in mind that subjecting embryos to treatment at various stages of development might produce alterations of structure and function in the central nervous system which could...

The Development of Fundulus heteroclitus Embryos in Solutions of Metrazol

Speidel1 has attempted to ascertain the specific effect of metrazol on the nervous system by immersing frog tadpoles in aqueous solutions of it and by studying, by direct microscopic and cine-photomicrographic methods, the outgrowth of living nerve fibers in the tadpoles'tails. Using a 2% solution for approximately 20 minutes, Speidel found that the growing nerve endings degenerated during the period of immersion, but recovered and subsequently regenerated when the tadpoles were returned to pond-water.The present communication reports the results of studying the development of certain aspects of gross behavior in whole embryos (Fundulus heteroclitus) which were immersed in solutions of metrazol at different stages of development and which remained in them for varying periods of time. The investigation was undertaken with the possibility in mind that subjecting embryos to treatment at various stages of development might produce alterations of structure and function in the central nervous system which could...

The Comparative Accuracy of the Direct Microscopic and Agar Plate Methods in Determining Numbers of Bacteria in Milk

Summary The results of this statistical analysis of counts made by the direct microscopic and agar plate methods show a high degree of variability in both methods, as applied comparatively to rating raw market milk supplies. In other words, there is such a relatively large experimental error in both methods that sanitarians interested in the bacteriological control of milk supplies must avoid the danger of making too fine distinctions in establishing the maximum numerical limits for the various, so-called, grades of milk. Variations of 200 to 300 per cent have been frequently reported in counts made in well controlled laboratories. While it is true that some technicians, under exceptionally well controlled conditions, have been able to obtain smaller percentage variations, it should not be forgotten that we must be governed by limitations arising through practical application where many samples of milk are daily prepared for counting by technicians who do not have time to exercise that extreme care which is possible only in research laboratories where there always exists ample time in which to plate the half dozen or so samples. The direct microscopic count of individual cells per cubic centimeter varied from 4.8 to 6.2 times greater than the count of the groups of bacteria under the microscope, and from 3 to 4.5 times greater than the agar plate colony count. The plate count on either plain or lactose agar was greater than the corresponding microscopic group counts. This relationship is to be expected in most samples due to the breaking up of the groups into smaller components during the process of plating the milk. The individual cell count averaged 4.5 times greater than the plain agar plate count but where lactose was added this difference was reduced to 3 times. There was likewise a reduction in the variability as well as a noticeable increase in the correlation. Undoubtedly the addition of lactose to the medium would materially increase the accuracy of the plating method. Although the microscopic group count was more variable than either the count of individual cells by the microscopic method or the agar plate count, yet the probable error of the coefficients of variability showed little or no significant difference. There was a better correlation between the counts of the individual bacterial cells and groups of cells per cubic centimeter by the direct microscopic method than between the individual cell count and the agar plate count. The poorest correlation obtained was that between the microscopic group counts and the agar plate counts. This is undoubtedly largely due to the variations introduced in the former count by the arbitrary manner in which separate groups are determined and by the counting of dead organisms and in the latter by variations in the adaptability of different organisms to growth in cultural media, and also by variations in the breaking up of colonies during plating. A few excessively high counts may have a marked effect upon the degree of correlation. For example, the elimination from 439 counts in all, of nine which were excessively high and which represented over-crowded microscopic fields and agar plates, resulted in a markedly lower correlation throughout (see table 7). It is conceivable that with raw milk uniformly low in bacterial content that there might be very little if any correlation between the two methods. Although, while showing more or less lack of correlation as applied to individual samples of milk, yet a close agreement may, in general, be expected in the results obtained by the two methods in rating the whole supply. Arithmetic averages of bacteria counts are apt to be misleading as a measure of the central tendency. One high count among several that were uniformly low would give an erroneous idea of the numerical bacterial quality of a given milk supply. The median, which is the middle number of a series of counts arranged in sequence, is a much fairer measure. One method gives as reliable a picture of the bacterial condition of any given raw milk supply as does the other. There is no reason for believing that one method is significantly more accurate than the other; nor that one lends itself more advantageously to the dividing of a raw milk supply into classes than the other. The plating method, however, is more applicable to the counting of bacteria in milk where there are only a few thousand present per cubic centimeter. Irrespective of the wide variations and possible sources of error that may be pointed out, both methods, if properly administered, are sufficiently accurate to insure a marked reduction in the amount of carelessly handled milk, without inflicting an undue hardship upon anyone.