Flow cytometry for determination of the efficacy of contact lens disinfecting solutions against Acanthamoeba spp

Flow cytometric analyses of cellular staining with fluorescent viability dyes and direct microscopic observations of methylene blue exclusion were compared for evaluation of the effects of a chlorhexidine gluconate-based contact lens disinfectant solution and a polyhexamethylene biguanide solution against cysts and trophozoites of Acanthamoeba castellanii and Acanthamoeba polyphaga. The flow cytometric procedure with propidium iodide (used to stain dead cells) indicated that more than 90% of trophozoites of both species (inocula of 10(5) to 10(6)/ml) at 22 degrees C lost their viability after 4 h of exposure to chlorhexidine. When propidium iodide was used in combination with fluorescein diacetate (for live cells), the apparent number of propidium iodide-stained cells was reduced, but the relative efficacies of the two biguanide solutions appeared unchanged from those evident with the single dyes; the chlorhexidine solution was more effective than the polyhexamethylene biguanide solution. Similar data were obtained with the more cumbersome methylene blue exclusion procedure. Flow cytometric analyses provided a statistically reproducible and rapid procedure for determining the relative antiamoebal efficacies of the disinfecting solutions.     

Flow Cytometry for Determination of the Efficacy of Contact Lens Disinfecting Solutions against Acanthamoeba spp.

Flow cytometric analyses of cellular staining with fluorescent viability dyes and direct microscopic observations of methylene blue exclusion were compared for evaluation of the effects of a chlorhexidine gluconate-based contact lens disinfectant solution and a polyhexamethylene biguanide solution against cysts and trophozoites of Acanthamoeba castellanii and Acanthamoeba polyphaga. The flow cytometric procedure with propidium iodide (used to stain dead cells) indicated that more than 90% of trophozoites of both species (inocula of 10⁵ to 10⁶/ml) at 22°C lost their viability after 4 h of exposure to chlorhexidine. When propidium iodide was used in combination with fluorescein diacetate (for live cells), the apparent number of propidium iodide-stained cells was reduced, but the relative efficacies of the two biguanide solutions appeared unchanged from those evident with the single dyes; the chlorhexidine solution was more effective than the polyhexamethylene biguanide solution. Similar data were obtained with the more cumbersome methylene blue exclusion procedure. Flow cytometric analyses provided a statistically reproducible and rapid procedure for determining the relative antiamoebal efficacies of the disinfecting solutions.     

Cryopreservation of Holospora undulata, a bacterial parasite of the ciliate Paramecium caudatum

We investigated cryopreservation of horizontal transmission stages of Holospora undulata, a micronucleus-specific bacterial parasite of Paramecium caudatum. Unlike in previous studies on related Holospora species, protocols using glycerol as cryoprotectant failed entirely. In contrast, freezing with dimethyl sulfoxide (Me2SO) conserved infectiousness of nearly all replicate inocula, although infection success was considerably lower than that of fresh inocula. Infection probability was enhanced by increasing the Me2SO concentration from 5 to 10%, and by freezing at -196 degrees C rather than -80 degrees C. Prolonged storage of up to 3 months had no significant effect on the viability of the inocula.     

Viability testing and characterization of germination of fungal spores by automatic image analysis

Fungal spores are used in the laboratory for culture maintenance and at laboratory and other scales as inocula for fermentations. The spore swelling and germination processes constitute a major part of the lag phase, and the subsequent culture morphology and productivity can be greatly influenced by the initial concentration and condition of the spores. An image analysis method has been developed for assessing the viability and the germination characteristics of fungal spores in submerged cultures. Structural variations during germination, i.e., swelling, germ tube formation, and germ tube elongation, are measured in terms of distributions of spore volumes and of germ tube lengths and volumes. These measurements are fully automatic and give a very rapid assessment of spore viability. This image analysis method might be used as a tool in culture maintenance and for determining the quality of inocula for fungal fermentations. (c) 1993 John Wiley & Sons, Inc.     

Effect of anoxic conditions on wood-decay fungi treated with argon or nitrogen

The effects of low-oxygen conditions, achieved with either argon or nitrogen gas, on the viability of wood-decay fungi Coniophora puteana and Antrodia vaillantii, grown on artificial growth medium, were tested. Initial tests for viability were run after 1, 2, 3 and 4 weeks of exposure to low oxygen conditions, at oxygen levels in the vessels maintained below 10 ppm. Treatment was later extended to 10 and 16 weeks. Tests included measurements of CO₂ production and determination of the ability of fungal tissue to regenerate. The effect of anoxic conditions on the mycelia of treated fungal species was expressed as an increased time needed for regeneration or as a complete absence of growth of inocula taken from the exposed cultures. The cultures that were retarded by the low-oxygen environment consequently produced less CO₂ per hour. For C. puteana cultures, the effects of anoxic treatment became apparent in the second week of the treatment. The number of affected cultures rose steadily with the prolongation of anoxic treatment. By the 16th week of the experiment, 80% of the inocula of C. puteana did not regenerate. A. vaillantii inocula regeneration was not affected until after the fourth week of treatment. The influence of anoxic treatment on the cultures of this species was more pronounced in the test on the 10th and especially on the 16th week, when 67% of inocula did not regenerate. Argon or nitrogen gas caused the same degree of loss of viability in both the fungal species tested. In general, A. vaillantii mycelial cultures proved to be less sensitive to anoxic conditions, caused by either argon and nitrogen gas.     

Long-term survival of Bordetella bronchiseptica in lakewater and in buffered saline without added nutrients

Abstract Bordetella bronchiseptica grew from small inocula, and retained viability for at least 24 weeks, in unsupplemented lakewater or phosphate-buffered saline. From washed inocula of around 10³ colony-forming units/ml, there was growth at both 10°C and 37°C to give 10⁶–10⁷ colony-forming units/ml. At 10°C, these counts were maintained with little diminution up to week 24 when observations ceased. In the tests at 37°C, two of three strains tested showed similar retention of viability. These results suggest that B. bronchiseptica may exist as hitherto unsuspected reservoirs of infection in freshwater habitats.     

Spent culture supernatant of Mycobacterium tuberculosis H37Ra improves viability of aged cultures of this strain and allows small inocula to initiate growth

Spent culture supernatant from early-stationary-phase Mycobacterium tuberculosis H37Ra cultures increased the viability of bacilli from aged cultures of this strain and allowed small inocula to initiate growth in liquid culture. The resuscitation factor was acid labile and heat stable, with a mass of less than 1,375 Da.     

Sheared-Root Inocula of Vesicular-Arbuscular Mycorrhizal Fungi

For efficient handling, vesicular-arbuscular mycorrhizal fungi should be processed into small and uniform inocula; however, processing can reduce the inoculum density. In this article we describe the preparation and use of sheared-root inocula of Glomus spp. in which inoculum densities were increased during processing. Our objectives were to determine inoculum viability and density after shearing and to ascertain if the sheared inocula could be pelletized or used with a gel carrier. Root samples were harvested from aeroponic cultures, blotted dry, cut into 1-cm lengths, and sheared in a food processor for up to 80 s. After shearing, the inoculum was washed over sieves, and the propagule density in each fraction was determined. Sheared inocula were also encapsulated in carrageenan or used in a gel carrier. Shearing aeroponically produced root inocula reduced particle size. Propagule density increased with decreasing size fraction down to a size of 63 μm, after which propagule density decreased. The weighted-average propagule density of the inoculum was 135,380 propagules g (dry weight) of sheared root material^-1. Sheared roots were encapsulated successfully in carrageenan, and the gel served as an effective carrier. Aeroponic root inoculum was stored dry at 4°C for 23 months without significant reduction in propagule density; however, this material was not appropriate for shearing. Moist roots, useful for shearing, began to lose propagule density after 1 month of storage. Shearing proved to be an excellent method to prepare viable root inocula of small and uniform size, allowing for more efficient and effective use of limited inoculum supplies.     

Production, Survival and Evaluation of Liquid Culture-produced Inocula of Coniothyrium minitans Against Sclerotinia sclerotiorum

Growth of Coniothyrium minitans on potato dextrose broth was compared with that on an inexpensive molasses-yeast liquid medium at 18-22°C in static culture. Biomass and conidial production were, in general, similar, although the rate of biomass production was quicker and conidial production was slightly greater per unit volume of medium in the molasses-yeast medium. Air-dried biomass from molasses-yeast liquid culture containing mycelia, pycnidia and conidia of C. minitans was mixed (12%, w/w) with kaolin to give a kaolin-biomass dust. The ability of C. minitans to survive and subsequently infect and reduce the viability of sclerotia of Sclerotinia sclerotiorum from this kaolin-biomass dust was found to be little affected by storage for 48 weeks between 4 and 15°C but was decreased by higher storage temperatures. The kaolin-biomass dust preparation did not differ from a standard maizemeal-perlite inoculum of C. minitans in its ability to infect sclerotia of S. sclerotiorum or reduce their viability or carpogenic germination in glasshouse and field pot bioassays. Further, when either inoculum was applied once to glasshouse soil naturally infested with S. sclerotiorum prior to planting three successive crops of lettuce, the pattern of disease control, reduction of sclerotial numbers/ plot, infection of sclerotia, reduction of sclerotial viability and survival in soil were similar for both inocula. The potential for the commercial development of liquid-culture-produced inocula of C. minitans is discussed.     

Evaluation of Gambierdiscus survival after exposure to ballast water

The dinoflagellate Gambierdiscus was exposed to ballast water from a trans-oceanic vessel, and maintained at a variety of temperatures in the dark to determine if viability would be maintained. Logarithmically growing Gambierdiscus inocula were admixed (1:6, vol:vol) with ballast water, maintained in the dark at 22.6 °C, 24.6 °C, 26.8 °C and 29.0 °C and assessed for numerical abundance over six days. Calculated growth rates from the biomass time series showed no indication that ballast water negatively impacted Gambierdiscus viability; accompanying microscopic inspections supported this conclusion. Filtration of large volumes of collected ballast water failed to show the presence of any Gambierdiscus cells contained therein. Recovery and microscopic examination of the experimental inocula after 10 weeks in the dark, failed to show cyst development at any temperature regime. This examination of ballast water showed no evidence of cytotoxicity to Gambierdiscus spp.     

Growth Physiology of Mycobacteria in Modified Dubos Liquid Medium

Although mycobacteria grow in Dubos liquid medium showing an arithmetic linear growth, the initial few days of growth were found to correspond to an ‘induction’ period. In this period, rapid increase of the amount of growth occurred, whereas increase of the number of colony-forming units remained at a low level. This finding shows that the rapid increase of the amount of growth is accompanied by rapid death of multiplied bacteria. In a successive period, which was considered to correspond to the logarithmic growth phase, a 1:1 correspondence existed between the amount of growth and the number of colony-forming units. The induction period is not considered to be a lag phase, in which the bacteria grow slowly, but a period of unbalanced relationship between the growth and the viability. Even when we inoculated different sizes of bacteria, the amounts of growth became similar in both inoculations after several days of incubation. However, the number of colony-forming units remained always smaller in the use of small inocula than in the use of large inocula. In the use of small inocula, much more rapid increase of the amount of growth occurred. However, this rapid increase gave rise to rapid death of bacteria.     

Morphology and viability analysis of<Emphasis Type="Italic"> Streptomyces clavuligerus</Emphasis> in industrial cultivation systems

The effects of varying inoculum age and production scale upon the morphology and viability of Streptomyces clavuligerus were studied by analyzing visible and fluorescent light images acquired throughout pilot-plant and pre-industrial scale fermentations. Changes in production scale reveal that in 5 m³ fermentors, the maximum hyphal area obtained is double the value obtained in 0.5 m³ fermentors. It is probably due to the higher shear stresses acting upon hyphae in the 0.5 m³ fermentor caused by higher tip speeds observed in these. The morphological quantification based on elongation and branching rates allowed fermentations to be pattern classified into distinct physiological time zones namely elongation, branching, fragmentation, etc. The general pattern observed for fermentations inoculated with late exponential phase inocula was similar to the pattern of fermentations run with stationary phase inocula except that both the elongation and branching periods started earlier in the former case. Using the available staining technique and image acquisition system, the viability seemed to be generally high and constant throughout the time course of all the studied fermentations.An erratum to this article can be found at     

The effectivity of arbuscular mycorrhizal fungi from high input conventional and organic grassland and grass-arable rotations

The effectivity of arbuscular mycorrhizal spores in promoting growth of Allium ameloprasum L. cv. Musselburgh and Trifolium repens L. cv. Menna was tested for inocula from three soil series under long term organic or intensive, conventional grass and grass-arable rotations. For two soil series, Allium responses to inocula from soils recently converted to organic fanning were also assessed. Finally, Trifolium root fragments were used to inoculate Allium so as to evaluate responses to this inoculation procedure. Plants were sown into previously sterilised, matched soils from organic farms with no nutrient input. Mycorrhizal treatments generally increased growth for Allium. However, for Trifolium, infection decreased growth in the most fertile soil and gave an increase only in the least fertile. In the least fertile soil, inocula from organic farms were more effective than those from conventional farms. For Trifolium (all soils) and for Allium (least fertile soil), there was evidence of more efficient uptake of phosphorus in plants inoculated with spores from organic farms. The pattern of Allium response to inoculation with spores from conventional, conversion and organic sources was not consistent between soil type, but there was evidence of lower root infection for conversion compared with organic inocula and of a trend towards higher infectivity as the time period under organic management increased. Inoculating Allium with AMF root fragments produced a plant response similar to that obtained when spores were used, confirming that spore viability was not the sole factor influencing AMF effectivity in earlier experiments. Intensive farming practices may reduce the effectiveness of indigenous arbuscular mycorrhizal populations, particularly where fertiliser inputs are high and inherent fertility is low. This could have practical implications where high input farms are converted to organic management.     

Improved aeroponic culture of inocula of arbuscular mycorrhizal fungi

We compared conventional atomizing disc aeroponic technology with the latest ultrasonic nebulizer technology for production of Glomus intraradices inocula. The piezo ceramic element technology used in the ultrasonic nebulizer employs high-frequency sound to nebulize nutrient solution into microdroplets 1 μm in diameter. Growth of pre-colonized arbuscular mycorrhizal (AM) roots of Sudan grass was achieved in both chambers used but both root growth and mycorrhization were significantly faster and more extensive in the ultrasonic nebulizer system than in the atomizing disc system. Shearing of the AM fungi (AMF) infected roots in both the systems did not reduce inoculum viability, as evident from the MPN data. However, sheared roots from the ultrasonic nebulizer system had significantly more infective propagules than those produced in the atomizing disc system. Thus, the latest ultra-sonic nebulizer aeroponic technology appears to be superior and an alternative to conventional atomizing disc or spray nozzle systems for the production of high-quality AMF inocula. These can be used in small doses to produce a large response, which is a prerequisite for commercialization of AMF technology. Accepted: 11 January 2000     

Effect of oscillatory high hydrostatic pressure treatments on Byssochlamys nivea ascospores suspended in fruit juice concentrates

The effect of continuous (689 MPa with holding times of 5, 15 or 25 min) and oscillatory (one, three or five cycles at 689 MPa with holding times of 1 s) high hydrostatic pressure treatments on the viability of Byssochlamys nivea ascospores suspended in apple and cranberry juice concentrates adjusted by dilution to water activities (a_w) of 0·98 and 0·94 was evaluated at 21 and 60 °C. Inactivation of the initial spore inocula was achieved after three or five cycles of oscillatory pressurization at 60 °C when the a_w was 0·98 in both fruit juices. With a_w 0·94, the initial inocula were reduced by less than 1 log-cycle after five pressure cycles. Inactivation was not observed within 25 min with continuous pressurization at 60 °C. In treatments at 21 °C, no effect on spore viability was observed with continuous or oscillatory treatments.     

Effect of Carbenicillin on Pseudomonas aeruginosa

The activity of carbenicillin against 200 strains of Pseudomonas aeruginosa was measured by a quantitative agar dilution method. Minimal inhibitory concentrations (M.I.C.''s) for five graded inocula were measured in terms of complete inhibition (CI) and reduced growth (RG). The M.I.C. decreased progressively as inocula were reduced, median values for the 200 strains ranging from 100 to 37.5 μg. per ml. by the CI criterion, and from 75 to 25 μg. per ml. by the RG definition. Ratios of M.I.C. obtained for large and small inocula were usually small. Identical M.I.C.''s by both CI and RG criteria were most often obtained when the inoculum for the RG criterion was 1 or 2 logs higher than that for complete inhibition.Population analysis of 15 strains of Ps. aeruginosa showed that one specific drug concentration usually caused a sharp drop in proportion of viable cells, ranging from 3 to 5 logs. None of the populations were completely non-viable even at 150 μg. per ml. There was evidence that the viability of different-sized populations was reduced disproportionately by carbenicillin.Carbenicillin 300 μg. per ml. exerted appreciable bactericidal effect against nine of 15 strains of Ps. aeruginosa after a 24-hour contact period; after only six hours the bactericidal effect was very small.Quantitative sensitivity measurements for carbenicillin should include M.I.C. values for both CI and RG criteria, using a range of inocula for testing. Such M.I.C. values may well be useful in monitoring carbenicillin therapy of tissue infections.     

Reproducibility of the Most Probable Numbers Technique for Determining the Sanitary Quality of Clams

The reproducibility of most probable numbers results in the bacteriological examination of clam meats, using the tentatively recommended procedure, is less than has been indicated in use with water analysis. Entrapped air bubbles in ground clam meats contribute to the variability of results by (i) lowering the density of the sample material, producing inaccurate aliquots for inocula, and (ii) interfering with the attainment of homogeneity in the brei.     

Immunization of mice with an avirulent pseudohyphal form of Cryptococcus neoformans

Pathogenicity tests with one dysgonic and six atypical strains of Microsporum canis were carried out on guineapigs. Five of the atypical strains were laboratory mutants from dysgonic strains isolated from living hosts. As sporulation and viability varied greatly between the strains, inocula consisted of suspensions of fungal fragments of known viable count. When a sufficiently active inoculum was used, lesions and fluorescent hairs were induced in the guinea-pigs by all but one of the strains tested. In each case the strain inoculated was reisolated from the lesions in pure culture. The significance of the results is discussed in the light of the unusual nature and origin of the strains.     

Efficacy of standard disinfectant test methods for contact lens-care solutions

Two disinfection systems based on hydrogen peroxide (0.5, 1.5 and 3%) and chlorhexidine gluconate (0.004%) were challenged in suspension tests using a modification of a standard method, with inocula containing 10⁶ or 10⁸ cfu/ml of the standard organisms Serratia marcescens, Pseudomonas aeruginosa and Staphylococcus warneri. The effect of an organic load on the shapes of microbial inactivation curves was also investigated. Although there were rapid declines in viability of 1–2 log units (cfu/ml) within the first minute of challenge in all cases, declines in viability subsequently levelled out rapidly, and with hydrogen peroxide viable counts increased slightly thereafter. These increases are unexplained, but may in part be attributable to disruption of cell clumps during challenge.     

Evaluation of biodegradability by the reduction of Tetrazolium Violet in Biolog microplates

A novel biodegradation test using Biolog MT microplates was developed. The method was based on the reduction of Tetrazolium Violet during mineralization of organic substrates. Both a microbial mixed culture (activated sludge) and pure culture of a bacterium (Pseudomonas aeruginosa) were used as inocula to evaluate its applicability. The procedure was successfully demonstrated with the ozonated samples of p-nitrophenol. Compared with previous methods, the proposed method is fast and convenient to use in practice.