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PubMed2005

On-farm production of AM fungus inoculum in mixtures of compost and vermiculite.

On-farm production of arbuscular mycorrhizal (AM) fungus inoculum can reduce the cost of the inoculum and increase utilization of this symbiosis in plant production. Bahiagrass (Paspalum notatum Flugge) seedlings, colonized by AM fungi, were transplanted into raised bed enclosures. Media within the enclosures was vermiculite mixed with either field soil or yard clippings compost in Experiment I and vermiculite mixed with yard clippings compost or dairy manure/leaf compost in Experiment II. Compost and vermiculite mixtures yielded more propagules of AM fungi than soil-based mixtures in Experiment I. Growth of plants in a 1:4 (v/v) mixture of yard clippings compost and vermiculite produced more inoculum (503 propagules cm(-3)) than growth in 1:9 and 1:99 (v/v) mixtures (240 and 42 propagules cm(-3), respectively). Water, inorganic nutrient solution minus P, and fish protein digest were added to inoculum production enclosures in Experiment II. Results indicated that supplemental nutrient addition was unnecessary. This method produces a concentrated inoculum of AM fungi in a form readily used as an amendment to horticultural potting media for the production of vegetable seedlings.

Agriculture↗

PubMed1998

Molecular analysis of yeast population dynamics: effect of sulphur dioxide and inoculum on must fermentation.

The effects of sulphur dioxide and a commercial starter inoculum on yeast population dynamics have been analysed by a molecular approach. Yeast identification from fermenting Carinyena grape musts was performed by RFLP's of mtDNA and rRNA-coding DNA. As expected, the use of a commercial inoculum speeded up the start of fermentation, while SO2 addition limited the development of non-Saccharomyces species. However, this effect was also observed with yeast inoculation. Further analysis of population dynamics could lead to a recommendation for the reduction of the dosage of SO2 by the addition of appropriate inoculum of yeasts in the must. Furthermore, the timing of inoculum addition could be modified to allow a proper contribution of non-Saccharomyces species. Molecular biology analysis of population dynamics could provide a tool to efficiently reduce the dosage of SO2 and adjust the timing of inoculum addition.

DNA, Mitochondrial↗

PubMed2000

Assay of prick test inoculum volume. II. Average values and individual variability.

INTRODUCTION: The amount of reagent introduced into the skin by a prick test is critical in studies on the reproducibility and standardization of the method. OBJECTIVE: The purpose of the present study was to measure the average volume and the individual variability of the inoculum performed with a prick test standardized technique. METHODS: Two hundred forty prick tests--16 inoculations per subject--were performed on the volar sides of arms of 15 healthy volunteers. The tests were performed by a skilled tester whose technical performance complied with the criteria of good reproducibility described in international guidelines. A 1-mm tip standard device and a 50% glycerosaline solution labeled with Tc99m were used for the test. The inoculum size was calculated using a direct assay method based on the gamma camera. RESULTS: The average volume of the prick test inoculum was equal to 0.016 microliters, with a remarkable dispersion of the values around the mean (median, 15906: range, 418 to 82253 picoliters). Further, we observed great variability from one subject to another and great variability in the same subject from one skin site to another. A statistical analysis of the data shows that this variability depends on the individual characteristics of the subjects examined. A skilled tester using a standardized technique is not responsible for significant variability. CONCLUSIONS: Even when performed by a skilled operator and with standardized techniques, the prick test shows great limits of reproducibility, at least as far as the size of the inoculum volume is concerned. The variability of the inoculum depends, in a statistically significant way, on the subject's individual characteristics and therefore can be reduced only within certain limits by the standardization and perfectibility of the technique.

Allergens↗

PubMed1980

Salmonella isolation with Rappaport's medium after pre-enrichment in buffered peptone water using a series of inoculum ratios.

The ability of malachite green/magnesium chloride broth (Rappaport's medium) to isolate salmonellas from 25 ml quantities of sewage-polluted natural water was investigated. Samples were first pre-enriched in buffered peptone water and varying volumes of inoculum from the pre-enrichment culture were inoculated into Rappaport's broth. Inoculum ratios in the range 1:2000 to 1:10 were examined. The inoculum ratio denotes the ratio of the volume of inoculum to the volume of fluid medium into which it is introduced. Optimum results were obtained with the 1:2000 ratio, although the salmonella isolation rate was only slightly less with the 1:500 and 1:100 ratios. The 1:2000 inoculum ratio was obtained with a graduated loop holding approximately 0.005 ml of fluid. Use of a loop for inoculation has advantages in speed of performance and safety of manipulation.

Bacteriological Techniques↗

PubMed1995

Inoculum size, incubation period and severity of malaria. Analysis of data from malaria therapy records.

The size of the infecting inoculum may influence the severity of malaria, but evidence is scarce. Malaria therapy records provide a unique source of information on induced malaria in people. The therapy was given to large numbers of neurosyphilis patients and the malaria was left untreated as long as possible. Data from patients treated at the Horton Hospital, Epsom 1923-60 with a single strain of vivax malaria were analysed to assess the influence of inoculum size on severity of disease. Malaria was induced by mosquito bite, blood inoculation or direct sporozoite inoculation. The different measures of inoculum size were inversely correlated with pre-patent period, as expected. Overall, information was available on a total of 563 non-immune patients who were not treated during the first 5 days of patent parasitaemia. No strong or consistent relationships were found between measures of inoculum size and any of the measures of severity used: neither parasitaemia levels, nor peak fevers, nor number of paroxysms of fever. In the largest data set, longer pre-patent periods were associated with tertian fever, spontaneous recovery and less use of modifying treatment. Difficulties in interpreting the results are discussed, particularly with respect to misclassification of both exposure and outcome variables. While an inoculum size-severity relationship cannot be ruled out, a strong relationship is very unlikely.

Adolescent↗

PubMed2001

Predicting vegetative inoculum performance to maximize phytase production in solid-state fermentation using response surface methodology.

Microbial phytase is used to reduce the environmental loading of phosphorus from animal production facilities. The limiting factors in the use of this enzyme in animal feeds can be overcome by solid-state fermentation (SSF), which is a promising technology for commercial enzyme production with lower production costs. Inoculum quality and the influence of inoculum quality on phytase production are important factors which need in-depth investigation before scaling-up of high-yielding fermentation process. A full factorial experimental design for 240 h with sampling at every 24 h was used to determine the effects of the treatments, inoculum age (plate and liquid culture), media composition and the duration of SSF on the production of fungal biomass and phytase in SSF systems using Aspergillus niger. The optimal treatment combination for maximal phytase production was determined by statistically comparing all treatments at each sampling time. Both 7- and 14-day plate cultures and M1 + medium composition with 72-h-old liquid inoculum treatments resulted in optimal phytase production at 144 h of SSF, which was the shortest duration observed for maximal phytase production. This resulted in maximal phytase production with a mean of 884 +/- 121 U/g substrate, while the maximal phytase production observed at 216 h of SSF (mean phytase activity of 1,008 +/- 121 U/g substrate), with the same treatment combinations, was not statistically significant from that at 144 h of SSF. Phytase production was strongly growth-associated with younger inocula. The significant treatment variables, age of liquid inoculum and the duration of SSF, were used to predict the system response for phytase production using response surface methodology. From the response surface model, the optimal response of the experiment was predicted and the reliability of the prediction was checked with the verification experiment.

6-Phytase↗

PubMed2001

beta-Lactam-susceptibility patterns of Plesiomonas shigelloides strains: importance of inoculum and medium.

The susceptibility of 10 Plesiomonas shigelloides strains to 30 beta-lactam antibiotics was examined. Susceptibility testing was carried out with a microdilution procedure using 3 media (Isosensitest broth, Mueller-Hinton broth and cation-adjusted Mueller-Hinton broth) and 4 inocula [1 x 10(4), 1 x 10(5), 1 x 10(6) and 1 x 10(7) colony-forming units (CFU)/ml]. A high inoculum dependency of MIC values was found for numerous beta-lactams. Each strain showed 2 completely different susceptibility patterns to several cephalosporins and aztreonam, but the same patterns were found with little variation in each strain. Using an inoculum of 1 x 10(4) CFU/ml the strains showed a high susceptibility to aztreonam and to all cephalosporins in all media (pattern 1), whereas they showed a decreased susceptibility to aztreonam and numerous cephalosporins with an inoculum of 1 x 10(7) CFU/ml (pattern 2). Using an inoculum of 1 x 10(6) CFU/ml, 4/10 strains in lsosensitest broth and 2/10 strains in Mueller-Hinton media showed pattern 1. Susceptibility testing of numerous penicillins revealed a medium-independent inoculum dependency, characterized by a step-to-step correlation between MICs and inocula. The beta-lactam susceptibility patterns arising from different inocula point to new beta-lactamase expression and regulation mechanisms in Plesiomonas. The potential to be resistant to a variety of beta-lactams under conceivable testing conditions should question the use of numerous beta-lactams for the treatment of Plesiomonas infections.

Animals↗

PubMed1994

Effect of inoculum size on the in-vitro susceptibility to beta-lactam antibiotics of Moraxella catarrhalis isolates of different beta-lactamase types.

The effect of inoculum size on the results of agar dilution MIC tests was assessed for 20 Moraxella catarrhalis isolates with BRO-1 enzyme, 20 with BRO-2 enzyme and 15 isolates that did not produce beta-lactamase. The compounds tested were ampicillin, coamoxiclav, cefaclor, cefixime and cefetamet, and the inocula were 10(4), 10(5), 10(6) and 10(7) cfu/spot. The MICs of ampicillin for BRO-1 and BRO-2 producers were consistently higher than those for non-producers at inocula of 10(7) cfu/spot but overlapped with those for non-producers at lower inocula. A small beta-lactamase-related inoculum effect was seen with coamoxiclav; small inoculum effects also occurred with cefaclor and cefixime but were not related to enzyme presence or type. MICs of cefetamet were the least affected by the inoculum size. For all the compounds, the degree of correlation between MICs and the inhibition zones observed in disk diffusion tests was independent of the inoculum used in the MIC tests. These data suggest that high inocula should be used to determine MICs of ampicillin for M. catarrhalis but that this precaution is unnecessary with the cephalosporins tested or with coamoxiclav.

Amoxicillin↗

PubMed1985

Effect of inoculum size on the induction of endophthalmitis in aphakic rabbit eyes.

A reproducible animal model is necessary to examine the use of antimicrobial agents for prophylaxis and treatment of bacterial endophthalmitis. We determined the minimum inoculum size of S. aureus and P. aeruginosa that consistently produced endophthalmitis when injected into aphakic rabbit eyes immediately following surgery. Both anterior chamber and intravitreal injections were examined. For S. aureus, an intravitreal inoculum of 19.3 +/- 7.5 CFU and an anterior chamber inoculum of 50.5 +/- 4.0 CFU were required. For P. aeruginosa, an intravitreal inoculum of 5.5 +/- 2.6 CFU and an anterior chamber inoculum of 97.5 +/- 10.7 CFU consistently produced a fulminant infection. Lower inocula of both bacteria produced endophthalmitis in both locations, but the effect was inconsistent.

Animals↗

PubMed1987

Inoculum effect with chloroquine and Plasmodium falciparum.

In the studies reported here, we examined the inoculum effect observed with chloroquine and Plasmodium falciparum. The 50% effective doses observed with both chloroquine-susceptible and -resistant parasites increased five- to sevenfold from their baseline values as the inoculum was increased from 2 X 10(5) to 2 X 10(7) parasitized erythrocytes per ml (parasitemias of 0.1 to 10% with a hematocrit of 2%). Increasing the inoculum also decreased the chloroquine uptake per parasitized erythrocyte 15- to 20-fold with both chloroquine-susceptible and -resistant parasites. However, because of the 100-fold increase in the inoculum, the total amount of chloroquine taken up actually increased sufficiently to reduce the extracellular chloroquine concentration in vitro by 60 to 90%. These studies suggest that a chloroquine uptake of greater than or equal to 2.0 pmol/10(6) parasitized erythrocytes is necessary for chloroquine to inhibit parasite growth. More marked reductions in the amount of chloroquine uptake per parasitized erythrocyte were observed with a hematocrit of 40% using similar parasitemias of 0.1 to 10% (inocula of 4 X 10(6) to 4 X 10(8) parasitized erythrocytes per ml). Thin-layer chromatography of [3H]chloroquine taken up by chloroquine-resistant P. falciparum revealed no evidence of drug alteration by the parasite. These studies define the mechanism responsible for the inoculum effect observed with chloroquine and P. falciparum in vitro.

Animals↗

PubMed1990

Effects of incubation temperature, inoculum size, and medium on agreement of macro- and microdilution broth susceptibility test results for yeasts.

We examined the effects of temperature and inoculum on the agreement of macro- and microdilution broth MICs of five antifungal agents against six isolates of Candida species or Torulopsis glabrata. Incubation temperature affected results with amphotericin B, flucytosine, fluconazole, and SCH 39304, producing better agreement at 35 degrees C than at 37 degrees C. Agreement between methods was better with an inoculum size of 10(2) than with one of 10(4) yeast cells per ml in testing fluconazole or SCH 39304, and the discrepancies seen with a higher incubation temperature and a larger inoculum appeared to be additive. However, inoculum size did not seem to affect agreement between methods in testing amphotericin B, flucytosine, or ketoconazole. Regardless of test conditions, macrodilution broth MICs of amphotericin B for different isolates were strikingly higher than microdilution test MICs, with mean differences being greater than ninefold under some test conditions. We conclude that for most currently available antifungal agents, an incubation temperature of 35 degrees C and a starting yeast inoculum of less than 10(4) cells per ml improve the agreement between macro- and microdilution broth tests.

Amphotericin B↗

PubMed1997

Effects of incubation temperature, inoculum size, and time of reading on broth microdilution susceptibility test results for amphotericin B fgainst Fusarium.

In vitro antifungal susceptibility testing for filamentous fungi remains unstandardized and is unreliable for determining adequate therapy. A study was performed to evaluate the effect of inoculum size (10(2), 10(3), 10(4), and 10(5) conidia/ml), incubation time (48 and 72 h), and temperature (25, 30, and 35 degrees C) on MICs of amphotericin B for Fusarium spp. (20 strains). The inoculum size showed the clearest effect: when the inoculum was varied from 10(2) to 10(5) conidia/ml, the geometric mean MICs showed increases of between 10- and 19-fold in all the combined conditions of temperature and incubation time assayed. Time of incubation had less effect (increases of between two- and threefold in approximately half of the geometric mean MICs), and temperature especially had little effect (the increases were no higher than twofold). The effects of interaction between inoculum size and temperature on MICs were not statistically significant, while the combined effects of inoculum size and time of reading and of time of reading and temperature produced systematic variation in MICs.

Amphotericin B↗

PubMed1983

Rapid inoculum standardization system: a novel device for standardization of inocula in antimicrobial susceptibility testing.

A rapid inoculum standardization system for antimicrobial susceptibility testing without incubation or the conventional turbidity adjustment has been developed. The rapid inoculum standardization system consists of a plastic rod with cross-hatched grooves on one end and a specific nutrient medium in a vial. The crosshatched grooves are designed to pick up and release a known number of viable microorganisms. In use, the end of the rod is touched to five colonies 1 to 2 mm in diameter from a primary agar plate, thus filling the grooves with bacteria. The rod is placed into the vial, and the bacteria are suspended in the medium by agitation with a Vortex Genie Mixer. The resulting suspension contains 5 X 10(7) to 5 X 10(8) CFU/ml for most gram-negative bacilli and gram-positive cocci. Microorganisms such as streptococci that have colonies less than 1 mm in diameter require as many as 10 colonies for an adequate inoculum suspension. Ninety-five commonly encountered bacterial isolates were tested in triplicate by agar plate counts. The resulting overall geometric mean of the agar plate counts was 1.52 X 10(8) CFU/ml for the species tested. We have found that the rapid inoculum standardization system provides a consistent and reproducible method for the standardization of inoculum for antimicrobial susceptibility testing without the incubation period and turbidity adjustment.

Anti-Bacterial Agents↗

PubMed2001

Utility of inoculum counting (Walshe and English criteria) in clinical diagnosis of onychomycosis caused by nondermatophytic filamentous fungi.

Opportunistic onychomycosis caused by nondermatophytic molds may differ in treatment from tinea unguium. Confirmed diagnosis of opportunistic onychomycosis classically requires more than one laboratory analysis to show consistency of fungal outgrowth. Walshe and English in 1966 proposed to extract sufficient diagnostic information from a single patient consultation by counting the number of nail fragments positive for inoculum of the suspected fungus. Twenty fragments were plated per patient, and each case in which five or more fragments grew the same mold was considered an infection by that mold, provided that compatible filaments were also seen invading the nail tissue by direct microscopy. This widely used and often recommended method has never been validated. Therefore, the validity of substituting any technique based on inoculum counting for conventional follow-up study in the diagnosis of opportunistic onychomycosis was investigated. Sampling of 473 patients was performed repeatedly. Nail specimens were examined by direct microscopy, and 15 pieces were plated on standard growth media. After 3 weeks, outgrowing dermatophytes were recorded, and pieces growing any nondermatophyte mold were counted. Patients returned on two to eight additional occasions over a 1- to 3-year period for similar examinations. Onychomycosis was etiologically classified based on long-term study. Opportunistic onychomycosis was definitively established for 86 patients. Counts of nondermatophyte molds in initial examinations were analyzed to determine if they successfully predicted both true cases of opportunistic onychomycosis and cases of insignificant mold contamination. There was a strong positive statistical association between mold colony counts and true opportunistic onychomycosis. Logistic regression analysis, however, determined that even the highest counts predicted true cases of opportunistic onychomycosis only 89.7% of the time. The counting criterion suggested by Walshe and English was correct only 23.2% of the time. Acremonium infections were especially likely to be correctly predicted by inoculum counting. Inoculum counting could be used to indicate a need for repeat studies in cases of false-negative results from laboratory direct microscopy. Inoculum counting cannot serve as a valid substitute for follow-up study in the diagnosis of opportunistic onychomycosis. It may, nonetheless, provide useful information both to the physician and to the laboratory, and it may be especially valuable when the patient does not present for follow-up sampling.

Aged↗

PubMed2004

Wide variations in herpes simplex virus type 1 inoculum dose and latency-associated transcript expression phenotype do not alter the establishment of latency in the rabbit eye model.

The latency-associated transcript (LAT) is required for efficient reactivation of herpes simplex virus type 1 from latent infection in the rabbit eye model, but LAT's mechanism of action is unknown. In addition to reactivation, the LAT region seems to correspond to multiple functions, with some LAT deletion mutants exhibiting increased virulence, increased neuronal death, and restricted establishment of latency. While a LAT promoter deletion mutant (17DeltaPst) seems to be primarily restricted in reactivation in the rabbit, subtle effects on virulence or the establishment of latency cannot be precluded at the normal high levels of virus inoculum used in the rabbit model. Since such additional LAT phenotypes may be more evident with lower doses of virus, we evaluated the influence of initial viral inoculum and LAT expression on the progression of acute infection and the establishment of latency. We have assayed both virus recovery rates and viral genome loads in rabbit corneas and trigeminal ganglia. Our results show that (i) in the corneas and trigeminal ganglia, the maximum amount of virus present during acute infection is independent of the LAT genotype and inoculum dose, although greater viral yields are obtained earlier with higher inoculum doses, and (ii) the range in numbers of latent genomes detected in the ganglia is independent of the inoculum dose and the LAT genotype and therefore no difference in establishment of latency is observed.

Animals↗

PubMed1988

Effect of inoculum size and frequency on the establishment of Streptococcus mutans in the oral cavities of experimental animals.

SPF Sprague-Dawley rats and ICR mice were inoculated with either Streptococcus mutans MT8148R (serotype c) or 6715 (g), and the influence of inoculum size, inoculum frequency, and sucrose on the establishment of S. mutans in the oral cavity was examined. Successful colonization of S. mutans in the experimental animals was absolutely dependent on the number of the cells introduced orally. Furthermore, inoculum frequency and sucrose seemed to act as secondary factors to modify the establishment of S. mutans, and it is suggested that high inoculum frequency may decrease the inoculum size necessary for the colonization of S. mutans in the oral cavity.

Animals↗

PubMed2004

Does smearing inoculum reflect the bacterial composition of the smear at the end of the ripening of a French soft, red-smear cheese?

The microbial community composition and dynamics during the production of a French soft, red-smear cheese were investigated. The colonization efficiency of the smearing inoculum was followed, and the parts played by the inoculum used and the resident microflora were tentatively estimated. Single-strand conformation polymorphism analysis (SSCP) was applied to 2 productions of a soft, red-smear cheese produced by the same dairy plant at 4-mo intervals. Microbial composition of the different cheese samples analyzed was found to be reproducible from one production to another. However, the composition of the surface flora of both cheeses at the end of the ripening did not reflect the composition of the smearing inoculum used, qualitatively as well as quantitatively. These results were confirmed by those obtained when assessing the microbial composition of the culturable flora by the spread plate technique. The inoculum used by the industry had low resiliency potentialities against colonization of cheeses by resident organisms. Therefore, fitness and colonization potential of smearing inocula should be carefully assessed by the industry before use. The use of Arthrobacter strains as part of the smearing inoculum should be evaluated.

Arthrobacter↗

PubMedDate not supplied

The response of ectomycorrhizal fungal inoculum to long-term increases in nitrogen supply.

The inoculum of ectomycorrhizal (EM) fungi was examined in a 16 y long nitrogen fertilization experiment maintained in a temperate oak savanna. To measure EM fungal inoculum, bur oak seedlings were grown in three types of bioassays: (i) intact soil cores that measure inoculum such as spores, mycelia and mycorrhizal roots; (ii) resistant propagule bioassays that measure inoculum types resistant to soil drying; and (iii) previously mycorrhizal root bioassays that measure the ability of EM fungi to colonize new roots from mycorrhizal roots. Colonization of bur oak seedlings was characterized by morphotyping and where necessary by restriction analysis and internal transcribed spacer (ITS) sequencing. Fourteen morphotypes were found in intact soil core bioassays with species of Cortinarius, Cenococcum and Russula abundant. Five morphotypes were found in resistant propagule bioassays with Cenococcum, a thelephoroid morphotype and a Wilcoxina-like ascomycete abundant and frequent. In intact soil core bioassays total percent root colonization and number of morphotypes were not affected by N supply in 2000 and 2001. However the composition of EM fungi colonizing oak seedling roots was different with increased N supply such that Russula spp. (primarily Russula aff. amoenolens) were most abundant at the highest level of N supply. Dominant Russula spp. did not colonize any roots in resistant propagule bioassays but did colonize oak seedling roots from previously mycorrhizal roots. Results suggest that in this savanna N supply can influence the kinds of inoculum propagules present and thereby might affect the dynamics of ectomycorrhizal communities by differentially influencing reproductive and colonization strategies.

Biodiversity↗