Inoculum Preparation

ABSTRACTThe influence of inoculum preparation in EUCAST broth dilution and Etest to detect the coexistence of resistant and susceptible Candida subpopulations (defined as polyresistance [PR]) was evaluated. Cocultures of two echinocandin-resistant and susceptible clinical C. glabrata strains were used to simulate the occurrence of mixed populations in clinical samples, and antifungal susceptibility testing was performed with standard and modified approaches of inoculum preparation. Polyresistant results manifested as microcolonies or double ellipses in Etest and in single reduced optical density (OD) values (dip in OD) in microdilution. The strict inclusion of five distinct colonies of 1:5 and 1:10 resistant and susceptible cocultures led to higher rates of PR and R results compared to including one to two colonies in inoculum preparation (30% and 26% for Etest and broth dilution, respectively). Modifying the inoculum preparation by increasing the turbidity from a 2 to a 4 McFarland standard before redilution to a 0.5 McFarland standard reliably enabled the detection of resistance, with better identification of PR by Etest than by broth dilution (82% versus 32%, respectively) and of resistant minimum inhibitory concentration (MIC) values in 18% of Etests and 67% of microdilutions. The highest identification of PR succeeded with Etest and a modified 3 McFarland standard approach of inoculum preparation. Our data demonstrate that inoculum preparation as recommended and practiced does not reliably identify resistant subpopulations in polyresistant Candida cultures. By increasing the inoculum size for Etest assays from a 2 to a 4 McFarland standard with subsequent redilution, we propose a simple adaptation to increase reliability.

The possible introduction of error into Staphylococcus aureus oxacillin intrinsic persister measurements by the agar dilution plate-count method resulting from technical factor variations associated with inoculum preparation was investigated. pH stability and exponential growth were shown to be present at the time of inoculum standardization. The use of glass or plastic tubes and the contamination of tube walls during inoculum broth culture produced no differences in test results. For S. aureus and oxacillin, the agar dilution plate-count method is a new and reliable approach to the quantitative study of bacterial inhibition and killing.

The effect of lovastatin on cholesterol synthesis is well known. Although lovastatin has been produced from several Aspergillus species and cultivation systems, the yield of lovastatin from Aspergillus sclerotiorum PSU-RSPG178 in a static liquid system has not yet been determined. Therefore, the objective of this study was to optimize lovastatin production from this strain using a central composite design developed through response surface methodology (RSM). The inoculum was prepared from mycelium or spore cultured in static liquid and shaking systems with potato dextrose broth (PDB) as the medium. The best inoculum preparation and cultivation systems were selected to study the optimization of the four fermentation parameters of medium concentration (X-5X), temperature (25-37°C), culture medium to flask volume ratio (2.5-12.5) and time (7-35 days). The best inoculum preparation was the mycelium method and the best cultivation system was the static liquid system. In the optimization procedure, a yield of 1315.69 mg/L lovastatin was obtained at a PDB concentration of 5X, a temperature of 25°C, a medium to flask volume ratio of 1:2.5 and a time of 21 days. In a model employing the optimized parameters, temperature was found to have the most effect on lovastatin production, which will decrease if temperature increases above 25°C. Based on this study, A. sclerotiorum PSU-RSPG178 can be a useful strain for producing lovastatin and RSM as an optimization technique not only reduced the number of experiments but also resulted in an optimized lovastatin yield.

Studying behaviors of four Italian lemon rootstocks towards Mal Secco leaf infection with the fungus Phoma tracheiphila, isolated in Tunisia in controlled environment, is the aim of this research work. It involves prospection of infected lemons fields in Tunisia, isolation and morphological identification of the fungus, preparation of inoculum and infection of leaves of four rootstocks. Following the artificial inoculation in three assessments after 10, 20 and 30 days post inoculation (dpi) by observing the appearance of disease symptoms, counting the percentage of positive inoculation and determining the average disease intensity according to the leaf empirical scale. All these parameters indicated about the behaviour of the rootstock towards disease in addition to allowing the classification of the four lemon rootstocks according to susceptibility to Mal Secco. Therefore Volkameriana which showed great sensitive behavior was considered as susceptible rootstock. However, Sour Orange showed an intermediate susceptibility to Mal Secco infection and was classified as tolerant rootstock. Nevertheless Flying Dragon and Citrange Troyer were considered as resistant rootstocks after showing a great resistance to the leaf infection by Phoma tracheiphila.

Production of Bacillus thuringiensis biopesticides using wastewater sludge as a raw material: effect of inoculum and sludge solids concentration

Detection of the Scrapie Agent in Tissues of Normal Mice with Special Reference to the Possibility of Accidental Laboratory Contamination

A variety of factors which might affect zone sizes were studied with strains of Corynebacterium diphtheriae; a standard disc method for antimicrobial sensitivity testing was used. Moderate variations in inoculum size, inoculum preparation, and pH of Mueller Hinton agar (MHA) did not appreciably affect zone sizes. The addition of blood to MHA was necessary to insure the growth of all C. diphtheriae strains on all lots of MHA. Zone diameters on MHA with blood were consistently 4 to 9 mm smaller than on plain MHA; however, zone diameters were within the sensitive range for seven antibiotic discs used on both media. Minimal inhibitory concentration (MIC) values for penicillin, erythromycin, and rifampin were determined by a plate dilution method. The geographical source, toxigenicity, and type of the strains showed no significant correlation with MIC values or zone diameters for eight antibiotic discs. When MIC values were compared to obtainable blood levels, all of the strains appeared to be sensitive with MIC values of </= 0.5 mug/ml for penicillin and </= 0.01 mug/ml for erythromycin and rifampin. Strains from persons who failed to have C. diphtheriae eliminated by one course of treatment with penicillin or erythromycin were examined. The susceptibility of these strains did not vary significantly from that of the other strains studied.

Infectious diseases, including urinary tract infections (UTIs), significantly impact global health, diminishing the quality of life and increasing clinical and economic burdens. Concerns arise due to the potential development of antimicrobial resistance (AMR) facilitated by biofilm formation. Nanotechnology, specifically silver and silver nanoparticles (1–100 nm), proves effective against bacterial strains, making them valuable in the biomedical field. Advances in microscopic imaging techniques, such as Confocal Laser Scanning Microscopy (CLSM), enhance our understanding of biofilms. This study aims to assess the effectiveness and optimal concentration of gold and silver nanoparticles in combating biofilms formed by uropathogenic Escherichia coli (UPEC) through CLSM analysis. The research comprises the following stages: rejuvenation of bacterial isolates and inoculum preparation, testing the anti-biofilm activity of gold and silver nanoparticles at concentrations of 25, 50, 75, and 100 ppm against UPEC using CLSM analysis. The findings reveal that both gold and silver nanoparticles exhibit anti-biofilm activity against UPEC at concentrations of 25, 50, 75, and 100 ppm, as confirmed by CLSM analysis. The optimal concentration for anti-biofilm activity of gold and silver nanoparticles against UPEC is identified as 100 ppm.

Antifungal susceptibility testing of yeasts has lacked reproducibility because of the absence of universally accepted guidelines. The major sources of variation in susceptibility testing have been attributed to the choice of medium and pH, inoculum preparation and size, incubation temperature and time, and end-point criteria. These parameters have been addressed by the National Committee for Clinical Laboratory Standards (NCCLS) Subcommittee on Antifungal Susceptibility Testing, and proposed guidelines have recently been published. Although major advances in antifungal susceptibility testing have been achieved, particularly through the efforts of the NCCLS subcommittee, the results of studies to date do not support a correlation between the results of in vitro susceptibility testing and in vivo response. Routine antifungal susceptibility testing of yeasts should therefore be discouraged until the methodology is standardized such that it predicts clinical outcome.

Exploring the biochar as a suitable carrier for a bioinoculant Aspergillus niger K7 and its consequence on Eleusine coracana in field studies

Consumers have been seeking to consume healthy products with functional properties. Probiotic foods and beverages are types of functional food that help with gut health; thus, they have been gaining popularity. Saccharomyces boulardii is one of the few yeasts recognized as having probiotic properties. Thus, studying its functionality and aspects in beverages, which can be good vehicles for these microorganisms, is necessary. In this review, articles concerning probiotic beverages supplemented with Saccharomyces boulardii are discussed in terms of the general characteristics of yeast, variety of matrices, viability, physicochemical characteristics and technological aspects, inoculum preparation and application methods in matrices and in vitro analysis. Saccharomyces boulardii is a potential yeast that can be added to beverages. The addition of prebiotic microcapsules, which can also protect yeast after in vitro digestion, seems to improve viability. pH can decrease with increasing acidity as a result of fermentation. The final product may have significant differences in sensory aspects, although it can present good acceptance.

SUMMARYThis review provides a comprehensive analysis of bacterial cellulose production, with a focus on the key factors influencing the bioprocess, including microorganism selection, substrate optimization and cultivation techniques. It addresses the challenges associated with bacterial cellulose production and proposes strategies to optimize upstream processes, such as microorganism preservation, inoculum preparation and culture medium formulation, which are critical for improving production efficiency. Additionally, the review explores the effects of fermentation parameters such as time, temperature, and oxygen availability on the yield and quality of bacterial cellulose. It also highlights the growing interest in utilizing alternative substrates, particularly agro-industrial waste, to reduce production costs and improve sustainability. By evaluating these factors, this review provides valuable insights for the further development of bacterial cellulose production in both research and industrial applications.

The purpose of the present study was to develop a quantitative relationship between the primary factors of state affecting pH control in a bicarbonate-buffered medium. Starting with the Henderson-Hasselbach equation, several assumptions led to the following equation: L = B(T)-s x dCO2 x 10(pH-pK) where L is the lactate concentration (mM), B(T) is the total amount of base added (mM), s is the solubility of CO(2) (mM/%), dCO(2) is the dissolved CO(2) concentration (%) and pK is the acid ionization constant for bicarbonate. This equation appropriately described the relationship of these factors when using bicarbonate, carbonate and HCl (as a lactic acid surrogate) in water. However, the equation required modification to describe the relationship in cell culture medium, due presumably to the presence of other buffers and components; the final form of the equation from an empirical fit in the absence of cells was: L = B(T)-0.88 x dCO2(0.79) x 10(pH-6038) This equation was tested against actual cell culture data, from inoculum preparation in a T-flask through a 10000-litre fed-batch bioreactor, by comparing the lactate concentration calculated from base, pH and dCO(2) data with that actually measured in the bioreactor using a YSI 8500 SELECT Biochemistry Analyzer (YSI Inc., Yellow Springs, OH, U.S.A.). In every case, the calculated and actual lactate concentrations were in good agreement. The equation was useful for isolating the mechanisms leading to varied base addition across 2-, 600- and 10 000-litre-scale bioreactors. This procedure enables a new approach for quantitatively evaluating and understanding factors associated with bioreactor pH control.

Coffee fermentation represents a natural bioreactor in which microbial metabolism shapes the aroma, acidity, and sensory identity of one of the most valued beverages in the world. This study optimized a semi-controlled fermentation process using commercial lactic acid bacteria (LAB) and yeasts under wet conditions to enhance the sensory quality of Coffea arabica var. Colombia at a smallholder scale. A sequential experimental strategy combined a definitive screening design (DSD) to identify key variables—fermentation time (X₁), inoculum concentration (X₂), water content (X₃), microorganism type (X₄), and inoculum preparation method (X₅)—with a factorial optimization step. Relative microbial growth was included as a covariate, and the sensory score (S) of the final coffee served as the main response variable, as determined by a certified Q-grader following the Specialty Coffee Association (SCA) cupping protocol. During fermentation, pH, titratable acidity, soluble solids, and microbial viability were monitored to characterize process dynamics. The DSD revealed that microorganism type, fermentation time, and inoculum preparation method were the most influential factors on sensory outcomes. Optimization predicted the highest S value (85.14) with a yeast inoculum prepared from fresh commercial Saccharomyces cerevisiae yeast (Levapan, Colombia) and a fermentation duration of 48 h, using minimal inoculum concentration (3%) and water content (50%). Experimental validation confirmed these conditions, achieving a relative error of 0.36%. The results demonstrate that accessible commercial microbial inoculum can significantly improve process control, microbial performance, and sensory quality without requiring specialized infrastructure. This biotechnological approach provides a cost-effective and scalable fermentation model for smallholder producers, integrating microbial optimization with sustainable practices to strengthen participation in the specialty coffee market.

ABSTRACTPhenotypic drug susceptibility testing (DST) requires a standardized amount of inoculum to produce reproducible susceptibility results. The most critical step in the application of DST in Mycobacterium tuberculosis isolates is the preparation of the bacterial inoculum. In this study, the effect of bacterial inoculum prepared in various McFarland turbidities on primary antituberculosis drug susceptibility of M. tuberculosis strains was investigated. Five standard ATCC strains (ATCC 27294 [H37Rv], ATCC 35822 [izoniazid-resistant], ATCC 35838 [rifampicin-resistant], ATCC 35820 [streptomycin-resistant], ATCC 35837 [ethambutol-resistant]) were tested. Inoculums of McFarland standard of 0.5, 1, 2, 3, and 1:100 dilutions of 1 McFarland standard of each strain were used. The effect of inoculum size on DST results was determined by the proportion method in Lowenstein-Jensen (LJ) medium and nitrate reductase assay (NRA) in the LJ medium. In both test methods, the increase in inoculum size did not affect the DST results of the strains. On the contrary, DST results were obtained more rapidly as a result of the use of dense inoculum. DST results obtained in all McFarland turbidities were found to be 100% compatible with the recommended amount of inoculum, 1:100 dilution of 1 McFarland standard (inoculum size of gold standard method). In conclusion, the use of a high amount of inoculum did not change the drug susceptibility profile of tuberculosis bacilli. Minimizing manipulations during the inoculum preparation phase of susceptibility testing, this outcome will decrease the need for equipment and make the test application easier, particularly in developing countries.IMPORTANCE During DST application, it can be challenging to evenly homogenize TB cell clumps with lipid-rich cell walls. These experiments must be carried out under Biosafety Level-3 (BSL-3) laboratory conditions with personal protective equipment and taking safety precautions because the procedures applied at this stage cause the formation of bacillus-laden aerosols and carry a serious risk of transmission. Considering this situation, this stage is important given that it is not possible to establish a BSL-3 laboratory in poor and developing countries. Reducing the manipulations to be applied during the preparation of bacterial turbidity will minimize the risk of aerosol formation. Perhaps there will be no need to do these steps for susceptibility tests in these countries or even in developed countries.