The effects of exposure to cold during incubation on developmental stability, fear, growth, and carcass traits in Japanese quails.

The aim of this study was to determine the effects of 6 h/day cold (35.0 °C) acclimatization between the 9th and 15th days of incubation of Japanese quail embryos on hatchability, livability, chick quality, developmental stability, fear response, live weight, and slaughter-carcass characteristics. Two homologous incubators and a total of 500 hatching eggs were used in the study. Randomly selected half of the eggs were exposed to cold according to the eggshell temperature. The cold acclimation of Japanese quail embryos had no adverse effects on all mentioned traits, except for chick quality. Chicks in the control group had higher Tona scores (99.46) than those exposed to cold (99.00) (P < 0.05). In addition, there were differences among the treatment groups in terms of the parameters of mature weight (ß0), instantaneous growth rate (ß2), and inflection point coordinates of the Gompertz growth model (P < 0.05 for all). It was found that exposing embryos to cold during the incubation changed the shape of the growth curve. As the development of embryos exposed to cold slows down, a compensatory growth occurs in the early posthatch period. Thus, the growth rate increased in the period before the inflection point of the growth curve.

Factors influencing the development of gastrointestinal tract and nutrient transporters' function during the embryonic life of chickens-A review.

Intestinal morphology and regulation of nutrient transportation genes during the embryonic and early life of chicks influence their body weight and feed conversion ratio through the growing period. The intestine development can be monitored by measuring villus morphology and enzymatic activity and determining the expression of nutrient transporters genes. With the increasing importance of gut development and health in broiler production, considerable research has been directed towards factors affecting intestine development. Thus, this article reviews (1) intestinal development during embryogenesis, and (2) maternal factors, in ovo administration, and incubation conditions that influence intestinal development during embryogenesis. Conclusively, (1) chicks from heavier eggs may have a better-developed intestine than chicks from younger ones, (2) in ovo supplementation with amino acids, minerals, vitamins or a combination of several probiotics and prebiotics stimulates intestine development and increases the expression of intestine mucosal-related genes and (3) the long storage period, improper incubation temperature and imbalanced ventilation can negatively influence intestinal morphology and nutrient transporters gene expression. Finally, understanding the intestine development during embryonic life will enable us to enhance the productivity of broilers.

Optimization of incubation conditions of Plasmodium falciparum antibody multiplex assays to measure IgG, IgG1-4, IgM and IgE using standard and customized reference pools for sero-epidemiological and vaccine studies.

BACKGROUND: The quantitative suspension array technology (qSAT) is a useful platform for malaria immune marker discovery. However, a major challenge for large sero-epidemiological and malaria vaccine studies is the comparability across laboratories, which requires the access to standardized control reagents for assay optimization, to monitor performance and improve reproducibility. Here, the Plasmodium falciparum antibody reactivities of the newly available WHO reference reagent for anti-malaria human plasma (10/198) and of additional customized positive controls were examined with seven in-house qSAT multiplex assays measuring IgG, IgG1-4 subclasses, IgM and IgE against a panel of 40 antigens. The different positive controls were tested at different incubation times and temperatures (4 °C overnight, 37 °C 2 h, room temperature 1 h) to select the optimal conditions. RESULTS: Overall, the WHO reference reagent had low IgG2, IgG4, IgM and IgE, and also low anti-CSP antibody levels, thus this reagent was enriched with plasmas from RTS,S-vaccinated volunteers to be used as standard for CSP-based vaccine studies. For the IgM assay, another customized plasma pool prepared with samples from malaria primo-infected adults with adequate IgM levels proved to be more adequate as a positive control. The range and magnitude of IgG and IgG1-4 responses were highest when the WHO reference reagent was incubated with antigen-coupled beads at 4 °C overnight. IgG levels measured in the negative control did not vary between incubations at 37 °C 2 h and 4 °C overnight, indicating no difference in unspecific binding. CONCLUSIONS: With this study, the immunogenicity profile of the WHO reference reagent, including seven immunoglobulin isotypes and subclasses, and more P. falciparum antigens, also those included in the leading RTS,S malaria vaccine, was better characterized. Overall, incubation of samples at 4 °C overnight rendered the best performance for antibody measurements against the antigens tested. Although the WHO reference reagent performed well to measure IgG to the majority of the common P. falciparum blood stage antigens tested, customized pools may need to be used as positive controls depending on the antigens (e.g. pre-erythrocytic proteins of low natural immunogenicity) and isotypes/subclasses (e.g. IgM) under study.

Influence of Incubation Conditions on the Nanoparticles Toxicity Based on Seed Germination and Bacterial Bioluminescence.

The effects of the modified incubation conditions of a conventional bioassay on the toxicity of partially soluble nanoparticles (NPs) were evaluated based on the activity of seed germination and bacterial bioluminescence. Different levels of toxicity were observed for seed germination (CuO > ZnO > NiO) and bacterial bioluminescence (ZnO > CuO > NiO). The NP inhibition of seed germination increased strongly under modified incubation conditions: sample volume from 5 mL to 10 mL, shaking from none to 70 rpm, and working vessel from a Petri dish (+/− filter paper) to an Erlenmeyer flask (no filter paper). In the case of seed germination, the toxicity levels of NPs under the modified conditions were 1.26 to 8.49 times higher than the conventional method according to the type of NPs and modified conditions (p-values < 0.05). No significant differences in bacterial bioluminescence were observed between conventional (130 rpm) and modified (160 rpm) conditions. These findings show that for an accurate assessment of partially soluble NPs toxicity in ecosystems, the conventional bioassay method, which is designed for soluble chemicals, needs to be performed under modified conditions because of their insolubility.

Removal of divalent cations and oxyanions by keratin-derived sorbents: Influence of process parameters and mechanistic studies.

Keratin has become a promising adsorbing material for the removal of heavy metals from polluted water due to its environmentally benign nature, unique chemical structure, and binding ability. We developed keratin biopolymers (KBP-I, KBP-IV, KBP-V) using chicken feathers, and assessed their adsorption performance against metal-containing synthetic wastewater at varying temperatures, contact times, and pH. Initially, a multi-metal synthetic wastewater (MMSW) containing cations (Cd2+, Co2+, Ni2+) and oxyanions (CrVI, AsIII, VV) was incubated with each KBP under different sets of conditions. Temperature results exhibited that KBP-I, KBP-IV and KBP-V showed higher metals adsorption at 30 °C and 45 °C, respectively. However, the adsorption equilibrium was achieved for selective metals within 1 h of incubation time for all KBPs. For pH, no significant difference was observed in adsorption in MMSW due to buffering of pH by KBPs. To minimize buffering, KBP-IV and KBP-V were tested further for single-metal synthetic wastewater at two different pHs i.e. 5.5 and 8.5. KBP-IV and KBP-V were selected due to their buffering capacities and high adsorption abilities for oxyanions (pH 5.5) and divalent cations (pH 8.5), respectively indicating that chemical modifications changed and enhanced the functional groups of the keratin. X-ray Photoelectron Spectroscopy analysis was performed to demonstrate the adsorption mechanism (complexation/chelation, electrostatic attraction, or chemical reduction) for the removal of divalent cations and oxyanions by KBPs from MMSW. Furthermore, KBPs exhibited adsorption behavior for Ni2+ (qm = 2.2 mg g-1), Cd2+ (qm = 2.4 mg g-1), and CrVI (qm = 2.8 mg g-1) best described by Langmuir model with the coefficient of determination (R2) values >0.95 while AsIII (KF = 6.4 L/g) was fitted well to the Freundlich model with R2 value >0.98. Based on these findings, we anticipate that keratin adsorbents have the potential to employ at a large scale for water remediation.

Effect of fire factors (smoke, ash, charcoal and heat) on seeds of plant species.

The effect of the main fire factors (smoke, ash, charcoal and heat) can influence the germination of species through their seeds. Hence, a methodology has been devised in order to have a common protocol for those who work in this area and serve as a valuable tool to compare different species that can be beneficial or detrimental to the conditions of a forest fire. Moreover, the methodology was completed with a check of the viability of the seeds before starting the germination process and another viability test with the non-germinated seeds (dormant or dead) after carrying out the germination test. In this way, the study of the main factors of fire, in combination with the study of the viability of the seeds, can be very useful to know the reproductive behaviour of herbaceous, shrub and tree species in a forest fire scenario. In addition, from the data obtained during the germination process of the seeds, a series of explained parameters can be obtained to better interpret the data acquired from the laboratory experiment and subsequent comparison. For all these reasons, the study of the germination of species in laboratory conditions can help us understand how they behave in the field.

Evaluation of oxygen partial pressure, temperature and stripping of antioxidants for accelerated shelf-life testing of oil blends using 1H NMR.

Lipid oxidation compromises the shelf-life of lipid-containing foods, leading to the generation of unpleasant off-flavours. Monitoring lipid oxidation under normal shelf-life conditions can be time-consuming (i.e. weeks or months) and therefore accelerated shelf-life conditions are often applied. However, little is known on their impact on the lipid oxidation mechanisms. In this study, different oxygen partial pressures (PO2; 10 and 21%), temperatures (20, 30 and 40 °C), and the removal of antioxidants through stripping of the oil were tested to accelerate lipid oxidation. Increasing the incubation temperature of stripped oil blends from 30 to 40 °C reduced the onset of lipid oxidation from 4 to 2 weeks, whereas the PO2 had no impact. Surprisingly, at room temperature, an increase in PO2 resulted in a longer onset time (10 weeks under 10% oxygen, 15 weeks under 21% oxygen). We hypothesize that this is due to a shift in (initiation) mechanism. In non-stripped oil, an increase in PO2 from 10 to 21% decreased the onset time from 16 to 10 weeks (40 °C). Temperature elevations and stripping led to a shift towards more trans-trans diene hydroperoxides, as compared to the cis-trans conformation. Additionally, oil stripping led to an increase in oxidized PUFAs with three or more double bonds in which the hydroperoxide group is located between the double bond pattern, instead of on the edge of it. Lastly, it was shown that small additions of LC-PUFAs (0, 0.3, 0.6, 1.2 and 2.3%, w/w) accelerate lipid oxidation, even in relatively stable stripped oils. In conclusion, increased PO2 and slightly elevated temperatures hold fair potential for accelerated shelf-life testing of non-stripped oils with a limited impact on the lipid oxidation mechanisms, whereas stripping significantly changes propagation mechanisms.

Evidence-based strategies for the characterisation of human drug and chemical glucuronidation in vitro and UDP-glucuronosyltransferase reaction phenotyping.

Enzymes of the UDP-glucuronosyltransferase (UGT) superfamily contribute to the elimination of drugs from almost all therapeutic classes. Awareness of the importance of glucuronidation as a drug clearance mechanism along with increased knowledge of the enzymology of drug and chemical metabolism has stimulated interest in the development and application of approaches for the characterisation of human drug glucuronidation in vitro, in particular reaction phenotyping (the fractional contribution of the individual UGT enzymes responsible for the glucuronidation of a given drug), assessment of metabolic stability, and UGT enzyme inhibition by drugs and other xenobiotics. In turn, this has permitted the implementation of in vitro - in vivo extrapolation approaches for the prediction of drug metabolic clearance, intestinal availability, and drug-drug interaction liability, all of which are of considerable importance in pre-clinical drug development. Indeed, regulatory agencies (FDA and EMA) require UGT reaction phenotyping for new chemical entities if glucuronidation accounts for ≥25% of total metabolism. In vitro studies are most commonly performed with recombinant UGT enzymes and human liver microsomes (HLM) as the enzyme sources. Despite the widespread use of in vitro approaches for the characterisation of drug and chemical glucuronidation by HLM and recombinant enzymes, evidence-based guidelines relating to experimental approaches are lacking. Here we present evidence-based strategies for the characterisation of drug and chemical glucuronidation in vitro, and for UGT reaction phenotyping. We anticipate that the strategies will inform practice, encourage development of standardised experimental procedures where feasible, and guide ongoing research in the field.

Sea turtle hatchling locomotor performance: incubation moisture effects, ontogeny and species-specific patterns.

Incubation conditions are critical in determining numerous traits in reptilian neonates. This is particularly significant in species with low offspring survival such as sea turtle species, because of the extremely high predation rates that hatchlings face during their initial dispersal from nesting beaches. Hatchlings that develop in suboptimal nest environments are likely to be smaller, slower and more susceptible to predation than hatchlings from optimal nest environments. Previous studies have focused on the effects of temperature on hatchling traits, but few have investigated the effects of moisture concentrations, despite moisture levels in nests influencing hatchling size, sex, incubation duration, and hatching success. Here, we incubated eggs of three sea turtle species at various moisture levels and tested the terrestrial and aquatic locomotor performance of the resultant hatchlings during the frenzy and post-frenzy period. We also compared and evaluated the ontogeny of early locomotor performance for each species over the first months of life. Drier incubation conditions produced hatchlings that crawled more slowly and took longer to self-right than hatchlings from wetter incubation conditions. There was no difference in swimming performance associated with moisture treatments. We suggest that moisture in the nest environment during incubation may influence hatchling performance via their initial hydration levels. Thus, nest moisture influences terrestrial performance (i.e., escaping from the nest and dispersing across the beach), although upon entering the ocean hatchlings have the opportunity to rehydrate by drinking and thus, differences in locomotor performance associated with moisture treatments cease.

Comparative assessment of growth media and incubation conditions for enhanced recovery and isolation of Acinetobacter baumannii from aquatic matrices.

Acinetobacter baumannii causes serious multidrug resistant nosocomial infections around the world. This comprehensive comparative study was designed to assess the effect of temperature (30, 37 and 42 °C), incubation (aerobic and microaerobic) condition and selective [CHROMagar Acinetobacter (CHR) and Leeds Acinetobacter Medium (LAM)] and non-selective [Modified Karmali Agar (MKA)] growth media on the enhanced recovery of A. baumannii from a variety of water (agricultural, recreational, raw drinking intake source, pre-chlorinated and post-chlorinated wastewater effluent) samples spiked with a known number of A. baumannii cells. After spiking each water type with a known number of cells in 10 mL volume, the sample was passed through a membrane filter (pore size 0.45 µm) and filters were placed on different selective media plates and subjected to incubate at various incubation conditions. The results reported in this study show that for all water types tested (except post-chlorinated wastewater effluent), LAM was the most effective selective growth medium in combination with variable temperature and incubation conditions for yielding high recovery rates of A. baumannii cells. Overall, A. baumannii showed that it has a high adaptive capacity to grow on selective and non-selective growth media at different temperature and incubation conditions. The data described in this study suggest that no single incubation condition and growth media would efficiently recover A. baumannii from all environmental water types tested. This data also indicate that selective growth media and incubation condition can significantly affect the recovery of A. baumannii. Differences in recovery of A. baumannii observed in this study which appeared to be dependent on the temperature and environmental characteristics of incubation as well as the sample type, suggest the need for caution when comparing recovery using different protocols.

Environmental and Personnel Monitoring Programs-A Risk-Based Case Study of Cutibacterium acnes.

In the aseptic manufacture of parenteral drug products and low bioburden, cell, and gene therapy products, the control and monitoring of environmental- and personnel-associated microorganisms is an imperative for the confirmation of controlled conditions and the assessment of microbial risks. Environmental and personnel monitoring programs exist to assure product quality and serve as one of the several means of removing the emphasis on finished drug product testing. Therefore, these programs must adequately assess these risks and identify situations in which increased microbial risks occur. The major source of microbial risks in the controlled clean room environments for parenteral drug product manufacture are personnel. Modern microbial analytical methods, including metagenomic analysis, have identified a greater abundance of Cutibacterium acnes; traditional culture-based monitoring fails to consistently recover and assist in the identification of the potential risk that this microorganism represents. This review provides a case-study assessment of this microorganism in the context of parenteral manufacture for the purpose of assisting in the deciding the necessary controls and the potential monitoring addressing this microbial risk.

Optimizing the Cell Culture Microenvironment.

The survival, proliferation, and differentiation of cells in culture are determined not only by their intrinsic potential but also by cues provided by the permissive or restrictive microenvironment in which they reside. The robustness and reproducibility of cell culture assays and endpoints relies on the stability of that microenvironment and vigilant attention to the control of variables that affect cell behavior during culture. These often underappreciated variables include, but are not limited to, medium pH and buffering, osmolarity, composition of the gas phase, the timing and periodicity of refeeding and subculture, and the impact of fluctuations in temperature and gas phase composition on frequent opening and closing of incubator doors. This chapter briefly describes the impact of these and other variables on the behavior of cultured cells.

Study of ex-ovo embryonic development of Gallus gallus domesticus / Estudio inicial del desarrollo embrionario ex-ovo de Gallus gallus domesticus

SUMMARY: The domestic chicken is a species of bird that has been extensively studied in regard to its biology and as a model organism for science. The reproduction of the species is by the laying of fertilized eggs, which in a period of 21 days will develop a chick inside. Several methods have been described to develop embryos ex-ovo, allowing the observation and manipulation of the organism. This work has the propose to standardize a method that allows the development of the embryos inside the artificial incubation system, which has a low cost and is easy to make. In this work, 100 chicken eggs were used to study the effects of humidity, mineral supplementation, and the preincubation time of the egg on the incubation ex-ovo of the embryos. Embryo development was documented through the different days. Pulverized eggshell was selected as an optimal source to provide calcium, magnesium, phosphorus, and other minerals to the developing embryo. By providing 900-1200 mg of pulverized eggshell, 40 mL of the 0.001 % solution of benzalkonium chloride, and a preincubation time of approximately 56 h, the embryos were able to develop until 19 days, and even though they did not reach hatching, the incubation conditions that allowed the survival and development of embryos until late stages were achieved. Thus, due to the conditions established for calcium, humidity and preincubation time, in the present work, the chicks reached 19 days of development.

El pollo doméstico es una especie de ave que ha sido ampliamente estudiada en cuanto a su biología y como organismo modelo para la ciencia. La reproducción de la especie es por la puesta de huevos fecundados, que en un período de 21 días desarrollarán un polluelo en su interior. Se han descrito varios métodos para desarrollar embriones ex-ovo, permitiendo la observación y manipulación del organismo. Este trabajo tuvo como objetivo estandarizar un método que permita el desarrollo de los embriones dentro del sistema de incubación artificial, el cual tiene un bajo costo y es fácil de realizar. En este trabajo se utilizaron 100 huevos de gallina para estudiar los efectos de la humedad, la suplementación mineral y el tiempo de preincubación del huevo sobre la incubación ex-ovo de los embriones. El desarrollo embrionario se documentó a través de los diferentes días. Se seleccionó la cáscara de huevo pulverizada como una fuente óptima para proporcionar calcio, magnesio, fósforo y otros minerales al embrión en desarrollo. Al suministrar 900-1200 mg de cáscara de huevo pulverizada, 40 mL de la solución de cloruro de benzalconio al 0.001 % y un tiempo de preincubación de aproximadamente 56 h, los embriones lograron desarrollarse hasta los 19 días, y aunque no llegaron a eclosionar, los embriones lograron desarrollarse hasta los 19 días. Se lograron condiciones de incubación que permitieron la supervivencia y desarrollo de los embriones hasta etapas tardías. Así, debido a las condiciones establecidas de calcio, humedad y tiempo de preincubación, en el presente trabajo los pollitos alcanzaron los 19 días de desarrollo.

Multicenter Study on Incubation Conditions for Environmental Monitoring and Aseptic Process Simulation.

Environmental monitoring and aseptic process simulations represent an integral part of the microbiological quality control system of sterile pharmaceutical products manufacturing operations. However, guidance documents and manufacturers practices differ regarding recommendations for incubation time and incubation temperature, and, consequently, the environmental monitoring and aseptic process simulation incubation strategy should be supported by validation data. To avoid any bias coming from in vitro studies or from single-site manufacturing in situ studies, we performed a collaborative study at four manufacturing sites with four samples at each location. The environmental monitoring study was performed with tryptic soy agar settle plates and contact plates, and the aseptic process simulation study was performed with tryptic soy broth and thioglycolate broth. The highest recovery rate was obtained with settle plates (97.7%) followed by contact plates (65.4%) and was less than 20% for liquid media (tryptic soy broth 19% and thioglycolate broth 17%). Gram-positive cocci and non-spore-forming Gram-positive rods were largely predominant with more than 95% of growth and recovered best at 32.5 °C. The highest recovery of molds was obtained at 22.5 °C alone or as the first incubation temperature. Strict anaerobes were not recovered. At the end of the five days of incubation no significant statistical difference was obtained between the four conditions. Based on these data a single incubation temperature at 32.5 °C could be recommended for these four manufacturing sites for both environmental monitoring and aseptic process simulation, and a second plate could be used, periodically incubated at 22.5 °C. Similar studies should be considered for all manufacturing facilities in order to determine the optimal incubation temperature regime for both viable environmental monitoring and aseptic process simulation. LAY ABSTRACT: Microbiological environmental monitoring and aseptic process simulation confirm that pharmaceutical cleanrooms are in an appropriate hygienic condition for manufacturing of sterile drug products. Guidance documents from different health authorities or expert groups differ regarding recommendation of the applied incubation time and incubation temperature, leading to variable manufacturers practices. Some recent publications have demonstrated that laboratory studies are not relevant to determine the best incubation regime and that in situ manufacturing site studies should be used. To solve any possible bias coming from laboratory studies or single-site in situ studies, we conducted a multicenter study at four manufacturing sites with a significant amount of real environmental monitoring samples collected directly from the environment in pharmaceutical production during manufacturing operations with four solid and liquid nutrient media. These samples were then incubated under four different conditions suggested in the guidance documents. We believe that the results of our multicenter study confirming recent other single-site in situ studies could be the basis of the strategy to determine the best incubation regime for both viable environmental monitoring and aseptic process simulation in any manufacturing facility.

Effects of Incubation Conditions on Cr(VI) Reduction by c-type Cytochromes in Intact Shewanella oneidensis MR-1 Cells.

It is widely recognized that the outer membrane c-type cytochromes (OM c-Cyts) of metal-reducing bacteria play a key role in microbial metal reduction processes. However, the in situ redox status of OM c-Cyts during microbial metal reduction processes remain poorly understood. In this study, diffuse-transmission UV/Vis spectroscopy is used to investigate the in situ spectral reaction of Cr(VI) reduction by c-Cyts in intact Shewanella oneidensis MR-1 cells under different incubation conditions. The reduced c-Cyts decreased transiently at the beginning and then recovered gradually over time. The Cr(VI) reduction rates decreased with increasing initial Cr(VI) concentrations, and Cr(III) was identified as a reduced product. The presence of Cr(III) substantially inhibited Cr(VI) reduction and the recovery of reduced c-Cyts, indicating that Cr(III) might inhibit cell growth. Cr(VI) reduction rates increased with increasing cell density. The highest Cr(VI) reduction rate and fastest recovery of c-Cyts were obtained at pH 7.0 and 30°C, with sodium lactate serving as an electron donor. The presence of O2 strongly inhibited Cr(VI) reduction, suggesting that O2 might compete with Cr(VI) as an electron acceptor in cells. This study provides a case of directly examining in vivo reaction properties of an outer-membrane enzyme during microbial metal reduction processes under non-invasive physiological conditions.

Comparison of different incubation conditions for microbiological environmental monitoring.

Environmental monitoring represents an integral part of the microbiological quality control system of a pharmaceutical manufacturing operation. However, guidance documents differ regarding recommendation of a procedure, particularly regarding incubation time, incubation temperature, or nutrient media. Because of these discrepancies, many manufacturers decide for a particular environmental monitoring sample incubation strategy and support this decision with validation data. Such validations are typically laboratory-based in vitro studies, meaning that these are based on comparing incubation conditions and nutrient media through use of cultured microorganisms. An informal survey of the results of these in vitro studies performed at Novartis or European manufacturing sites of different pharmaceutical companies highlighted that no consensus regarding the optimal incubation conditions for microbial recovery existed. To address this question differently, we collected a significant amount of samples directly from air, inanimate surfaces, and personnel in pharmaceutical production and packaging rooms during manufacturing operation (in situ study). Samples were incubated under different conditions suggested in regulatory guidelines, and recovery of total aerobic microorganisms as well as moulds was assessed. We found the highest recovery of total aerobic count from areas with personnel flow using a general microbiological growth medium incubated at 30-35 °C. The highest recovery of moulds was obtained with mycological medium incubated at 20-25 °C. Single-plate strategies (two-temperature incubation or an intermediate incubation temperature of 25-30 °C) also yielded reasonable recovery of total aerobic count and moulds. However, recovery of moulds was found to be highly inefficient at 30-35 °C compared to lower incubation temperatures. This deficiency could not be rectified by subsequent incubation at 20-25 °C. A laboratory-based in vitro study performed in parallel was inconclusive. We consider our results potentially conferrable to other pharmaceutical manufacturing sites in moderate climate zones and believe that these should represent a valuable reference for definition of the incubation strategy of microbiological environmental monitoring samples. LAY ABSTRACT: Microbiological environmental monitoring confirms that pharmaceutical cleanrooms are in an appropriate hygienic condition for manufacturing of drug products. Guidance documents from different health authorities or expert groups differ regarding recommendation of the applied incubation time, incubation temperature, or nutrient media. Therefore, many pharmaceutical manufacturers perform studies that aim to identify the optimal incubation setup for environmental monitoring samples. An informal survey of the results of such studies, which had been performed at Novartis or European manufacturing sites of different pharmaceutical companies, highlighted no consensus regarding the optimal incubation conditions for microbial recovery. All these studies had been conducted in the laboratory using selections of cultured microbial strains. We tried to solve this disagreement by collecting a significant amount of real environmental monitoring samples directly from the environment in pharmaceutical production and packaging rooms during manufacturing operation. These samples were then incubated under different conditions suggested in the regulatory guidelines. We believe that the results of our study are more meaningful than laboratory-based experiments because we used environmental samples with microorganisms directly isolated from the manufacturing area. Therefore, we believe that our results should represent a valuable reference for definition of the incubation strategy of microbiological environmental monitoring samples.