Cultivation Plates Research Articles

Design and Experiment of Automatic Transport System for Planting Plate in Plant Factory

In the plant factories using stereoscopic cultivation systems, the cultivation plate transport equipment is an essential component of production. However, there are problems, such as high labor intensity, low levels of automation, and poor versatility of existing solutions, that can affect the efficiency of cultivation plate transport processes. To address these issues, this study designed a cultivation plate transport system that can automatically input and output cultivation plates, and can flexibly adjust its structure to accommodate different cultivation frame heights. We elucidated the working principles of the transport system and carried out structural design and parameter calculation for the lift cart, input actuator, and output actuator. In the input process, we used dynamic simulation technology to obtain an optimum propulsion speed of 0.3 m·s−1. In the output process, we used finite element numerical simulation technology to verify that the deformation of the cultivation plate and the maximum stress suffered by it could meet the operational requirements. Finally, operation and performance experiments showed that, under the condition of satisfying the allowable amount of positioning error in the horizontal and vertical directions, the horizontal operation speed was 0.2 m·s−1, the maximum positioning error was 2.87 mm, the vertical operation speed was 0.3 m·s−1, and the maximum positioning error was 1.34 mm. Accordingly, the success rate of the transport system was 92.5–96.0%, and the operational efficiency was 176–317 plates/h. These results proved that the transport system could meet the operational requirements and provide feasible solutions for the automation of plant factory transport equipment.

Разработка перфорированного элемента рабочего органа глубокорыхлителя: топологическая оптимизация

The development or modernization of tillage machinery aims to increase its service life and efficiency and reduce the negative impact on the soil. In order to increase the efficiency of agricultural machines, the author attempted to optimize the working element of a subsoler - its plate - through topological and parametric modeling. The subsoiler plate with the mass of 1.925 kg with maximum stresses 176.8 MPa was used as a base model. The author determined the plate zones having the least influence on the stiffness and strength of the implement structure. When designing the modernized plate, the following parameters were taken into account: safety factor on the ultimate strength - 1.5 to 2; maximum mass reduction - less than 50%, minimum reduction - more than 10%. The plate is manufactured by laser cutting from 09G2S or 30XGSA steel. The manufacturing process, strength and wear characteristics, and cost were considered to be not lower than the base variant. The algorithm developed in the SysML system modeling language helped systematize the process and establish functional and non-functional requirements and limitations. Using Autodesk Fusion 360 automatic design system, the author developed the rational geometric shape of the soil cultivation plate of the subsoiler with a mass of 1.585 kg and a maximum stresses of 169.5 MPa according to this algorithm. Topological optimization resulted in a 17.67% reduction in the part’s weight at the second iteration while maintaining strength and wear resistance characteristics. By determining the thickness of the element, the author established compliance with the safety factor requirement. Samples of subsoiler plates with thickness of 16 mm were made of 09G2S steel. To confirm the calculated strength characteristics it is necessary to carry out laboratory and field tests of the prototype of the lightweight design.

Assembly and Operation of a Cooling Stage to Immobilize C. elegans on Their Culture Plates.

High-resolution in vivo microscopy approaches can reveal subtle information and fine details inside the model animal Caenorhabditis elegans (C. elegans), but require strong animal immobilization to prevent motion blur in the images. Unfortunately, most current immobilization techniques require substantial manual effort, rendering high-resolution imaging low-throughput. Immobilization of C. elegans is greatly simplified by using a cooling approach that can easily immobilize entire populations directly on their cultivation plates. The cooling stage can establish and maintain a wide range of temperatures with a uniform distribution on the cultivation plate. In this article, the whole process of building the cooling stage is documented. The aim is that a typical researcher can build an operational cooling stage in their laboratory following this protocol without difficulty. Utilization of the cooling stage following three protocols is shown, and each protocol has advantages for different experiments. Also shown is an example cooling profile of the stage as it approaches its final temperature and some helpful tips in using cooling immobilization.

High-throughput submicron-resolution microscopy of Caenorhabditis elegans populations under strong immobilization by cooling cultivation plates

Despite its profound impact on biology, high-resolution invivo microscopy largely remains low throughput because current immobilization techniques require substantial manual effort. We implement a simple cooling approach to immobilize entire populations of the nematode Caenorhabditis elegans directly on their cultivation plates. Counterintuitively, warmer temperatures immobilize animals much more effectively than the colder temperatures of prior studies and enable clear submicron-resolution fluorescence imaging, which is challenging under most immobilization techniques. We demonstrate 64× z-stack and time-lapse imaging of neurons in adults and embryos without motion blur. Compared to standard azide immobilization, cooling immobilization reduces the animal preparation andrecovery time by >98%, significantly increasing experimental speed. High-throughput imaging of a fluorescent proxy in cooled animals and direct laser axotomy indicate that the transcription factor CREB underlies lesion conditioning. By obviating individual animal manipulation, our approach could empower automated imaging of large populations within standard experimental setups and workflows.

Influence of glucose on swarming and quorum sensing of Dickeya solani.

Dickeya solani is a pathogen most frequently responsible for infecting potato plants in Europe. As in the case of most plant pathogens, its ability to colonize and invade the host depends on chemotaxis and motility. The coordinated movement of Dickeya over solid surfaces is governed by a quorum sensing mechanism. In D. solani motility is regulated by ExpI-ExpR proteins, homologous to luxI-luxR system from Vibrio fisheri, in which N-acyl-homoserine lactones (AHLs) serve as signaling molecules. Moreover, in many Gram-negative bacteria motility is coupled with central metabolism via carbon catabolite repression. This enables them to reach more nutrient-efficient niches. The aim of this study was to analyze the swarming motility of D. solani depending on the volume of the medium in the cultivation plate and glucose content. We show that the ability of this bacterium to move is strictly dependent on both these factors. Moreover, we analyze the production of AHLs and show that the quorum sensing mechanism in D. solani is also influenced by the availability of glucose in the medium and that the distribution of these signaling molecules are different depending on the volume of the medium in the plate.

In Situ Agarose Microfabrication Technology Using Joule Heating of Micro Ionic Current for On-Chip Cell Network Analysis.

Agarose microfabrication technology is one of the micropatterning techniques of cells having advantages of simple and flexible real-time fabrication of three-dimensional confinement microstructures even during cell cultivation. However, the conventional photothermal etching procedure of focused infrared laser on thin agarose layer has several limitations, such as the undesired sudden change of etched width caused by the local change of absorbance of the bottom surface of cultivation plate, especially on the indium-tin-oxide (ITO) wiring on the multi-electrode array (MEA) cultivation chip. To overcome these limitations, we have developed a new agarose etching method exploiting the Joule heating of focused micro ionic current at the tip of the micrometer-sized capillary tube. When 75 V, 1 kHz AC voltage was applied to the tapered microcapillary tube, in which 1 M sodium ion buffer was filled, the formed micro ionic current at the open end of the microcapillary tube melted the thin agarose layer and formed stable 5 μm width microstructures regardless the ITO wiring, and the width was controlled by the change of applied voltage squared. We also found the importance of the higher frequency of applied AC voltage to form the stable microstructures and also minimize the fluctuation of melted width. The results indicate that the focused micro ionic current can create stable local spot heating in the medium buffer as the Joule heating of local ionic current and can perform the same quality of microfabrication as the focused infrared laser absorption procedure with a simple set-up of the system and several advantages.

Single Spheroid Metabolomics: Optimizing Sample Preparation of Three-Dimensional Multicellular Tumor Spheroids.

Tumor spheroids are important model systems due to the capability of capturing in vivo tumor complexity. In this work, the experimental design of metabolomics workflows using three-dimensional multicellular tumor spheroid (3D MTS) models is addressed. Non-scaffold based cultures of the HCT116 colon carcinoma cell line delivered highly reproducible MTSs with regard to size and other key parameters (such as protein content and fraction of viable cells) as a prerequisite. Carefully optimizing the multiple steps of sample preparation, the developed procedure enabled us to probe the metabolome of single MTSs (diameter range 790 ± 22 µm) in a highly repeatable manner at a considerable throughput. The final protocol consisted of rapid washing of the spheroids on the cultivation plate, followed by cold methanol extraction. 13C enriched internal standards, added upon extraction, were key to obtaining the excellent analytical figures of merit. Targeted metabolomics provided absolute concentrations with average biological repeatabilities of <20% probing MTSs individually. In a proof of principle study, MTSs were exposed to two metal-based anticancer drugs, oxaliplatin and the investigational anticancer drug KP1339 (sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)]), which exhibit distinctly different modes of action. This difference could be recapitulated in individual metabolic shifts observed from replicate single MTSs. Therefore, biological variation among single spheroids can be assessed using the presented analytical strategy, applicable for in-depth anticancer drug metabolite profiling.

Antioxidant Characterization and Biological Effects of Grape Pomace Extracts Supplementation in Caenorhabditis elegans

The aim of this work was to evaluate the biological activity of four grape pomace (GP) extracts that are rich in polyphenols using C. elegans as an in vivo model. Different concentrations of the GP extracts were assessed for their effects on the resistance of C. elegans against thermally induced oxidative stress, accumulation of reactive oxygen species (ROS), and lifespan. The cultivation of C. elegans with relatively low concentrations of GP extracts increased their resistance against thermal stress and prolonged their lifespan, while high levels displayed detrimental effects. In the studied extracts, maximum protection was observed for levels of polyphenols around 7 to 9 µg gallic acid equivalents per cultivation plate. The obtained results suggested that small changes in the ROS levels could have beneficial effects, although further studies are required to fully understand the impact of the extracts and assayed doses on ROS levels to explain the mechanism that is involved in the observed effects.

Rapid and reliable identification of waterborne Legionella species by MALDI-TOF mass spectrometry

Detection and enumeration of Legionella bacteria in drinking water is regulated in Germany by ISO 11731-2. The mandatory method for species identification employs parallel subculturing of suspicious colonies on selective media requiring the handling of a large number of cultivation plates. After changes to the drinking water quality regulation in Germany in 2012 the demand for Legionella contamination testing increased drastically. A more reliable, faster and less laborious method for species identification is therefore desirable. Matrix-assisted laser desorption ionization followed by time of flight detection mass spectrometry (MALDI-TOF MS) promises an accelerated identification of bacteria with high reliability and reduced expenditure. Our study shows that MS-based species identification results are in full concordance with cultural and biochemical detection and differentiation and that valuable additional information can be gained, even though the ISO regulation demands an extended incubation period for primary bacterial cultures that is actually in contrast to the prerequisites of the MALDI Biotyper system. In addition, the established identification algorithm is very economical and improves time-to-result. Based on our findings, the amendment of MALID-TOF MS identification to ISO11731-2 as an alternative identification method should be taken into consideration.

Aerosolized Microbes from Organic Rich Materials: Case Study of Bat Guano from Caves in Romania

Cave air, bat guano, and swabs of bat fur from caves with bat guano in Romania were analyzed by using RIDAHCOUNT cultivation plates and standard selective media for Staphylococcus and Streptococcus. The samples of guano varied in concentration of cultivable chemoheterotrophic bacteria (max. 1.9310 10 CFU/g), coliforms (max. 2.2310 8 CFU/g), Escherichia coli (max. 1.0310 8 CFU/g), and yeasts and molds (max. 1.7310 7 CFU/g). The gravity-settling principle was applied to sample airborne microorganisms, and a new method was developed for evaluation of aerosolization potential. In cave air, the concentration of total bacteria was higher than yeast and molds. In addition to coliforms, enterobacteria, E. coli, and unidentified cultivable bacteria in the air samples, we also identified Chryseomonas luteola, Klebsiella pneumoniae, Micrococcus, Salmonella, Staphylococcus, and Streptococcus. In the experiment that provoked microbial aerosolization from guano, 3.35% of total cultivable fungi were aerosolized, up to 0.10% of bacteria, and 0.00% of E. coli. The concentration of Staphylococcus in the air exceeded counts of Streptococcus. The highest concentrations of airborne microorganisms were on the ground level. Using cultivation plates as a robust method we demonstrated that the relative proportion of microbial subgroups in the air remained constant in different seasons, with lower concentrations of airborne microbiota in the autumn. Caves as simplified natural systems demonstrated complex relationships between atmospheric parameters and microorganisms. Bats introduce into caves varying, but not negligible, concentrations of microbes on their fur. Caves with guano had relative high concentration of airborne microbes that may represent a biohazard for animals and humans.

Shift of Bacterial Community in Synanthropic Mite Tyrophagus putrescentiae Induced by Fusarium Fungal Diet

Background Tyrophagus putrescentiae (Acari: Astigmata) and Fusarium sp. co-occur in poorly managed grain. In a laboratory experiment, mite grazing resulted in significant reduction of fungal mycelium on cultivation plates. The destruction of mycelium appeared to be a result of an interaction between the mites, fungi and associated bacteria.Methodology and Principal FindingsA laboratory experiment was performed to simulate a situation of grain multiinfested by mites and Fusarium fungi. Changes of mite-associated bacterial community in T. putrescentiae were described in 3 habitats: (i) T. putrescentiae mites from a rearing diet prior to their transfer to fungal diet; (ii) fungal mycelium before mite introduction; (iii) mites after 7 day diet of each Fusarium avenaceum, F. culmorum, F. poae and F. verticillioides. Bacterial communities were characterized by 16 S rRNA gene sequencing. In total, 157 nearly full-length 16 S rRNA gene sequences from 9 samples representing selected habitats were analyzed. In the mites, the shift from rearing to fungal diet caused changes in mite associated bacterial community. A diverse bacterial community was associated with mites feeding on F. avenaceum, while feeding on the other three Fusarium spp. led to selection of a community dominated by Bacillaceae.Conclusions/SignificanceThe work demonstrated changes of bacterial community associated with T. putrescentiae after shift to fungal diets suggesting selection for Bacillaceae species known as chitinase producers, which might participate in the fungal mycelium hydrolysis.

Direct mass spectrometric screening of antibiotics from bacterial surfaces using liquid extraction surface analysis

There is a need to find new antibiotic agents to fight resistant pathogenic bacteria. To search successfully for novel antibiotics from bacteria cultivated under diverse conditions, we need a fast and cost-effective screening method. A combination of Liquid Extraction Surface Analysis (LESA), automated chip-based nanoelectrospray ionization, and high-resolution mass or tandem mass spectrometry using an Orbitrap XL was tested as the screening platform. Actinobacteria, known to produce well-recognized thiazolyl peptide antibiotics, were cultivated on a plate of solid medium and the antibiotics were extracted by organic solvent mixtures from the surface of colonies grown on the plate and analyzed using mass spectrometry (MS). LESA combined with high-resolution MS is a powerful tool with which to extract and detect thiazolyl peptide antibiotics from different Actinobacteria. Known antibiotics were correctly detected with high mass accuracy (<4 ppm) and structurally characterized using tandem mass spectra. Our method is the first step toward the development of a novel high-throughput extraction and identification tool for antibiotics in particular and natural products in general. The method described in this paper is suitable for (1) screening the natural products produced by bacterial colonies on cultivation plates within the first 2 min following extraction and (2) detecting antibiotics at high mass accuracy; the cost is around 2 Euro per sample.

The use of glass beads cultivation system to study the global effect of the ppk gene inactivation in Streptomyces lividans

The glass beads cultivation system developed in our laboratory for physiological studies of filamentous microorganisms supports differentiation and allows complete recovery of bacterial colonies and their natural products from cultivation plates. Here, we used this system to study the global effect of ppk gene disruption in Streptomyces lividans. The ppk encoding the enzyme polyphosphate kinase (P) catalyses the reversible polymerisation of gamma phosphate of ATP to polyphosphates. The resulting are phosphate and energy stock polymers. Because P activity impacts the overall energetic state of the cell, it is also connected to secondary metabolite (e.g. antibiotic) biosynthesis. We analysed the global effects of the disruption of this gene including its influence on the production of pigmented antibiotics, on morphological differentiation, on the levels of ATP and on the whole cytoplasmic protein expression pattern of S. lividans. We observed that the S. lividans ppk mutant produced antibiotics earlier and in greater amount than the wild-type (wt) strain. On the other hand, we did not observe any obvious effect on colony morphological development. In agreement with the function of Ppk, we detected much lower levels of ATP in ppk- mutant than in the wt strain. Proteomic analysis revealed that the genes that were influenced by ppk inactivation included enzymes involved in carbon or nitrogen metabolism, phosphate transport and components of the cell translational machinery. We showed that the synthesis of translation elongation factor Tu is during sporulation much higher in ppk- mutant than in wild-type strain.

Problems with detection of proteolytic microorganisms and their undesirable activities in milk

Occurrence of proteolytic enzymes in milk is often associated with technological problems and sensory, rheological and functional defects of final dairy products. Thus, the simple, cost-effective and available laboratory method for evaluation of undesirable proteolysis risk is needed. In our work we have tested cultivation plate methods and chemical methods (formol titration, ammonium reflectometric determination, the Kjeldahl method, the agar-well diffusion assay and spectrofotometry after cleavage of azo-casein) to choose the proper ones which can provide information on undesirable proteolytic changes especially in raw milk. Although the microbiological analyses cannot detect enzymes indigenous to milk, but only the quantity of producers of microbial enzymes, they seem to be the most acceptable, particularly usage of the Glucose-Trypton-Yeast Extract agar with 10% vol. of sterile milk added before pouring onto plates (incubation at 30&amp;deg;C for 72 h). The chemical methods are not sensitive enough to analyse the real milk samples.

Impact of regenerated silk protein membrane on the cytokine expression of transfected human corneal epithelium cells

The purpose of this research was to study the influence of the regenerated silk fibroin film (SF) on cytokines expression of transfected human corneal epithelial cells (HCECs) and to investigate the possibility of constructing biomaterial complex using SF, modified by transgenic cells. Empirical study.Ad-VEGF(165) was injected into the limbus of a rabbit's cornea to induce cornea neovascularization (CNV). CNV was evaluated by growth areas and VEGF characteristic was evaluated by immunohistochemistry. HCECs was cultivated on silk protein membrane in the cell cultivation plate. Modality of cells, activity of cell proliferation and infection efficiency of Ad-VEGF(165) were monitored to evaluate the biocompatibility of silk fibroin. The angiogenesis-related cytokines in the cell cultivation supernatant was measured using ELISA method such as vascular endothelial growth factor (VEGF), angiogenin 1 (Ang1), epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta) in the supernatant (Two-way analysis of variance). (1) The area of corneal neovascularization was observed to be (7.60 +/- 1.12) mm(2) at 1 week after Ad-VEGF(165) was injected and it became (12.28 +/- 2.54) mm(2) another three weeks later. Positive expression of VEGF in corneal stromal was observed by immunohistochemistry at 3 d, 1 week and 1 month after injection. (2) There was no difference noticed in amorphous, growth curve and infection efficiency of Ad-VEGF(165) between both cells culture conditions of silk protein membrane and plate cultivation. (3) After transfection, the concentration of VEGF, Ang1, EGF and TGF-beta expressions in the corneal epithelium cell cultivation supernatant with silk protein membrane as carriers was (721.67 +/- 66.97) ng/L, (1042.67 +/- 315.81) ng/L, (2421.00 +/- 0.00) ng/L, and (313.33 +/- 34.06) ng/L respectively; and the concentration of each of the aforementioned expression was (721.67 +/- 66.97) ng/L, (860.33 +/- 315.81) ng/L, (1960.33 +/- 797.90) ng/L, and (278.00 +/- 53.11) ng/L without using silk protein membrane as carriers. The increase of VEGF (F = 168.16, P < 0.0001), EGF (F = 52.76, P < 0.0001), Ang1 (F = 12.47, P = 0.001), and TGF-beta (F = 0.008, P = 0.932) in the Ad-VEGF(165) group was considered statistically significant; however, there was no evident change in the concentration of VEGF (F = 0.071, P = 0.793), EGF (F = 0.563, P = 0.465), Ang1 (F = 0.14, P = 0.714), and TGF-beta (F = 0.008, P = 0.932)expressions in the corneal epithelium cell cultivation supernatant both with or without using silk protein membrane as carriers. Same as cell HCECs culturing in the cultivation plate, through SF application, VEGF(165) destination gene could be high-level expressed in the supernatant having which the HCECs is cultured on SF, and in addition, the angiogenesis-related cytokines content of Ang1, EGF, and TGF-beta autocrine in the HCECS cultivation supernatant could be high-level expressed as well.

Diversity of marine gliding bacteria in Thailand and their cytotoxicity

Eighty-four marine gliding bacteria were isolated from specimens collected in the Gulf of Thailand and the Andaman Sea. All exhibited gliding motility and swarm colonies on cultivation plates and they were purified by subculturing and micromanipulator techniques. Their 16S rRNA genes were amplified by the polymerase chain reaction (PCR) technique. The phylogenetic analysis indicated that the represented isolates can be separated into six different clads (gr 1 - gr 6) within the Cytophaga-Flavobacterium-Bacteriodes (CFB) group. Group 1 formed a remote linear, with only 90% sequence similarity, from Flavobacteriaceae bacterium which indicated a potentially novel taxonomic group. Groups 2 and 3 were identified as the recently proposed Tenacibaculum mesophilum and Fulvivirga kasyanovii respectively. Groups 4, 5 and 6, consisting of the largest number of the members, were identified as Rapidithrix thailandica , Aureispira marina and Aureispira maritima respectively. The isolates were cultivated in four different cultivation media (Vy/2, RL 1, CY and SK) and the crude extracts were submitted to screen cytotoxicity using a sulphorodamine B (SRB) assay. The results from cytotoxic screening showed that groups 2, 4 and 6 were capable of producing the cytotoxic metabolites against selected human cell lines (breast adenocarcinoma (MCF-7), colon cancer (HT-29), cervical cancer (HeLa) and oral cancer (KB)). However, groups 1, 3 and 5 did not produce metabolites with cytotoxicity when cultivated in the same cultivation media as the previous groups. CY medium was the only cultivation medium which could yield the cytotoxic metabolites against MCF-7.

THE RATE OF SHOOT REGENERATION FROM APPLE (MALUS DOMESTICA) LEAVES DEPENDING ON THE IN VITRO CULTURE CONDITIONS OF THE SOURCE PLANTS

Efficiency of somatic organogenesis depends to a great degree on the physiological status of the in vitro source plants. Important factors are the cultivation conditions – nutrient media, cultivation plates, gas-exchange possibilities, temperature and light regime. The aim of the present study was to investigate the effect of conditions for cultivating the source plants (cultivation plates and nutrient media) on the regeneration capacity of the apple (Malus domestica) leaf explants. Studies were carried out with in vitro propagated plants of the apple cultivar ‘Chadel’. The source microplants were cultivated on MS nutrient media with the addition of sucrose 10 g/L and modified MS with added ВАP 2.5 μM, IAA 0.0571 μM and sucrose 30 g/L. Two types of cultivation plates were used in the experiment – glass jars and plastic vessels with a gas-permeating cover. Leaf segments of the source plants that had been cultivated in each of the four combinations of nutrient media/cultivation plates were set for regeneration. The nutrient media for regeneration were based on MS with added TDZ (7.5 μM), sucrose 10 g/L, sorbitol 30 g/L and different IAA content – 5, 10, 15 and 20 μM. The explants were cultivated in darkness for 15 days, after which – at a photoperiod of 16/8 hours (40 μmol m -2 s -1 PAR) at a temperature of 22±2°С for 25 days. The best results (over 80% regeneration) were achieved when using explants of plants grown in plastic vessels with a gas-permeating cover in modified MS nutrient medium.

Evaluation of errors and limits of the 63-μm house-dust-fraction method, a surrogate to predict hidden moisture damage

BackgroundThe aim of this study is to analyze possible random and systematic measurement errors and to detect methodological limits of the previously established method.FindingsTo examine the distribution of random errors (repeatability standard deviation) of the detection procedure, collective samples were taken from two uncontaminated rooms using a sampling vacuum cleaner, and 10 sub-samples each were examined with 3 parallel cultivation plates (DG18). In this two collective samples of new dust, the total counts of Aspergillus spp. varied moderately by 25 and 29% (both 9 cfu per plate). At an average of 28 cfu/plate, the total number varied only by 13%.For the evaluation of the influence of old dust, old and fresh dust samples were examined. In both cases with old dust, the old dust influenced the results indicating false positive results, where hidden moist was indicated but was not present. To quantify the influence of sand and sieving, 13 sites were sampled in parallel using the 63-μm- and total dust collection approaches. Sieving to 63-μm resulted in a more then 10-fold enrichment, due to the different quantity of inert sand in each total dust sample.ConclusionThe major errors during the quantitative evaluation from house dust samples for mould fungi as reference values for assessment resulted from missing filtration, contamination with old dust and the massive influence of soil. If the assessment is guided by indicator genera, the percentage standard deviation lies in a moderate range.

Culture media for enterococci and group D-streptococci

Lancefield group D-streptococci are contaminants of various food commodities, especially those of animal origin. They encompass the new genus Enterococcus comprising 13 known species and some species of streptococci which have their habitat in the intestine of animals, e.g. Streptococcus bovis, suis and equinus. The serologically based grouping may no longer constitute the best definition for streptococci from the food chain. Food hygiene monitoring systems using enterococci as indicators need reliable methods for selective cultivation and identification of marker strains. Up to now more than 100 modifications of selective media have been described for isolating streptococci or enterococci from various specimens. The selection of a medium requires either experience or consultation. It depends on the kind of specimen, the method of cultivation (plate count or membrane filter) and whether or not the habitat is heavily contaminated with other organisms. The choice of media is made more difficult as commercial versions of the same culture medium may vary in recipe and/or performance from producer to producer. Therefore, reviewing the literature may help in the choice of medium and confirmation tests. The selectivity and productivity of some commonly used or cited media are reported here, partly based on our own experience: citrate azide tween carbonate agar (CATC), kanamycin aesculin azide agar (KAA) and M-enterococcus agar (ME) including earlier results with aesculin bile azide agar (ABA), and thallous acetate tetrazolium glucose agar (TITG). No medium was completely selective for all group D-streptococci or for all enterococci but some media were highly selective for a single Enterococcus species, e.g., for E. faecalis which serves as indicator of human pollution. Confirmatory tests must be carried out when experience in the evaluation procedure is limited. Selective media for enterococci should be used only after or while checking in parallel their selectivity and productivity against appropriate test organisms.

Incubation time for the cultivation of Renibacterium salmoninarum from Atlantic salmon, Salmo salar L., broodfish

Abstract. Cultivation plates for the primary isolation of Renibacterium salmoninarum were incubated for 12 or more weeks over 3.5 years. The incubation time for sample of two groups of Atlantic salmon, Salmo solar L., broodfish on a selective agar medium is reported. Samples turned positive over a long time‐span. but the longest incubation time recorded for growth of the bacterium was 19 week. In one group, 79% of all positive samples form fish with macroscopic signs of bacterial kidney disease were positive after 6 weeks of incubation and the highest incidence of positive samples occured during the sixth week. From covertly infected fish within the same group.42% of sample were positive after 6 weeks of incubation, and the highest incidence of positive sample occured during the sixth and the ninth weeks of incubation. The growth pattern of the bacterium at primary isolation was characterized by a prolonged lag phase followed by a relatively growth in one week from microscopic to macroscopic size of the colonies.