Escherichia coli and Agrobacterium tumefaciens strains were cultivated in liquid or on solid lysogeny broth (LB) medium [84 (link)] according to previous descriptions [69 (link),70 (link)]. V. dahliae cultures were incubated at 25 °C as previously described [60 (link),68 (link),70 (link)]. Conidia were harvested and concentrations determined as described [69 (link)] or using a Thoma counting chamber (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany).
Thoma counting chamber
Manufactured by Marienfeld
Sourced in Germany
The Thoma counting chamber is a microscope slide-based device used for cell counting and concentration measurement. It features a defined grid pattern and depth to allow for accurate quantification of cells or particles within a sample volume.
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Lab products found in correlation
6 protocols using thoma counting chamber
1
Cultivating Bacterial and Fungal Strains
2
Cultivation of Pyrococcus furiosus
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Pyrococcus furiosus was cultivated under anaerobic conditions at 85°C in ½ SME medium as described previously (Waege et al., 2010 (link)). Gelrite (1%) was added for solidification of the medium. For the comparison of the two strains, Pfu pYS3 containing the empty shuttle vector and Pfu pYS5 with tfb-rf1 under the control of a gluconeogenic promoter, a slightly modified medium without peptone and with 0.025% yeast extract was used. Growing on starch was performed in the presence of 0.1% starch and for growing on pyruvate, the starch was replaced with 40 mM Na-pyruvate. For the selection of the shuttle vector the antibiotic simvastatin was used at a concentration of 10 μM. Each strain was cultivated with starch or with pyruvate in three independent experiments until a cell density of approximately 1 × 108 cells per ml. Cell numbers were analyzed with a Thoma counting chamber (0.02 mm depth; Marienfeld, Lauda-Königshofen, Germany) using phase-contrast microscopy.
3
Cultivation and Analysis of Pyrococcus furiosus
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P. furiosus was cultivated under anaerobic conditions at 85°C–95°C in SME medium with 0.1% starch, yeast extract and peptone, as described previously [34] (link), [35] (link). For solidification, gelrite was added to a final concentration of 1%. The antibiotic simvastatin (Toronto Research Inc., North York, Canada) was dissolved in ethanol and sterilized by filtration. For a more detailed analysis of the growth behaviour of Pyrococcus strains with modified rpoH genes, bottle flasks with 20 ml ½ SME-starch medium (supplemented with 10 µM simvastatin) were used and incubated at 95°C. Cell numbers were analyzed with a Thoma counting chamber (0.02-mm depth; Marienfeld, Lauda-Königshofen, Germany) using phase-contrast microscopy. All experiments were done in triplicate and the statistical mean value was used for plotting.
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P. furiosus was cultivated under anaerobic conditions at 85°C–95°C in SME medium with 0.1% starch, yeast extract and peptone, as described previously [34] (link), [35] (link). For solidification, gelrite was added to a final concentration of 1%. The antibiotic simvastatin (Toronto Research Inc., North York, Canada) was dissolved in ethanol and sterilized by filtration. For a more detailed analysis of the growth behaviour of Pyrococcus strains with modified rpoH genes, bottle flasks with 20 ml ½ SME-starch medium (supplemented with 10 µM simvastatin) were used and incubated at 95°C. Cell numbers were analyzed with a Thoma counting chamber (0.02-mm depth; Marienfeld, Lauda-Königshofen, Germany) using phase-contrast microscopy. All experiments were done in triplicate and the statistical mean value was used for plotting.
4
Inoculum Preparation for Microbial Fermentations
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The following microorganism strains were used for the experimental fermentations: Lachancea thermotolerans Concerto™ (Hansen, Horsholm, Denmark), Saccharomyces bayanus EC1118 (Lallemand, Montreal, QC, Canada), Lactiplantibacillus plantarum (former Lactobacillus plantarum) ML Prime™ (Lallemand, Montreal, QC, Canada) and Oenococcus oeni Alpha® (Lallemand, Montreal, QC, Canada).
The inocula were prepared by rehydrating 200 mg of the corresponding commercial strain product in 20 mL of sterilized water under sterile laboratory conditions. The number of cells was evaluated by cell counting using a Thoma counting chamber (Paul Marienfeld, Lauda-Königshofen, Germany) in a Leica DM 750 microscope (Wetzlar, Germany) in the initial solution of 10 g/L of the commercial products. The initial inocula volume was adjusted for the different treatments taking into account the initial population of the rehydrated commercial product and the objective population of the different treatments.
The inocula were prepared by rehydrating 200 mg of the corresponding commercial strain product in 20 mL of sterilized water under sterile laboratory conditions. The number of cells was evaluated by cell counting using a Thoma counting chamber (Paul Marienfeld, Lauda-Königshofen, Germany) in a Leica DM 750 microscope (Wetzlar, Germany) in the initial solution of 10 g/L of the commercial products. The initial inocula volume was adjusted for the different treatments taking into account the initial population of the rehydrated commercial product and the objective population of the different treatments.
5
Microalgae Cell Counting Protocol
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Cell counting was performed before inoculation and at the end of experiments. A Marienfeld Thoma counting chamber was used with an inverted microscope in phase contrast imaging mode. First, a glass coverslip was placed over the counting chamber such that there is 100 µm between the counting grid and the glass slide. Then, 10 µl of control‐diluted samples was injected under the coverslip with a precision pipette. The Thoma chamber has 16 large squares in the counting grid in which the cells are counted per square and summed up. Each sample was counted three times and the average is used to calculate the original concentration.
On the last day of the culture experiments, samples are extracted from the wells. They were first actuated at 2000 rpm for 1 min to resuspend microalgae into the medium. All content was then extracted with a sterile syringe and stored in a 0.5 ml vial. To remove the remaining attached algae as thoroughly as possible, 0.5 ml nutrient medium was used to wash the well with repeated in and out motion using a pipette and is also stored in a 0.5 ml vial. Finally, the same counting procedure was applied to determine the final microalgae concentration in each well.
On the last day of the culture experiments, samples are extracted from the wells. They were first actuated at 2000 rpm for 1 min to resuspend microalgae into the medium. All content was then extracted with a sterile syringe and stored in a 0.5 ml vial. To remove the remaining attached algae as thoroughly as possible, 0.5 ml nutrient medium was used to wash the well with repeated in and out motion using a pipette and is also stored in a 0.5 ml vial. Finally, the same counting procedure was applied to determine the final microalgae concentration in each well.
6
Growth of P. furiosus under CuSO4 stress
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P. furiosus was cultivated under anaerobic conditions in 40 ml ½ SME medium supplemented with 0.1 % yeast extract, 0.1 % peptone and 40 mM pyruvate at 95°C, as described previously (Fiala and Stetter, 1986 (link); Waege et al., 2010 (link)). For growth comparison experiments, the medium was supplemented with different CuSO4 concentrations (compare Figure 2 ) and each condition for MURPf52 (parental strain) and MURPf74 (ΔcopR strain) was recorded in biological triplicates during 48 hours of incubation by measuring the turbidity changes in situ using a photodiode and a LED with 850 nm as light source. The recorded values were converted to cell/ml by using a calibration curve with known cell concentrations, calculated in a Thoma counting chamber (0.02-mm depth; Marienfeld, Lauda-Königshofen, Germany) using phase-contrast microscopy.
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