The cyanobacterium Synechocystis sp. PCC6803 was obtained from the Freshwater Algae Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences. Synechocystis sp. PCC6803 was cultivated in BG-11 medium at 30°C (Chen et al., 2014 (link)). For solid BG-11 medium, 1.5% (w/v) Difco Bacto-agar (Becton Dickinson, Sparks, MD, United States), 0.3% (w/v) sodium thiosulfate, and 10 mM TES 2- [(2-hydroxy-1, 1-bis (hydroxylmethyl) ethyl) amino] ethanesulfonic acid pH 8.2 were added to BG-11 medium. The culture was bubbled with air under a light intensity of 40 μmol⋅m–2⋅s–1 (Teruo and Kaplan, 2003 (link)). Transformed strains were selected by adding 50 μg/mL kanamycin (Dingguo Company, Beijing, China) to both liquid and solid BG-11 medium. The mutant and wild type in the logarithmic growth phase were added to the liquid medium without kanamycin. Under a light intensity of 40 μmol⋅m–2⋅s–1, 30°C, after waiting for a period of growth (about 3–5 days), the OD730 value was uniformly adjusted to 1.0. Samples were collected and measured at the same time each day. Cell density was determined by measuring the optical density (OD) of the suspension at 730 nm (OD730) with a spectrophotometer (DU-70, Beckman Coulter, Brea, CA, United States).
Difco bacto agar
Manufactured by BD
Sourced in United States
Difco Bacto agar is a microbiology laboratory product used as a solidifying agent in culture media. It is a purified agar derived from red seaweed, which provides a firm, stable gel to support the growth of microorganisms.
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11 protocols using difco bacto agar
1
Cultivation and Characterization of Synechocystis sp. PCC6803
2
Culturing Synechocystis sp. PCC6803
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The cyanobacterium Synechocystis sp. PCC6803 was obtained from the Freshwater Algae Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences. Synechocystis sp. PCC6803 was cultivated in BG-11 medium (5 mM glucose) at 30°C [64 (link)]. For solid BG-11 medium, 1.5% (w/v) Difco Bacto-agar (Becton Dickinson, Sparks, MD, USA), 0.3% (w/v) sodium thiosulfate, and 10 mM TES (2-[(2-hydroxy-1, 1-bis (hydroxymethyl) ethyl) amino] ethanesulfonic acid pH 8.2) were added. The culture was bubbled with air under a light intensity of 40 μmol photon/m2/s. Transformed strains were selected by addition of 50 μg/ml kanamycin (Dingguo Company, Beijing, China) to the liquid and solid BG-11 media.
3
Motility and Nitrogen Fixation in Cyanobacteria
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The motility of baeocytes acts as a discrimination feature between unicellular pleurocapsalean and chroococcidiopsidalean taxa (Waterbury and Stanier, 1978 (link)). Thus, a motility test was applied to all isolates by transferring small proportions of the cultures on one side of agar plates with BG 11 medium. This side of the plate was fully covered with a black adhesive tape and placed in a culture cabinet (CLF Plantclimatics, Percival), as described above, in a way that the uncovered side was directed orthogonally to the light source. Over the course of 3 months, it was regularly checked if the cells are motile at any stage of their developmental cycles and move toward the light.
The nitrogen fixing abilities of the strains were also checked when they were transferred to agar plates (Difco Bacto Agar, Becton Dickinson) with nitrogen-free BG 11 medium and kept under the same cultivation conditions as described above for at least 4 months. During this time, they were regularly inspected by light microscopy and transferred to fresh BG 11 agar plates with nitrogen to check for their recovery.
The nitrogen fixing abilities of the strains were also checked when they were transferred to agar plates (Difco Bacto Agar, Becton Dickinson) with nitrogen-free BG 11 medium and kept under the same cultivation conditions as described above for at least 4 months. During this time, they were regularly inspected by light microscopy and transferred to fresh BG 11 agar plates with nitrogen to check for their recovery.
4
Arsenic Stress Response in Arabidopsis
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Surface-sterilized seeds were germinated and grown for 5 days in modified Hoagland medium containing 1% (w/v) sucrose and 1% (w/v) agar (Difco Bacto agar, BD Laboratories; http://www.bd.com ) in a growth chamber (NK system, LH-70CCFL-CT, Japan) at 23°C under continuous white light (at an irradiance of 80–100 μmol m−2 s−1; Shibasaki et al., 2009 (link)). The seedlings were grown vertically for 5 days and then transferred to new plates with or without arsenite and arsenate, and incubated for various lengths of time under continuous light at irradiance of 80–100 μmol m−2 s−1 (NK system, LH-1-120.S, Japan). After the incubation, pictures of the seedlings were taken using a digital camera (Canon Power Shot A 640; http://canon.jp/ ) and root elongation of the seedlings was analyzed by the image-analysis software ImageJ (http://rsb.info.nih.gov/ij/ ). pin1-3 was maintained as heterozygous, and homozygous seedlings were selected using the fused cotyledon phenotype as described earlier (Aida et al., 2002 (link)).
5
Inhibition of P. oryzae Mycelial Growth
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To investigate the effect of cell-free supernatants on the growth of P. oryzae mycelium, in vitro tests were carried out using plastic slides with five replications of each treatment. Sterile microscopic plastic slides covered with a thin, flat layer of water agar (Difco Bacto agar; BD Diagnostics, Le Pont-deClaix, France) were placed in a plastic Petri dish containing sterile, moist absorbent paper [39 (link)]. Subsequently, an agar plug (diameter = 8 mm) taken from an actively growing Complete Media (CM) plate of P. oryzae VT5M1 was inoculated at the center of each plastic slide. Two droplets (15 µL each) of a cell-free supernatant of the isolates were placed on two sides of the plastic slide (about 2 cm from the P. oryzae plug) while for the control treatment, two droplets of LB broth were used on both sides of the plug. All plates were incubated at 28 °C for five days. Microscopic slides were assessed by using an Olympus BX51 Microscope. Additionally, the diameter of mycelial growth was determined, and converted to the percentage growth inhibition based on the following formula:
6
Agar-based Media Preparation Protocol
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Difco Bacto Agar was purchased from BD Biosciences, San Jose, CA, USA. Other chemicals were from Wako Pure Chemical Industries, Osaka, Japan.
7
Salmonella Motility Assays: Swimming and Swarming
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Bacterial strains were grown overnight at 37°C in LB broth with or without antibiotic, as appropriate. LB broth was used because nutrient-rich broth is required for Salmonella swarming. To prepare inocula, cells were adjusted to an OD600 of 0.5 in phosphate-buffered saline (PBS). Motility plates were prepared (the night before inoculation to allow time to set) by combining LB broth and Difco Bacto agar (BD); for swimming assays, an agar concentration of 0.3% was used, and for swarming assays an agar concentration of 0.6% was used with the addition of 5 g/liter glucose. To inoculate swimming plates, a straight loop was immersed in the inoculum and then stabbed into the center of the swimming plate. These plates were then incubated for 7 h at 37°C. For swarming assays, 5-µl aliquots of inoculum were spotted on the top of swarming plates, which were incubated for 16 h at 37°C. Images were taken using a Syngene Transilluminator box, and ImageJ software was used to quantify the area covered. Data were analyzed using a two-tailed Student’s t test, and P values of ≤0.05 were considered significant.
8
Mung Bean Sprout Enumeration via Selective Agars
9
Soft Agar Colony Formation Assay
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Base layers of 7 mL of DMEM containing 0.5% Difco Bacto agar (BD Bioscience, Franklin Lakes, NJ, USA) were set in each well of 6-well plates before the addition of a second layer with 1 × 103 cells in 1.5 mL of medium containing 0.3% agar. The plates were incubated for 10 days at 37 °C in the incubator to form clones. Colony counts were made with the aid of a microscope. Five fields were selected randomly, and only clones containing over 50 cells were counted to determine the clone-forming ability of the cells.
10
Root Growth Assay for Arabidopsis
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Surface-sterilized seeds were placed on modified Hoagland medium (Baskin and Wilson, 1997) containing 1% (w/v) sucrose and 1% (w/v) agar (Difco Bacto agar; BD Laboratories, Sparks, MD, USA) (http://www.bdbiosciences.com , accessed on 30 May 2021). Two days after stratification at 4 °C in the dark, plates were transferred to a growth chamber (NK System; LH-70CCFL-CT) at 23 °C under continuous white light at an irradiance of 80–90 µmol m−2 s−1. The seedlings were grown vertically for 5 days. For root growth assay, 5-day-old seedlings were transferred to new Hoagland plates and kept at 23 °C (NK System; LH-70CCFL-CT, Tokyo, Japan) and 29 °C growth chamber (NK System; LH-1-120.S, Tokyo, Japan) for 72-h treatment under continuous white light at an irradiance of 80–90 µmol m−2 s−1. Root growth elongation at different time point was carried out based on the procedure described earlier.
All phenotypic images were taken by Canon Power Shot A640 (Canon, Japan) without flash and using micro-focus function. Root elongation was measured using ImageJ (https://imagej.nih.gov/ij/ , accessed on 30 May 2021) software.
All phenotypic images were taken by Canon Power Shot A640 (Canon, Japan) without flash and using micro-focus function. Root elongation was measured using ImageJ (
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