Oryza sativa cv. Nipponbare was used for all experiments. Rice grain was surface sterilized with 70% (v/v) ethanol (Chem-Supply, SA, Australia) for 1 min and 30% (v/v) bleach (White King, NSW, Australia) with a few drops of Tween-20 (Sigma-Aldrich, MO, United States) for 30 min and washed three times with sterile dH2O. Surface sterilized rice grain was germinated in a petri dish with moist sterile filter paper (Whatman, United Kingdom) for 7–9 days prior to transplanting to 1 l pots filled with potting mix in a glasshouse maintained at 26°C and 70% relative humidity at The University of Melbourne (Melbourne, VIC, Australia). Lighting was provided through natural lighting supplemented with a mixture of high-pressure sodium and metal halide lamps for 12 h during the day. The potting mix was prepared by mixing one part washed fine sand (Col Smith, VIC, Australia), one part propagating sand (Brunnings, VIC, Australia), two parts premium vermiculate (Exfoliators, VIC, Australia), and one part General Mix potting media (Australian Growing Solutions, VIC, Australia) fertilized with Osmocote Exact Standard 8–9 M (ICL, NSW, Australia) at a rate of 6 g/L.
Sterile filter paper
Manufactured by Cytiva
Sourced in United Kingdom, United States
Sterile filter paper is a laboratory equipment used to filter and separate liquids from particles or solids. It is designed to maintain a sterile environment during the filtration process. The core function of sterile filter paper is to provide a reliable and controlled means of filtration while preventing the introduction of contaminants.
Automatically generated - may contain errors
Lab products found in correlation
Petri dishes, by SPL Life Sciences (2 mentions)
Tween 20, by Merck Group (1 mentions)
Ethanol, by Chem-Supply (1 mentions)
Leica dm500, by Leica Microsystems (1 mentions)
Polypropylene round bottom tube, by BD (1 mentions)
Thujopsene, by Merck Group (1 mentions)
β caryophyllene, by Merck Group (1 mentions)
Hplc grade, by Carl Roth (1 mentions)
Beckman j2 21, by Beckman Coulter (1 mentions)
Bacto, by BD (1 mentions)
Potato dextrose broth (pdb), by BD (1 mentions)
14 protocols using sterile filter paper
1
Rice Cultivation Protocol for Experiments
2
Fungal Morphology Observation Protocol
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The morphology of the isolates was observed following the conventional slide culture technique. Sterile PDA was cut into small squares of (approximately 1 cm) and a block was placed on a sterile glass slide inside Petri plates (100 × 15 mm) underlaid with sterile filter paper (Whatman 1). Each agar block was inoculated with a fungal colony on the four corners using sterile needles and a coverslip aseptically placed over it. The filter paper underlay was wetted with distilled water and incubated in the dark at 25 °C for 3 days. The coverslip was gently taken off and placed on a glass slide containing a drop of lactophenol cotton blue (LPCB) and examined under the light microscope (Leica DM500, Leica Microsystems, Wetzlar, Germany) with a 40× objective lens [39 (link)].
3
Germination and Growth of Rice Seedlings
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The sterilized seeds were pregerminated in Petri dishes (90 × 20 mm; SPL Life Sciences) containing a sterile filter paper (90 mm diameter; Whatman) by complete submergence in 2.0 mM CaSO4 [4 (link), 118 (link)]. The Petri dishes were closed with 3 M Surgical Tape (Micropore) and kept in continuous darkness at a 12-h/12-h day/night cycle and 28 °C/26 °C temperatures. After 3 days, seedlings with a similar radicle and coleoptile development were selected. These seedlings had an elongated coleoptile, but no apparent first leaf yet and a radicle of at least 5 mm. The selected seedlings were transferred under sterile conditions to 14-mL polypropylene round-bottom tubes (Falcon) containing 1 mL of 2.0 mM CaSO4 either supplemented with a test agent (pure compound or extract of interest) or none (control). Each test tube contained one seedling. Test tubes were loosely capped to allow adequate aeration of the rice seedlings and were placed in the growth chamber at a 12-h/12-h day/night cycle and 28 °C/26 °C temperatures and light intensity of 170 µmol m−2 s−1.
4
Extraction of Cinnamon Bark Bioactive Compounds
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Fresh cinnamon bark was taken from the botanical garden. It was ground to a fine powder in a mechanical grinder. Ten grams of this finely powdered cinnamon was mixed with 100 ml of sterile deionized water and kept in a water bath in a round-bottomed flask at 55°C–60°C for 5 h and then filtered through sterile filter paper (Whatman®, UK). The aqueous extract was decanted, clarified by filtration through a muslin cloth, and evaporated in a porcelain dish at 40°C, which resulted in the dried extract. This dried extract was suspended in polyethylene glycol 400 (20% w: v) and sterile DW to give a final concentration of 20% w/v. The entire procedure was performed under proper aseptic conditions.[24 (link)]
5
Tajan Cultivar Seed Disinfection and Fungal Inoculation
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Tajan cultivar seeds were disinfected by applying 70 % ethanol for 1 min and then washed 5 min with sterile water, followed by 1.5% sodium hypochlorite for 1 min immediately.
Then, the seeds were washed with sterile water for 1, 3, and 5 min respectively. The seeds were placed in 9 cm diameter petri dishes containing sterile filter paper (Whatman),
after which 5 mL distilled water was added and incubated in darkness at 25 ˚C for 72 h. Germinated wheat seeds were inoculated with spore suspension of Serendipita indica(5×105 spore /1 mL) and stored for 6 h at room temperature ( 11 ).
In order to prepare the spore suspension, S. indica was cultured by transferring a plug of the young culture on complex medium (CM) in 25 ˚C and darkness for 21 days.
CM contained Glucose 20g, Peptone 2g, Salt solution: (NaNO3, Kcl, MgSO4.7H2O, KH2PO4) 50 mL, Yeast extract 1g, plus micronutrient mineral
solution Casemic acid 1g, agar 15g, and then dissolved in 1000 mL distilled water. Twenty-one days after inoculation, the Chlamydospore would be visible on the culture medium.
Mycelia were harvested by scraping the surface of the culture medium by a scalpel and filtered by lace fabric. Spore cell suspension 5×105 was prepared by distilled water
and 0.05% tween 20. The spore concentration was adjusted by a hemocytometer ( 11 ).
Then, the seeds were washed with sterile water for 1, 3, and 5 min respectively. The seeds were placed in 9 cm diameter petri dishes containing sterile filter paper (Whatman),
after which 5 mL distilled water was added and incubated in darkness at 25 ˚C for 72 h. Germinated wheat seeds were inoculated with spore suspension of Serendipita indica(5×105 spore /1 mL) and stored for 6 h at room temperature ( 11 ).
In order to prepare the spore suspension, S. indica was cultured by transferring a plug of the young culture on complex medium (CM) in 25 ˚C and darkness for 21 days.
CM contained Glucose 20g, Peptone 2g, Salt solution: (NaNO3, Kcl, MgSO4.7H2O, KH2PO4) 50 mL, Yeast extract 1g, plus micronutrient mineral
solution Casemic acid 1g, agar 15g, and then dissolved in 1000 mL distilled water. Twenty-one days after inoculation, the Chlamydospore would be visible on the culture medium.
Mycelia were harvested by scraping the surface of the culture medium by a scalpel and filtered by lace fabric. Spore cell suspension 5×105 was prepared by distilled water
and 0.05% tween 20. The spore concentration was adjusted by a hemocytometer ( 11 ).
6
Symbiotic nitrogen fixation in Lotus spp.
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Lotus burttii B-303 (seed bag numbers: 91091, 91101, and 91103) and Lotus japonicus MG-20 (seed bag number: 92147) seeds were surface sterilized with a 1.2% NaClO solution, rinsed, and soaked in water at room temperature for 2 h. Seeds were then transferred to 1/2 B5 medium agar plates and kept at 24 °C for 3 d in the dark and 3 d under a long-day photoperiod (16 h:8 h, light:dark). For shoot growth, nodulation, and infection quantification, three independent time-course experiments were conducted with 20 plants per condition and per time point. Six-day-old seedlings were transferred to sterile jars containing 300 ml of a sand:vermiculite mixture supplemented with 40 ml of FAB medium. After 2 d, each plant was inoculated with 1 ml of bacterial suspension (A600=0.005). For root hair phenotypic analysis and infection thread quantification, four independent experiments were conducted with 20 plants per condition. Six-day-old seedlings were gently placed over sterile filter paper (Whatman) on square Petri plates containing FAB medium. After 2 d, vertically grown plants were inoculated with bacterial suspensions (A600=0.05), covered with a second sheet of sterile filter paper, and incubated under a long-day photoperiod. Plants were inspected 1, 2, and 3 weeks post-inoculation (wpi).
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Lotus burttii B-303 (seed bag numbers: 91091, 91101, and 91103) and Lotus japonicus MG-20 (seed bag number: 92147) seeds were surface sterilized with a 1.2% NaClO solution, rinsed, and soaked in water at room temperature for 2 h. Seeds were then transferred to 1/2 B5 medium agar plates and kept at 24 °C for 3 d in the dark and 3 d under a long-day photoperiod (16 h:8 h, light:dark). For shoot growth, nodulation, and infection quantification, three independent time-course experiments were conducted with 20 plants per condition and per time point. Six-day-old seedlings were transferred to sterile jars containing 300 ml of a sand:vermiculite mixture supplemented with 40 ml of FAB medium. After 2 d, each plant was inoculated with 1 ml of bacterial suspension (A600=0.005). For root hair phenotypic analysis and infection thread quantification, four independent experiments were conducted with 20 plants per condition. Six-day-old seedlings were gently placed over sterile filter paper (Whatman) on square Petri plates containing FAB medium. After 2 d, vertically grown plants were inoculated with bacterial suspensions (A600=0.05), covered with a second sheet of sterile filter paper, and incubated under a long-day photoperiod. Plants were inspected 1, 2, and 3 weeks post-inoculation (wpi).
7
VOC Effects on Root Growth
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Arabidopsis seedlings were grown on AM and poplar plantlets on P20 medium in one half of the bi-compartmented Petri dishes, as described above. The adjacent compartment was equipped with sterile filter paper (1 × 1 cm; Whatman, Maidstone, UK). (–)-Thujopsene (≥97%; Sigma) and β-caryophyllene (≥80%; Sigma) were diluted to final concentrations of 1, 10, 100 and 1,000 p.p.b. in n-pentane (HPLC grade, Carl Roth GmbH & Co. KG). Thirty microlitres of the solution was dropped on the filter paper. The control plates were equipped with 30 μl of n-pentane. The treatment was performed once at the beginning of the experiment. The Petri dishes were immediately sealed with Parafilm and the plants were grown under controlled environmental conditions as described before. The LRs were quantified after 0, 4, 7 and 10 days of treatment. VOCs were collected for 6 h after 3, 7 and 10 days. Because no significant changes of the VOCs patterns were observed, only data for 10 d.h.c.c. are shown. Furthermore, Arabidopsis seedlings or poplar plantlets were grown in bi-compartmented plates with or without L. bicolor with lovastatin, as described above. The compartment with the inhibited fungus was supplemented with 30 μl of 100 p.p.b. (–)-Thujopsene solution or n-pentane as the control. The LRs were quantified after 10 days (n=5–10 plates per treatment).
8
Inhibition Assay of P. larvae by LAB
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The inhibition assay was conducted according to a previous method [62 (link)] with some modifications. The vegetative form of P. larvae ATCC 9545 strain, enterobacterial repetitive intergenic consensus (ERIC) genotype I, was cultivated at 35 ℃ for 48 h in a BHI medium. The concentration adjustment of OD600 = 0.7 was made, and the cultivated medium of P. larvae was spread on a BHI agar plate using a sterile cotton swab, and three replicate plates were used. The isolated strains of LAB were cultured on MRS agar at 37 ℃ for 3 days. Afterwards, a single colony was selected and cultured in MRS broth for 18 h at 37 ℃ in an anaerobic condition. The LAB in cultured MRS broth were collected by centrifugation at 13,000 ×g for 5 min, and the supernatant was discarded. The pellet was washed twice by suspending it in PBS solution and centrifuging under the same condition. Finally, the pellet was suspended by PBS and adjusted to OD600 = 0.7; subsequently, 10 µL of the suspended bacterial solution was dispensed onto a sterile filter paper (6 mm, Whatman, USA) and placed on a BHI agar plate, where the P. larvae were spread. The inhibition zone was observed after 48 h incubation at 37 ℃, microaerobic condition, 5% CO2.
9
Agrobacterium-Mediated Genetic Transformation of Sugarcane Axillary Buds
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Axillary bud on each sterilized billet was pricked (1 mm in depth) five times randomly using a sterile needle (Dispovan, India). Six billets carrying pricked axillary buds (PABs) were placed in a sterile beaker (1000 ml, Borosil, India) and immersed in diluted Agrobacterium suspension (600 ml i.e. 30 ml suspension in 570 ml liquid MS medium) for 5 h at 28°C. The agro-infected PABs were blotted dry using sterile filter paper (Whatman, USA) and co-cultivated on basal MS medium in jam jars at 28°C for 72 h. The co-cultivated PABs were washed in sterile water containing cefotaxime (500 mg/l) for 20 min, blotted dry and sown (one billet per bag) in polythene bags (12 cm x 20 cm) containing potting mixture (soil and farm yard manure in 3:1 ratio) with buds facing upwards. The PABs in polythene bags were incubated in transgenic glass house, watered twice a day during summers and once a day during winters for plant development. The three month-old putative T0 plants were transferred to earthen pots (22.5 cm diameter) containing potting mixture and grown to maturity. Subsequently, first clonal generation (CG1) and second clonal generation (CG2) of T0 plants were raised in plant to row fashion through billet planting in earthen pots as the crop does not set seed in this geographical region.
10
Seed Germination and Growth in CaSO4
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The sterilized seeds were pre‐germinated in Petri dishes (90 × 20 mm; SPL Life Sciences) containing a sterile filter paper (90‐mm diameter; Whatman) by complete submergence in 2.0 mM CaSO4 (Köhl, 2015 (link); Magneschi et al., 2009 (link)). The Petri dishes were closed with 3 M Surgical Tape (Micropore) and kept in continuous darkness at a 12‐h/12‐h day/night cycle and 28°C/26°C temperatures. After 2 days, seedlings with a similar coleoptile development were selected and transferred under sterile conditions in the dark under green safety light to 50‐ml conical centrifuge tubes (SPL Life Sciences). These tubes contained 50 ml 2.0‐mM CaSO4, either supplemented with the CA isomers or not (see the next section). Each 50‐ml tube contained five seedlings, and for each treatment, three tubes were filled. The tubes were closed, placed in a 50‐ml centrifuge tube rack, wrapped in four layers of tin foil, and placed back in the growth chamber at a 12‐h/12‐h day/night cycle and 28°C/26°C temperatures.
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