Protocol
- Culturing and maintenance of Penium margaritaceum
- Crofixation and freeze substitution of Penium for transmission electron microscopy
Culturing and maintenance of Penium margaritaceum
Penium is easily maintained in the laboratory in both liquid and agar-solidified cultures. It grows best at temperatures between 18oC and 24oC but can survive temperatures as high as 33°C. Using the culturing guidelines described below, log phase cultures may be obtained within 10 to 21 days of sub-culture initiation.
1) General considerations: Prepare all solutions using ultrapure water (18 megaohm) and analytical-grade reagents. Store stock solutions and medium in a refrigerator unless otherwise stated. Follow all institutional and government waste-disposal protocols for the laboratory.
2) Obtaining Penium margaritaceum: Penium is available from the Skidmore College Algal Culture Collection (contact D. Domozych, Department of Biology, ddomoz@skidmore.edu) or the Coimbra Culture Collection of Algae (http://acoi.ci.uc.pt/index.php).
3) Hardware required for culturing: The following hardware is required for culturing Penium: Laminar-flow hood for maintaining aseptic conditions, 200 mL Nunc tissue culture flasks (non-coated) or 125/250 mL glass flasks with cotton stoppers, 15 mL sterile centrifuge tubes, 50 mL sterile centrifuge tubes, refrigerated tabletop centrifuge that accommodates 15 mL and 50 mL centrifuge tubes, 10 mL sterile plastic pipettes and pipetor, sgerile plastic petri dishes, cell culture spreader or glass rod hockey stick.
4) Medium types: The basic medium for Penium is Woods Hole (WH) medium or MBL medium; see http://www.marine.csiro.au/microalgae/methods/Media%20CMARC%20recipes.htm#MBL for recipe. The sodium silicate macronutrient is not necessary when growing Penium. The pH of the medium should be adjusted to 7.2 and using 0.1N NaOH or HCl. Penium may also be grown at pH 6. To 100 mL of WH, add 0.5 g of MES and dissolve. Adjust pH to 6 with 0.1N NaOH.
All medium is dispensed in 500 mL stoppered glass bottles or into 125 or 250 mL glass flasks (fill glass flasks 1/3 full with medium) that are stoppered with cotton. The media is autoclaved at 120oC for 20 min. After cooling, 5 mL of Penium stock solution is aseptically added to each 125 or 250 mL flask using a sterile pipette. If tissue culture flasks are used, pour the cooled WH from the autoclaved glass bottles into the tissue culture flask to the maximum volume indicated on the side of the flask. Add 5 mL of Penium stock culture to each flask. Always use aseptic technique when culturing, including performing cell culture transfers under a laminar flow hood.
Enhancement of Penium cultures used for many experiments, i.e., larger cell yields, can be enhanced using soil extract as an additive to the basic medium, Woods Hole (WH) medium. To make soil extract: Fill a 1 L glass flask with 2.5 cm of garden soil (without pesticides or fertilizers) and cover with 800 mL of deionized water. Place the flask on a hot plate and heat to boiling. Turn heat down and let simmer for 6 h. Let cool overnight and repeat the heating process for the second day. On the third day, filter the extract through multiple layers of filter paper. The extract should be brown and clear and may be stored for up to a year in a refrigerator. WH medium is supplemented with this soil extract to make Woods Hole Soil (WHS) medium as follows: 50 mL of soil extract is added to 900 mL of WH medium. Mix, adjust pH to 7.2 and bring volume to 1,000 mL.
5) General culture conditions: Penium is grown at 20°–22oC under a 16h light/8h dark photocycle with 0.5 kilolux of cool white fluorescent light.
6) Special—solidifying medium with agarose: Prior to autoclaving, 5 g of agarose is added to 500 mL of WH or WHS in a stoppered glass bottle and shaken. After autoclaving and 10 min of cooling, the warm medium is poured into petri dishes (fill each dish to roughly a third of the height of the dish) and allowed to cool. After the agarose solidifies, 0.5 mL of Penium liquid culture (see above) is placed into the center of the dish with a sterile pipette. The cells are then spread across the surface of the dish with a sterile commercial cell spreader or with a flame-sterilized glass rod “hockey” stick. The dish is sealed with parafilm and placed in the culture facility. Colonies will be obvious on the agarose surface in 14–21 days.
Crofixation and freeze substitution of Penium for transmission electron microscopy
Penium’s unicellular phenotype and small size allow it to be easily and inexpensively cryofixed via spray freezing. A comparison of various freezing techniques including high-pressure freezing and jet freezing revealed that simple atomization of Penium cell suspensions into liquid propane using a commercial artist’s airbrush provided excellent preservation of cell ultrastructure. Other major equipment that is needed include a -80°/-90oC freezer, fume hood for propane gas liquification, a 60°–70oC oven, air compressor and portable UV light source.
Protocol
Pre-fixation: An acetone-based substitution solution is used for osmicated preparations, while an ethanol-based solution is employed for non-osmicated preparations. One to two days before cryofixation, prepare freeze substitution vials (20 mL glass screw cap scintillation vials; EMS, Ft. Washington, Pennsylvania, USA) as follows: a) Wash approximately 50–100 g of molecular sieves (Sigma) with either 100% acetone or 100% ethanol. b) With a spatula, cover the bottom of the 20 mL substitution vials with a layer of washed molecular sieves. To acetone-washed sieves, add 10 mL of 100% acetone, cap, shake and let sit for 24–48h. Repeat for ethanol-washed sieves with 100% ethanol. c) 24 h before fixation, place 10 mL of 70% glutaraldehyde into the bottom of a clean scintillation vial. Add dry molecular sieve and mix until no standing glutaraldehyde is observed above sieves. Let sit for 24 h under a fume hood. This process will dry out the glutaraldehyde (i.e., remove residual water). A clearly sticky mass of glutaraldehyde will cover the sieves when residual water is removed. 3–4 h before cryofixation, weigh out under a fume hood 0.5g of the “dried” glutaraldehyde-molecular sieves and place into either a vial containing dried acetone or ethanol. This amount will provide a final 1% glutaraldehyde acetone or ethanol substitution medium. Cap and swirl the vials for 1 min and let sit for 1 h with occasional swirling to dissolve the glutaraldehyde. Place the vials into a -80°C to -90oC freezer for at least 2 h. These vials represent the freeze substitution chambers and can be kept in the freezer for up to two weeks.
Cryofixation: The following procedures must be performed under a fume hood and no flames should be ignited in the laboratory. Place a 20 mL porcelain dish into a shallow liquid nitrogen-tolerant dewar so that its opening is slightly above that of the dewar. The height of the vials can be adjusted placing aluminum foil wads under the vials. Cool the porcelain dish with liquid nitrogen. Attach a plastic hose to the outlet of a propane gas source. We have successfully used propane from high-purity pressure tanks, a commercial bottle of camping stove propane or the outlet of building bunsen burner gas. Attach the large opening of a glass pasteur pipette to the other end of the hose. Gently turn on the outlet of the propane tank/bottle/outlet and place the tip of the pipette onto the bottom of the cooled porcelain dish. In 30–60 sec, a gurgling will be heard as the propane liquifies. Increase the flow of gas release and in a few minutes, the porcelain dish will fill with propane. Once filled, cover the porcelain dish with its porcelain cover or a piece of thick aluminum foil. Be sure to continually add more liquid nitrogen around the dish (not into the dish). In 3–5 min, the temperature of the propane will reach -180°C or lower. Position the artist’s airbrush approximately 20 cm above the center of the porcelain dish using a bunsen burner stand and clamps. Connect the air inlet port of the airbrush to an air compressor (or a building air source). Adjust the spray setting on the airbrush to a fine spray. Test that liquid will spray into the dish by covering the dish with a paper towel and using water as the test solution. Adjust the positioning of airbrush as needed. Collect Penium suspensions and wash 3x with fresh-growth medium or test solution. After the last centrifugation, add 500 µL of growth medium or test solution to resuspend the pellet. Place a 100–200 µl drop of cell suspension into the side port of the airbrush. Quickly remove the cover off the porcelain dish. Spray the cell suspension from the airbrush into the propane. Repeat until all of the cell suspension is used and then recover the porcelain dish. Under a fume hood, pour off the top 2/3 of the propane into a waste container. Pour the remaining liquid propane containing the cryofixed cells into either a cooled glutaraldehyde-acetone scintillation vial or glutaraldehyde-ethanol vial. Tighten the cap of the vial, gently swirl and then open the cap to release any residual propane. Place the vial in a -80° to -90oC freezer for substitution.
Osmicated preparations: 24 h after cryofixation, add 1/10 g of osmium tetroxide (EMS) to each glutaraldehyde-acetone vial under a hood. Swirl and place back into the freezer. Every 12 h, swirl the vials and let sit in the -80° to -90oC freezer for 48–96 h. This period of time constitutes the freeze substitution period. After freeze substitution, warming of the vials to room temperature may be performed in specialized equipment designed for warming or may be accomplished by pulling the plug of the freezer and allowing the vials to warm to room temperature. However, we have obtained good results by taking the vials and placing them in a -20oC freezer (commercial freezer) for 4 h, then in a 4oC chamber (refrigerator) for 2 h and then at room temp for 1 h. After this time, the glutaraldehyde-acetone medium containing the cells is poured into a 12 mL glass centrifuge tube and centrifuged (400 x g for 1 min) under a fume hood. The supernatant is poured off into a waste container, leaving the freeze substituted and osmicated cells in a pellet. The pellet is washed 3x with 100% acetone. Several plastics can now be used for infiltration and embedding, but we have found Spurrs Low Viscosity resin (EMS) the best. The pellet is resuspended in a solution of 25% Spurrs resin/75% acetone and gently shaken for 2 h. The cells are then concentrated by centrifugation and placed in a 50% Spurrs resin/50% acetone solution for 2 h, a 75% Spurrs resin/25% acetone solution for 4 h or ON and then in 100% Spurrs resin for 12–24 h. Cells may then be placed into the bottom of beem capsules and the capsules filled with fresh Spurrs resin. Or they may be suspended in a thin layer of Spurrs resin sandwiched between two sheets of Aclar plastic (EMS). The capsules or plastic sandwiches are polymerized for 9–12 h at 70oC. For the thin sheets of plastic formed in the sandwich, individual cells may be chosen using a dissecting microscope, cut out with a razor blade and then super-glued to the tip of a beem capsule.
Non-osmicated preparations: The glutaraldehyde-ethanol vials are kept at -80°C to -90oC for 48 h and are then warmed to -20oC by being placed in a commercial freezer. The substitution medium with cells is poured into 12 mL glass centrifuge tubes pre-cooled to -20°C and centrifuged. The cell pellet is then washed 3x with pre-cooled 100% ethanol. The cell pellet is then infiltrated in a solution of 50% London Resin White medium (LRW) /50% ethanol cooled to -20oC for 12 h and then 100% LRW for 24 h with two replacements of the 100% LRW over that time. The cells are then collected into pre-cooled beem capsules and polymerized with a UV light for 24 h at -20oC.
Sectioning: For routine ultrastructural observation, we use osmicated cells and for immunolabeling, either osmicated or non-osmicated cells. For immunolabeling, 50–70 nm sections are collected on Formvar (EMS)-coated gold or nickel grids. For routine observations, copper grids are employed. Sections are typically stained with conventional 1% uranyl acetate for 7–10 min and 0.1% lead citrate for 2 min before viewing on a TEM.