From: PO4::"dwalsh@CAPECOD.NET" "Dennis T. Walsh" 26-MAR-1996 12:44:12.45 To: Multiple recipients of list DIATOM-L CC: Subj: Re: Axenic cultures >I was wondering if anybody has any sugggestions regarding treating unialgal >cultures to render them axenic ?. I have tried filtering a sample of >culture through an 8 micron filter and backwashing the sample that remained >on the filter, i.e the phytoplankton, into sterile media but there is still >large concentrations of bacteria present after a few days. I realise it >is impossible to get rid of all bacteria but any suggestions as to how I >could reduce the numbers would be appreciated. > >Thanking you in advance > >Eric Mortimer > >Eric Mortimer, >Martin Ryan Marine Science Institute, >Department of Microbiology, >University College Galway. Eric: I have used the method below for the last 10 years to produce axenix algae cultures at a commercial shellfish hatchery. Algal Isolation with Agarose 1. FMC Bioproducts (5 Maple St., Rockland, ME 04841-2944. Telephone: 1-800-341-1574; technical service: 1-800-341-1574) has SeaPlaque and SeaPrep agarose that have low gelling and melting temperatures (Table 1.) that are ideal for isolating algae. SeaPlaque is usually fine for isolation of diatoms and flagellates; if the alga is particularly sensitive to temperature use SeaPrep. Table 1. Low Gelling/Melting Temperature Agarose Specifications: Gel Temp. Melting Temp. EEO Gel Strength Sulfate Mois= ture =B0C 1%,oC -Mr g/cm2 % = % SeaPlaque agarose 26-30 (1%) <65 <0.15 <200 (1%) <0.15 <1= 0 SeaPrep agarose 8-17 (0.8%) <50 <0.05 <75 (2%) <0.10 <1= 0 Current Price: SeaPlaque agarose 25g #50101 $75 ('95) SeaPrep agarose 10g #50301 $56 2. Take 0.2ml (diatom) or 1ml (flagellate) from a five day old primary and transfer to new sterile 50ml primary tube; allow culture to grow overnight under continuous light and +20oC. 3. Prepare a test tube rack containing dilution tubes (9ml of primary media) and Sea Plaque tubes (5-6ml of 0.8% Sea plaque dissolved in primary media). To prepare Sea Plaque, measure 0.8 gm. and mix it into 100ml of primary media in a 125ml flask or beaker. Heat this mixture to dissolve the agar-do not let it boil rapidly or it will foam and boil over. Dispense 5-6ml of liquid agar into test tubes and cap; sterilize dilution tubes and agar tubes for 15 min. I am assuming you have sterile prepackaged 25mm diameter petri dishes. If you have 47mm petri dishes, you may need 10-12ml of agar per tube. You can make up a lot of dilution tubes and 10-20 agar tubes at one time. The agar will solidify after autoclaving and should be good for several weeks if kept capped; it will eventually dry out. 5. Take 1.0 ml of the 24 hr. culture (step 2) and do a 2-3 step serial dilution. By count, I would estimate you want to dilute your algae to 102-103/ml before plating. Add 0.2ml from each dilution tube to separate test tubes containing 5-6 ml of the liquid 0.8% Sea Plaque. To liquefy the sterile agar tubes, place them in boiling water. Remove and allow to cool to near gelling temperature (Table 1.) before adding algal innoculum (critical step). Immediately mix the algae/agar and pour into a labeled petri dish. Gently swirl/tilt petri dish to cover bottom of petri dish with a thin layer of agar; don't get on top cover. Place petri dish on a flat surface and let gel solidify. This should occur quickly if your gel was already near the solidification temperature. You may want to place the petri dish in a refrigerator in order to assure gelling, but don't leave in refrigerator too long. Invert the petri dishes once the agar has solidified and place the dishes in a zip-locked bag that has a few drops of tap water in it (prevents dessication). Place the bag on a shelf in the algal room near the primaries. The objective is to get only a few algal colonies on/in the thin agar layer in the 25mm dish. Ideally, the brown(diatoms) or yellow/green(flagellates) algal colonies are separated not only from other algal colonies but also from any bacterial colonies (appear as white, yellow or pink aggregations). With experience you will know which dilution tube to plate and will not have to plate all dilution tubes. 6.The algae colonies should appear within 5-10 days; discard plates after 14 days if nothing appears. You want to pick the colonies as early as possible, so look for colonies under stereo scope at 25-50X. Prepare in advance a test tube rack containing capped test tubes with 10ml of primary media; sterilize and allow to cool overnight. Sterilize some disposable Pasteur pipettes which will be used for the picks. Draw out/sterilize tips of pipette into long, fine hollow needle point over flame. Pick a single colony; you can see it being drawn up the pipet by capillary action. Immediately transfer to 10ml media tube and snap off tip containing algae into media. Flame neck of test tube, label and store in innoculum room. If successful, you should have a new axenic algal clone bloom in this 10ml within 7-14 days. Transfer ~0.5ml to 50ml primary tube. You can repeat the procedure if bacteria or other contaminants are present. Bigelow can give you recipe for simple sterility test for algae. 7. You can use this same procedure for cleaning and/or storing newly received algal clones. Algal colonies on agarose can be stored at low light for 1-2 months if the culture was axenic. 8. I avoid antibiotics unless the above procedures fail and I'm sure its not my technique. You could add 5-10ppm of Neomycin to the agarose solution and this should eliminate the appearance of bacterial colonies. 9. I use the full strength nutrients because I want to select for algae that can grow well in a highly enriched media; however, you can experiment with various nutrient levels and obtain very different algal clones which may grow well in your full strength media. 10. Diatoms rarely undergo sexual reproduction; however, vegetative reproduction leads to smaller and smaller cells. Isolating diatoms on agarose will often induce sexual reproduction in diatoms. Sexual reproduction results in the production of a large cell containing a chloroplast and vacuole. The cell looks like a signet ring. If there is no evidence of sexual reproduction in the newly isolated diatom clones, you should use silicon-free media to grow several generations. This procedure may cause sexual reproduction. Finally, manipulation of the light/dark cycle may induce sexual reproduction in the diatoms. Reference: Stein,J.R. 1973. Handbook of Phycological Methods, Culture Methods and Growth Measurements. Cambridge University Press, London. @/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/ @/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@/@ Walsh Consulting Phone: (508) 385-2415 21 Pilgrim Rd. FAX: (508) 385-2415 Dennis, MA 02638 Internet: dwalsh@capecod.net Consulting firm offering over 19 years experience in commercial shellfish aquaculture and algal biotechnology. Your Internet resource to commercial expertise in aquaculture engineering, site selection and permitting, shellfish pathology, hatchery design, shellfish marketing and distribution, new anti-fouling paints and cost effective utilization of wetlands and aquatic resources. @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@