f/2 agar medium recipe

Welcome to Marine Aquarium Co-Op. We hope you enjoy your visit.

You're currently viewing our forum as a guest. This means you are limited to certain areas of the board and there are some features you can't use. If you join our community, you'll be able to access member-only sections, and use many member-only features such as customizing your profile, sending personal messages, and voting in polls. Registration is simple, fast, and completely free.

Join our community!

If you're already a member please log in to your account to access all of our features:

Pages:
1
2
3

f/2 agar medium recipe
Topic Started: Jan 12 2009, 08:31:22 AM (2,192 Views)
Post #1 Jan 12 2009, 08:31:22 AM	jpiotrowski
This is the same recipe I used years ago to make my plates. I can't remember what % of agar I used but mine wasn't solid )10-15% is most common to make it solid). I kept agar plates inocculated with phyto for 1/2 to 1 year. CULTURING ALGAE Prepared by Santiago Perez; 3/22/06 f/2 agar plates (for culturing/purifying zooxanthellae) Need: 35ppt Millipore-filtered seawater (MFSW) Agar (DIFCO BactoAgar; Fisher DF0140-15-4) Concentrated silica-free f/2 media (Sigma No. G0154) see appendix for original recipe (or see here: http://ccmp.bigelow.org) Petri dishes • Calculate the volume of MFSW to use based on the number of plates to be filled. Each plate is typically filled ˝ to ľ of the way. • You may wish to make different agar concentrations (e.g. 0.5, 1.0, 1.5 or 2.0%). Add the necessary amount of agar to the MFSW • Heat agar solution until it dissolves using a hotplate stirrer/stirbar) • Autoclave at 121°C, 15psi for at least 15 minutes. If you are autoclaving several large bottles it is recommended to autoclave for longer times (e.g. 30min). • Have sterile Petri dishes ready. • Let cool a bit but without causing the agar to solidify. You can use a water bath set at 50°C. • If using Sigma solution, add 50X f/2 media to a final concentration on 1X (1:50 dilution). Note: This media comes sterile. Be careful to maintain its sterility. If you suspect that this step is contaminating the preparation, filter-sterilize the media first. – However it may mean that the media is ruined. Do not autoclave f/2 media • Quickly dispense enriched agar onto the plates on a sterile surface next to a flame. • Cover dishes and stack to let cool. • Once the agar solidifies, store plates in the refrigerator. Some precipitate in the plates is normal and doesn’t affect the growth of algae. To culture algae from anemones: • Homogenize anemones and isolate algae by repeated centrifugation followed by resuspension in sterile f/2 media until little host tissue remains. This takes about 2-4 times with Aiptasia. Other 'bulkier' species may take more. • I then add the algal suspension to your culture vessels with the F/2 media. I usually inoculate 1ml (approx 1 million cells per ml. -I count algae with a hemocytometer) into 50ml sterile clear plastic conical tubes. 250ml sterile flasks work well too. • I keep them at 25C chamber with a 12/12 hour light/dark cycle. Cool white fluorecent bulbs work well - I try to get 50-100 uE of light intensity. • It can take 1-3 months for them to get going -be patient. • I turn cultures over once a month in the 50ml tubes. To get clones you can either try to plate algae from the freshly isolated algal suspension or from algae that have been growing already. • I inoculate these with varying amounts of algae and wait patiently to see if they form colonies. • Keep plates in the light (same environmetal conditions as above). Sometimes they don't like to grow on plates. You'll get all kinds of 'unwanted' growth of cyanobacteria and other. I have found that the zoox usually grow best on plates when some of these 'contaminants' are present. Perhaps they are getting some nutrient from them -who knows (?!) For f/2 liquid media • Filter natural seawater through 0.45uM Millipore filter • Sterilize filtered seawater in autoclave. • Let sterile seawater cool to room temperature (or colder) overnight • Add concentrated f/2 media using asceptic technique and mix well • Note: use silica free f/2 for culturing algae other than diatoms. • Dispense enriched seawater to sterile containers to be used for growing algae • Inoculate as necessary. For maintenance cultures of zoox: • You can use 50ml sterile falcon tubes and inoculate with up to 1ml of 1 month old culture. • Maintain a 2 month-old culture, a 1 month-old culture together with the newly started culture. On the next round discard the oldest culture (the now 3 month-old culture) • Grow at 25 C and adequate light on a 12h/12h Light/Dark cycle. APPENDIX f/2 Medium and Derivatives (Guillard & Ryther 1962, Guillard 1975) Below are recipes for f/2 medium, its derivatives (e.g, f/2 agar, f/2-Si, f/2 + Se, f/4, f/50) and related media (e.g., Black Sea). F/2 is listed first, followed by derivatives of f/2. f/2 Medium (Guillard & Ryther 1962, Guillard 1975) To 950 mL filtered seawater add: Quantity Compound Stock Solution Molar Concentration in Final Medium 1 mL NaNO3 75 g/L dH2O 8.83 x 10-4 M 1 mL NaH2PO4 • H2O 5 g/L dH2O 3.63 x 10-5 M 1 mL * Na2SiO3 • 9H2O* 30 g/L dH2O* 1.07 x 10-4 M* 1 mL f/2 trace metal solution (see recipe below) - 0.5 mL f/2 vitamin solution (see recipe below) - Make final volume up to 1 L with filtered seawater and autoclave. *Note: Autoclaved f/2 medium produces extensive silica precipitate. We delete silicate when it is not required by the alga (see f/2-Si medium below). 3 2 1 Discard f/2 Trace Metal Solution (Guillard & Ryther 1962, Guillard 1975) To 950 mL dH2O add: Quantity Compound Stock Solution Molar Concentration in Final Medium 3.15 g FeCl3 • 6H2O - 1 x 10-5 M 4.36 g Na2EDTA • 2H2O - 1 x 10-5 M 1 mL CuSO4 • 5H2O 9.8 g/L dH2O 4 x 10-8 M 1 mL Na2MoO4 • 2H2O 6.3 g/L dH2O 3 x 10-8 M 1 mL ZnSO4 • 7H2O 22.0 g/L dH2O 8 x 10-8 M 1 mL CoCl2 • 6H2O 10.0 g/L dH2O 5 x 10-8 M 1 mL MnCl2 • 4H2O 180.0 g/L dH2O 9 x 10-7 M Make final volume up to 1 L with dH2O. Autoclave. f/2 Vitamin Solution (Guillard & Ryther 1962, Guillard 1975) To 950 mL dH2O add: Quantity Compound Stock Solution Molar Concentration in Final Medium 1 mL Vitamin B12 (cyanocobalamin) 1.0 g/L dH2O 1 x 10-10 M 10 mL Biotin 0.1 g/L dH2O 2 x 10-9 M 200 mg Thiamine • HCl - 3 x 10-7 M Make final volume up to 1 L with dH2O. Autoclave and store in refrigerator. Note: Vitamin B12 and biotin are obtained in a crystalline form. When preparing the vitamin B12 stock solution, allow for approximately 11% water of crystallization (for each 1 mg of Vitamin B12, add 0.89 mL dH2O). When preparing the biotin stock solution, allow for approximately 4% water of crystallization (for each 1 mg of biotin, add 9.6 mL dH2O). f/2 Derivatives Black Sea Medium: For brackish water organisms (16 psu, half-strength nutrients). Combine 500 mL f/2 medium and 500 mL dH2O. Autoclave. f/2 agar: Prepare 1 liter of f/2 medium and dissolve 9g Bacto-agar (heat and mix). For test tubes, dispense dissolved agar medium into tubes, autoclave, and then cool with tubes slanted at an angle. For Petri plates, autoclave in a flask, cool almost to the gelling point, and then aseptically dispense into sterile Petri plates. Note: Agar can be added to other media (e.g., f/50 agar), and agar concentration can be varied to produce softer or firmer substrates. f/2-Si: Prepare as for f/2 medium but omit Na2SiO 3 • 9H2O. This is preferred over f/2 medium for organisms with no silica requirement because less precipitation forms. f/2 + Se: Extra silicon and selenium are beneficial to several diatom strains. Prepare 1 L of f/2 medium but use 2 mL of silicate stock, then add 1.0 mL of selenium stock solution (1.29 mg H2SeO 3 /L distilled H2O). Autoclave. f/2 (11 psu): For brackish water organisms. Mix 650 mL distilled H2O and 350 mL filtered seawater. Add f/2 medium nutrients and autoclave. f/2-Si (24 psu): Mix 750 mL distilled H2O and 250 mL filtered seawater. Prepare as for f/2 medium but omit Na2SiO 3 • 9H2O. f/4: Add 500 mL f/2 medium to 500 mL filtered seawater, then autoclave. f/4-Si: Autoclave 1 L of filtered seawater. When cool, aseptically add f/2-Si nutrients at half concentration (i.e., 0.5 mL). f/20-Si: Autoclave 1 L of filtered seawater. When cool, aseptically add f/2-Si nutrients at one tenth concentration (i.e., 100 μL). f/50-Si: This is more than a 1/25 dilution of f/2-Si medium. We autoclave 1 L of seawater in a Teflon-lined bottle. Wait for the autoclaved seawater to cool to room temperature (important). Aseptically add 40 μL of sterile f/2 nutrients (20 μL of vitamins). f/50-Si + CCMP1320 as food: Prepare f/50 and aseptically add 50 μL of healthy, moderately dense culture of CCMP1320. f/2m: To 1L f/2 medium add 1 g methylamine • HCl, mix until dissolved and autoclave. This medium is used to test for contamination by methylaminotrophic bacteria. f/2p: To 1 L f/2 medium, add 1 g Bacto-peptone, mix until dissolves and autoclave. This medium is used to test for contamination by non- methylaminotrophic bacteria and fungi. f/2pm: To 1L f/2 medium add 1 g Bacto-peptone and 1 g methylamine • HCl, mix until dissolved and autoclave. This general medium is used to test for contamination by bacteria and fungi. f/2 + NPM: Add f/2 nutrients to 900 mL of seawater and autoclave. After cooling, aseptically add 100 mL of the following organic stock solution. Dispense aseptically into test tubes.	breeding stock Posts: 85 Group: Members Member #48 Joined: September 16, 2008



Post #2 Jan 12 2009, 12:22:12 PM	Clint
I fresh out of autoclaves. I have a home canning pressure cooker at 15 psi. I thinking that would work. Do you think so?	breeding stock Posts: 377 Group: Members Member #69 Joined: November 22, 2008
Midvale (435) 213-6215


Post #3 Jan 12 2009, 03:15:09 PM	jpiotrowski
I have an autoclave that just sits in it's room...it's time we used it! I don't have any of the ingredients. If you could mix it up in 1L bottles and bring it over we could cook it, go to lunch then cool it down/keep it warm in an igloo water batch and pour out some plates. A pressure cooker would work just fine. Just cook it for 20 minutes. I think there is some anit-microbial properties in the medium itself, so no real worries other than getting the agar into solution. At my previous job I had a couple plate pouring machines which made life easy. Unfortunately, this is going to have to be the sloppy way! I would like to bum, buy or trade a few, maybe a whole sleeve for storing different strains. Again I would add 5-10% agar. John	breeding stock Posts: 85 Group: Members Member #48 Joined: September 16, 2008



Post #4 Jan 12 2009, 04:13:28 PM	Clint
I would like to make some broad range phyto media then do some isolation streak plates and see how many different varieties we could isolate. If it works like it should, we could keep fresh plates of culture in perpetuity. Could be quite a resource. I we would not need an autoclave for dissolving the agar. If I remember correctly it readily dissolves in boiling water. The autoclave would only be needed to get everything sterile. I'll have to check on ingredients....	breeding stock Posts: 377 Group: Members Member #69 Joined: November 22, 2008
Midvale (435) 213-6215


Post #5 Jan 12 2009, 04:13:56 PM	Amie
This is great. How many plates can you make and how much money needs to be invested. I'm all in. Do I need to run to the chemistry shop and buy some petri dishes?	stormy, stormy nights Posts: 2,362 Group: Admin Member #4 Joined: June 29, 2008
http://www.spawar.navy.mil/sandiego/technology/mammals/interns.html Tell them Adam sent you.


Post #6 Jan 12 2009, 05:48:35 PM	Clint
Is this a full service chem shop? if so I'm sure we could get any needed ingredients from them. Where are they located. From the looks of it, concentrated f/2 silica free media, agar, and plates are all we need. We can sterilize salt water on our own. It only gets complicated if we have to make the f/2 media.	breeding stock Posts: 377 Group: Members Member #69 Joined: November 22, 2008
Midvale (435) 213-6215


Post #7 Jan 12 2009, 09:04:28 PM	Clint
Amie, that f/2 you have, what is that exactly. That might be what we need.	breeding stock Posts: 377 Group: Members Member #69 Joined: November 22, 2008
Midvale (435) 213-6215


Post #8 Jan 12 2009, 10:16:03 PM	Amie
Clint Jan 12 2009, 09:04:28 PM Amie, that f/2 you have, what is that exactly. That might be what we need. I'm not sure what it is. I can find out though. It came from BSD.	stormy, stormy nights Posts: 2,362 Group: Admin Member #4 Joined: June 29, 2008
http://www.spawar.navy.mil/sandiego/technology/mammals/interns.html Tell them Adam sent you.


Post #9 Jan 12 2009, 11:24:37 PM	Amie
Oh, and Clint, yes it's a full service chem shop, they should have everything you need. Here's their website http://www.hvchemical.com/ Okay, the fertilizer I have is Guillard f/2 Medium. It containers N, P, trace elements and vitamins. Is that all you needed to know?	stormy, stormy nights Posts: 2,362 Group: Admin Member #4 Joined: June 29, 2008
http://www.spawar.navy.mil/sandiego/technology/mammals/interns.html Tell them Adam sent you.


Post #10 Jan 13 2009, 07:31:05 AM	jpiotrowski
Amie is it dry f/2? If so we can make it any concentration we want. Clint- If I remember right you would want to sterilize the basal media (salt water and agar) and add filtered f/2. I am not sure what will happen to the f/2 if it is heated, though there really isn't anything in it that would break down. Just the same. This will be interesting...I am not sure how algae will streak, but if it works that would be awesome!	breeding stock Posts: 85 Group: Members Member #48 Joined: September 16, 2008



1 user reading this topic (1 Guest and 0 Anonymous)

Fully Featured & Customizable Free Forums Learn More · Register Now
Go to Next Page
« Previous Topic · Nutrition: The Most Important Thing? · Next Topic »

Pages:
1
2
3