Vessel Preparation for Small-Scale Microalgae Culture

This guide is designed to present an easy-to-follow and stepwise method to establishing a microalgae culture.  If you would just like a simplified list of steps to follow without the additional discussion of “how and why”, the basic protocol can be found here.

Introduction

Don’t let anyone tell you how hard culturing microalgae is.  Once you get the hang of it, it is really quite easy and the time commitment is minimal.  Using the methods described here, you can expect around an hour per month to clean and sterilize a set of culture bottles, and around 30 minutes per week for culture maintenance and harvesting.  These are the methods we use to produce relatively small amounts of live microalgae (five to ten gallons per week).  This quantity is sufficient for the “greenwater technique” of larval rearing in which small amounts of live microalgae are added to larval rearing tanks to keep prey items such as rotifers enriched, and to allow the live microalgae to consume fish waste, keeping the tanks healthier.  Many hobbyists also use microalgae produced using these techniques to feed corals and other filter and suspension feeders in their reef aquariums.  While there are many variations on how to culture microalgae, the methods presented here are largely the same as those used in hatcheries for many decades and can easily be adapted to individual needs and to the materials available.   

Materials

• Glass pickle jars (1 gallon) with plastic lids
• Guillard’s f/2 fertilizer
• Artificial seawater (salinity 28-33ppt, although the desired salinity will vary depending on species being cultured)
• Starter culture of desired algae
• 5% w/v solution of sodium hypochlorite
• 5% w/v solution of sodium thiosulfate
• Chlorine test strips
• Air pump
• Clean flexible airline tubing
• Electric drill
• Appropriate size drill bit (11/64” works well for the pipettes I use, but may vary)
• Sterile 1ml volumetric pipets
• 2 or 5ml pipet pump
• Plastic pot scrubbing pads
• Sodium metasilicate (if culturing diatoms)
• Muriatic acid (if culture vessels need mineral deposits removed)
• Sandpaper, 220 grit or finer (if needed to clean up drill hole)
• Isopropyl alcohol (if needed for cleaning exterior of bottle or lids
• Permanent marker

Methods

Vessel selection

Nearly any clear vessel can be used to culture microalgae and other planktonic organisms, some better than others.  Glass pickle jars were selected because their wide mouths make cleaning and liquid transfer easy.  Additionally, they withstand multiple acid cleanings and chemical sterilization without degradation.  Unfortunately, they do easily crack when bumped into each other, so plastic bottles might be a better option if you are rough with your materials. A smooth-walled bottle works better than those with ribbed walls (like drinking water bottles), as the ribs cause uneven circulation patterns and can collect detritus and exacerbate contamination issues.  Other commonly used vessels for hobbyists include the huge bottles that puffed cheese balls come in, so if you know someone that can stomach mass quantities of puffed cheese balls, you’re already on your way.  Those bottles have some texture, but it is significantly less obtrusive than other options. 

Vessel preparation

• If the lid of your desired vessel has an inner liner, remove it. The foam inner liners can trap material making cleaning and disinfection ineffective.  If you are ordering culture bottles, see if there is an option for unlined lids.   
• Drill a hole in the center of the lid, appropriately sized to insert a 1ml pipet. This is in leu of standard rigid airline tubing.  Standard rigid airline tubing can be used, provided it can be properly sterilized, top-to-bottom, inside and out.  We prefer the ease of using 1ml pipets because they arrive pre-sterilized, are inexpensive enough to use disposably, and come plugged with sterile cotton which will help reduce airborne contamination.  If your cultures are set up in an environment with a high chance of airborne contamination, or are for commercial production, your air supply should also have an inline 0.45µm or 0.22µm filter. 
  The hole drilled should precisely fit your airline or pipet such that it takes a little pressure to push the tube through.  Too loose of a fit and your airline won’t stay put, and any gaps allow for contaminates to enter.  Try a test hole or two in a piece of scrap plastic to make sure your tube/drill bit combination gives a proper fit.     
• Use sandpaper if needed to remove burs and rough patches from the drill hole. The vessel should be cleaned before use even if new.  I try to avoid using detergent cleaners, if possible, for any aquatic equipment.  If stubborn organic deposits necessitate use of detergents, I only use low-residue laboratory cleaners such as Alconox ®.  For cleaning algae culture jars, detergent use is generally unnecessary.  Instead, a 50/50 mix of water and muriatic acid, combined with thorough scrubbing with a plastic pot scrubber will remove manufacturing residues and will leave your glassware sparkling.  This cleaning is also useful periodically for refreshing your glassware since calcium deposits will collect, reducing light penetration and providing substrate for contaminants to utilize.
  
  With any laboratory solutions such as chlorine and acid, wear gloves, goggles, and ventilate the area. Follow all manufacturer’s directions for safe handling and disposal.  Never mix chlorine and acids, as this will produce toxic chlorine gas.
  
  
• Once cleaned, rinse bottles with RODI, distilled, or other water of similar purity. Cleanliness is of utmost importance for long-term success of microalgae cultures.
  Your algae culture vessels are ready to fill and sterilize.

Assembly, filling, and sterilization of microalgae culture vessels.   

1. Fill the bottles with approximately 3l of freshly made artificial sea water. Don’t fill the bottle completely full, you will need the extra room for adding your inoculum.  Pretty much any marine salt mix will do.  You don’t need any fancy specialty mixes.  I’ve been using regular Instant Ocean for my microalgae cultures for the past 25 years and haven’t had any problems with the salt.  If available, use RODI or distilled water for the mix.  If culture problems develop at some point, using the purest source water available will at least allow you to push source water quality to the bottom of the list of possible issues. 
   1. The 3l suggestion assumes that you have a culture established. If you are first establishing a culture, you may need to work with smaller volumes at first to allow the culture to gain a toehold as you upscale your cultures.  See the next section Establishing and Maintaining Microalgae cultures.
2. Screw the lid fully onto the bottle.  If the outside face of the lid is dirty, wipe it down with a paper towel moistened with isopropyl alcohol.  I do this for all lids regardless of whether they appear dirty. 
3. Open the pipette by pulling back the paper sheath at the end of the pipet with the cotton plug.
4. Slide the plugged end of the pipette into the pipet pump and remove the pipet from the packaging.
5. Place the pointed tip of the pipet in the chlorine solution and draw up 600µl of chlorine.
   1. Keep the pipet vertical. If liquid runs back and touches the plug, the plug essentially seals itself to prevent gas/liquid passage and the pipette will need to be replaced. 
   2. 600µl of 5% chlorine is a good starting point. This gives a 1:5000 dilution resulting in a final chlorine concentration of 10ppm, sufficient to sterilize clean source water.  If you are unsure how clean your source water is, go ahead and add a full 1 ml.  The important thing here is to make sure that you have enough free chlorine to sterilize your water, and just as important, to precisely measure how much chlorine you have added so that you know how much sodium thiosulfate to add to neutralize the chlorine. 
   3. You can use unscented laundry bleach instead of the lab-grade hypochlorite solution. I did for many years, but no longer do.  The lab-grade solution is inexpensive enough to use, and I don’t worry about what additional additives might be lurking in the bleach.  If you do use bleach, make sure to adjust the concentration or quantity of your sodium thiosulfate neutralization solution since bleach is available at various concentrations. For more information about using lab-grade chlorine instead of bleach see this article.
   4. If you are starting a hatchery, or will be doing a lot of dispensing of small, measured quantities of liquids, you might want to invest in a good quality 1000µl micropipette. A nice micropipette and a case of pre-sterilized tips can set you back $500 or more, but the precision and time-savings are well worth it.  Plus, I find the action of shooting the used tip into the waste receptacle to be immensely satisfying. 
6. Place the pointed tip into the hole in the vessel lid.
7. Use the pipet pump to dispense the chlorine solution into the culture vessel.
8. Gently push the pipet into the bottle until the tip is just above the bottom of the bottle.
   1. About ¼ inch from the bottom is good starting point, but it will vary depending on your air flow. Too high and it won’t effectively suspend cells at the bottom, too close to the bottom it will make a high-pitched resonance that will drive you to madness.   
9. Use the pipet pump to draw water in and out of the pipet a few times to make sure the chlorine is vented from the pipet and mixed with the culture water.
10. Swirl the water in the bottle to thoroughly mix the chlorine. Make sure that all internal surfaces including the lid are contacted by the chlorinated culture water to ensure full sterility. 
    1. Since you are using an unlined lid there may be a bit of water that dribbles out as you swish it. Not a problem, but since this is chlorinated water, be aware it may bleach anything that it contacts. 
11. *Optional* Check chlorine residual with a test strip. Chlorine solutions lose potency over time.  If your chlorine stock is relatively fresh, and you are confident you have measured correctly, this step can be omitted.  Otherwise, using clean hands, open the lid and carefully dip a chlorine strip into the vessel, taking care not to touch the sides.  Replace the lid and give the bottle another swirl to eliminate any contaminants that may have fallen in.  Chlorine should be ~10ppm.
12. I prefer to cover the exposed end of the pipet tip with a bit of the packaging the pipet came in until immediately before use.
13. Your microalgae culture vessel is ready for use!
    1. I prefer to let my vessels stand overnight before use to ensure the chlorine has had sufficient contact time.
    2. Once chlorinated and capped, the vessels can be stored almost indefinitely, although I swap mine out if they have been sitting for more than a few months.
    3. As a time-saver, I modify this slightly.  I prepare 20 gallons of water at a time, chlorinate it en-masse, and use a small powerhead to fill 20 bottles at a time.  The left-over water is dechlorinated and used for water changes on rotifer cultures.