Algae Plating Protocol

[Cameron Clarke]

Algae Plating Protocol

Algae (especially spirulina) can be difficult to plate. These two methods of plating, with the agar recipe below, work for many of the algae species in the lab, but do not work for spirulina. With both methods, you will begin to see growth within 4-7 days, two weeks at most.

Materials:

1.5 mls of each algae culture

Inoculation loops

Sterile petri dishes  

Plain agar flakes

sea salt

distilled or deionized water

F/2 nutrient media or other appropriate nutrient mix

Plating Media:

2% agar dissolved into the normal liquid media for an algal species

For example: to plate Nanno, use a media of 2% agar, 3% salt, 3ml/L F/2 nutrient mix, in sterile water.

To Prepare Plating Media:

1. For salt water species, add 3% sea salt to distilled or deionized water, shake until thoroughly mixed.

2. For all species, add 2% plain agar flakes (we buy ours from asian grocery store and grind up in a blender) to the salt/fresh water and shake until thoroughly mixed.

3. Pour into autoclave-safe containers until the containers are half-full (more than half-full will overflow in the autoclave and potentially contaminate the autoclave)

4. Set the containers on an aluminum tray in the autoclave and sterilize using the liquid media setting.  This will take about an hour on Biocurious’ machine.  Don’t forget to sign in/out on the autoclave log.

5. Use the red oven mitts to carefully pull the media solution out of the autoclave.  Let cool briefly, then add 3ml/L of the F/2 nutrient mix. (The F/2 must be added after autoclaving because some of the vitamins will degrade at high temperatures)

6. As Geoff says it, the agar media is ready to poured into the petri dishes just at the point when you can finally touch the container without burning your fingers.  (If you wait longer than this, the agar will cool to the point where it begins to gelatinize -- if this happens, you’ll really need to start over.  The media can be microwaved but you won’t know how much of the F/2 solution has degraded.)

First method of plating: Streak Method

1. Pour agar media into sterile petri dishes and let cool for 30 minutes.

2. Follow standard protocols for streak plating here, available all over the internet.

3. Dip an inoculation loop into a liquid sample of the algae, make sure that you’ve got something on the loop before you begin streaking gently across the agar.

3. Either flame the inoculation loop in between streaks, or use a new disposable loop for each streak.  3 streaks per plate is enough.

4. Seal the petri dishes with parafilm and leave upside-down under the algae lights on the heating pad.

Second method of plating: Liquid Inoculation

1. Pour 1.5 ml of algal culture into an empty sterile petri dish.

2. Fill the bottom of the dish with the hot agar media.

3. Swirl gently to mix the algae through the agar media.

4. Let set for 30 minutes.

5. Cover petri dish with parafilm and leave upside down on the heating pad under the grow lights.