As science teachers, I believe we are stronger when we work as a team. Together, we can influence our youth to incorporate the “Reduce, Reuse, and Recycle” mindset wherever possible. Doing so will not only save money and help protect our environment, but can also serve as a model while we teach sustainability principles to our students. Working toward sustainability within science departments, while including our students in the process, is a perfect example of collaborative work for the greater good.  

An opportunity

My first-hand experience teaching around the concept of sustainability started when a local research and manufacturing company, Cell Signaling Technology (CST), funded a grant to expand inquiry at Gloucester High School (GHS), where I teach, in Gloucester, Massachusetts. Among many other results of the grant was the purchase of some agar for an inquiry biology lab.

Several of my colleagues wanted to run a particular Agar Diffusion Cube lab for their biology students. In this lab, students begin the agar diffusion by placing various-sized colorless and opaque agar cubes into a pink solution. The pink solution diffuses into the agar to various degrees depending on the size of the agar cube (also assuming contact time of agar cubes in the solution is constant). The pink solution consists of sodium hydroxide (NaOH) to which the indicator phenolphthalein has been added; students can easily measure the degree of diffusion of the solution into the agar cubes when the cube is sliced open. Phenolphthalein turns pink in a base and is colorless in an acid. 

Figure 1. Agar cubes before diffusion in sodium hydroxide solution (left) and after (right).

After the lab, one biology teacher brought bags of pink agar (basic pH) back to my chemistry lab and asked me what I could do with it. One of my AP Chemistry students, Cameron (Cam) Muniz, was present during that conversation, and he was excited about the possibility of trying to recycle the agar.


Agar-agar, also known as simply agar, is a plant-based gelatin obtained from red algae. This heterogeneous polysaccharide is composed of the linear polysaccharide agarose and agaropectin polymers. It is a semi-translucent vegetable gelatin that can be used in food as a thickening agent or stabilizer, or as a growth medium for organisms.

In the biology lab activity, students investigated how surface area impacted diffusion. The students placed 1cm, 2cm, and 3cm opaque agar cubes into a 0.1M NaOH solution with phenolphthalein for 10 minutes. As the NaOH (basic) solution with indicator (pink colored solution) diffused into the opaque agar cube, it turned the agar the same brilliant pink color. Students sliced apart their agar cubes and measured the distance the NaOH solution had diffused into the cubes.

There was a stark difference between the agar used at the start of the lab and the agar after some diffusion took place. I tasked the students with calculating the percent diffusion of each cube. By calculating the surface area of each cube and the surface area-to-volume ratio (surface area/volume), students could come to some conclusions about the size of the cubes and the diffusion that occurs. Students could confirm that the higher the surface area of the agar, the more NaOH solution would diffuse into the agar (indicated by the distance the pink color traveled into the agar). After the activity, students were asked to transfer knowledge about surface area/volume ratios, and then extend their thinking to how human bodies adapt to help exchange gases.

Safety Precautions

  • Consult Safety Data Sheets for all chemicals used.

  - SDS for 0.1M hydrochloric acid
  - SDS for 0.1M sodium hydroxide
  - SDS for 5% acetic acid  

  • Wear appropriate Personal Protective Equipment, including goggles, apron, and nitrile disposable gloves.
  • Agar that contains sodium hydroxide is alkaline and has a soapy feel. Wear disposable gloves when handling.
  • When heating the agar to recycle it, use caution with hot agar.

Investigating recycling as a possibility

To investigate if and how we could recycle the agar diffusion cubes after they were used in the lab, we began by heating the agar, to liquefy it. We then brought its pH down by adding drops of 0.1M hydrochloric acid to the liquefied agar until the pink color disappeared.

After one full cycle of recycling and reusing the agar, we determined that the agar did turn pink again once it was re-exposed to a basic solution containing phenolphthalein. The result, however, was a pale pink, rather than the bold pink that was evident the first time the agar was used in the process. Additionally, the integrity of the agar seemed to be compromised, because its consistency was increasingly less gelatinous with each cycle of using hydrochloric acid to neutralize the base.

With this evidence in mind, we decided to decrease the concentration of hydrochloric acid used to neutralize the agar and try grocery store vinegar (acetic acid with 5% acidity) to neutralize the agar in some additional trials. As soon as we began using trials with vinegar to neutralize the agar, it was apparent that the integrity of the agar was much better preserved than when we used any concentration of hydrochloric acid. Perhaps because acetic acid is a relatively weak acid, the agar seemed to retain its integrity better when compared to the neutralization process with hydrochloric acid.

Figure 2. Agar result after neutralization with 0.1M HCl (left) and in vinegar (right).

Once we saw some success with the neutralization process, we decided to repeatedly cycle the agar through acidic and basic conditions (from colorless to pink and back again) by recreating the lab conditions of putting the agar into the basic sodium hydroxide solution with indicator and then using vinegar to remove the pink color. Some of the agar was recycled 19 times before our school closed due to the COVID-19 pandemic!

Agar has traditionally been used to grow bacteria, and it should be noted that we did not recycle any agar that was used as a growth medium. However, by using vinegar we were able to successfully recycle the agar from a basic pH (pink-colored agar) back to its original opaque non-pink color multiple times. In order to count as a “cycle,” the agar was exposed to base (it turned pink) and was then treated with acetic acid to return it to its non-pink color. The non-pink colored agar is the starting material used in the Agar Diffusion Cube Lab.

Steps to recycle agar

  1. Use 10-second intervals of time in a microwave to liquefy the solid, pink-colored agar cubes after the diffusion lab. The time required will vary with the amount of agar being recycled and the microwave’s power. In our small batches, we generally used about 30 seconds of time to liquefy about 300ml of the agar.
  2. Neutralize the liquefied agar using vinegar sparingly, until the pink color disappears.
  3. Pour the neutralized agar into molds (ice cube trays don’t produce a perfect cube, but served our purposes well) to make recycled cubes. Alternatively, you may use a tray or pan and then cut the cooled agar into cubes.
  4. Cool to solidify the agar and store in the refrigerator, either in molds or in zipper closure storage bags.

Note: During our trials, we cooled the agar by putting it into a freezer for about 10 minutes when we wanted a quicker turnaround time to begin our next cycle.

Because our school building closed unexpectedly, we did not reach an endpoint for the maximum number of times the agar could be recycled. We will likely continue to experiment as COVID-19 restrictions lift. Additionally, we stored our recycled agar in the science department refrigerator; however, at some point between the school closure in March 2020 and the fall reopening, the fridge stopped working. Interestingly, despite the agar being exposed to higher temperatures during the summer months, only a small portion of it in one storage bag grew some mold.

Outcomes and thanks

Recycling the agar cubes after each use in the diffusion lab is a great way to stretch budget dollars, increase the life cycle of the agar and the phenolphthalein, while also expanding the number of labs we do with our biology students. Agar is fairly expensive, costing about $70 per pound at our local health food store, so the ability to reuse the agar is a great benefit.

A special thanks to my AP Chemistry student, Cameron Muniz, who ran the trials, collected data, and made a short video of the recycling process as part of his application for an internship with Cell Signaling Technology. Unfortunately, the timing was such that due to COVID-19, CST’s internship program was interrupted. Through Cam’s contribution of time and effort, he turned CST’s initial investment into a sustaining one that also reduces waste. Cam will be attending Cornell University in the fall, majoring in Chemistry and Chemical Biology.

Due to budgetary constraints at my school, I am well versed at recycling as many chemicals and supplies as I can in my chemistry lab. However, if my colleague hadn’t asked me if I could do something with the pink agar, it would not have occurred to me to try to recycle it. I will consider it a tribute to Cam and all science students if other teachers begin to recycle the agar they use in diffusion cube activities. I encourage teachers to look for new ways to recycle, reduce, reuse, and promote sustainability. Moreover, if students are willing to get involved in the process of creating more opportunities to be green, that’s a double win for the environment! 

Photo credit:
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