December 3, 2019
Dyeing Textiles with Bacteria: Making LB-Agar Plates
In order to dye textiles with fluorescent bacteria, the first thing you’re going to need is an agar plate. A common thing people ask is why would I need to make an agar plate when I could simply just buy one? Well, for a lot of bacteria, you need to add specific antibiotics to the agar solution in order for the bacteria to grow properly. A lot of times, this agar solution doesn’t come pre-made.
But let’s back up a minute. Why do we make agar plates in the first place?
Agar plates provide a healthy environment for microorganisms to grow in. Think of your agar plate as a blank canvas, and the bacteria you’re using as your paint. However, this paint is a little different in that in order for us to be able to see it on our canvas, it needs some extra help. That’s what this LB/agar plate is for.
Just like people say that baking is a science, science is also similar to baking. There are these nutrient broth “recipes” that are different depending on the type of bacteria you want to grow. In this case, we’re using LB as our media, which is what we call the liquid solution we’re about to make; I’ll get more into the specifics of that later. Agar is simply the setting agent for plates. It allows our media to change from liquid to a solid “jelly”.
Eventually, the bacteria will grow on these plates after an incubation period. They will form what we call bacterial colonies. This will be the visible mass of microorganisms on your plate, all originating from a single mother cell. They should look kind of like polka dots. However, since there is a textile on the agar plate that we’re hoping to dye, the bacteria might not grow exactly in this fashion. Instead, the bacteria will hopefully grow enough to coat the textile completely.
Before using any materials (even items like graduated cylinders and glass bottles), be sure to autoclave them. An autoclave is basically a big oven that helps to sterilize liquids, instruments, and materials that are going to be used in a lab. A basic autoclave is similar to a pressure cooker, if you’re familiar with that device. It uses the power of steam to kill any unwanted bacteria.
The agar solution will go in the autoclave once it is made. Alongside killing unwanted bacteria, this heats the media up enough so that all the chemicals are able to dissolve properly.
Now, you may ask, how do we know what nutrients to use in the agar solution? In this case, we’re using LB. LB is also known as Luria broth. It’s nutritionally rich, and using LB as a media has been an industry standard for the cultivation of E. coli since the 1950s. Nutrient broth made for plates must include some combination of the following: carbohydrates for energy, nitrogen for protein synthesis, other minerals for growth and survival, and agar to set the plates. You can find pretty much anything online nowadays. In general, using a nutrient agar broth or LB is a safe bet to get your bacteria growing. I found information on NCBI’s page in regards to what media to use for the specific bacteria for this project.
You’ll see later in the protocol that what is added to the agar solution depends on the bacteria used. There are a couple different strains being used. For normal fluorescent bacteria, such as GFP or RFP, the LB-agar solution is more simplistic. However, you also have the option of dyeing a textile with melanin.
But why are we using melanin? What does melanin do? What we are using is a genetically engineered strain of E. coli bacteria that expresses melanin. Melanin, as you know, is a pigment that is present in our hair and skin. As it turns out, it can also be used to dye textiles. We will grow this bacteria on our plates in hopes that it will color the textile completely.
Besides the LB and agar, you might have noticed the addition of chloramphenicol to the nutrient broth. This is an antibiotic. The use of antibiotics in a cell culture is simply to minimize the risk of contamination of our bacteria, just like how antibiotics help stop the growth of harmful bacteria in terms of human health. Which antibiotics we need to add depends on the type of bacteria we are growing. Again, everything is online. If you know what bacteria you’re using, you’ll be able to find what antibiotic other people have used with it on resources such as NCBI.
If you notice, the concentration of the antibiotic, chloramphenicol, is extremely small. In order to be able to use this in the LB agar nutrient solution, it is necessary to make what is called a stock solution. This involves diluting the chloramphenicol to 1000x by using 10 mL of distilled water. Then, the concentration must be adjusted to it’s dilution, and you should be able to use a measurable amount of the antibiotic in your nutrient media. Here’s a video that details a bit more about sterile antibiotic stock solutions if you are interested.
The tyrosine and copper sulfate are just additional nutrients to help the bacteria grow.
When pouring plates, you must be VERY careful handling the liquid bottles as they come out of the autoclave. Make sure to wear what we call “hot hands”, or pot holders, to make sure you don’t burn yourself. Making sure that the plate is situated with the lid side up, carefully pour the liquid until it completely covers the bottom of the plate. Do not fill the plate all the way up to the brim. It should be less than halfway full- you just want enough liquid to completely cover the bottom. Place the lid back on quickly to avoid any contamination of the plates, and let rest to set. I’ve linked a video here that shows you in depth how to do this.
It can take anywhere between 30 minutes to an hour for plates to set properly. Normally, it’s recommended that plates sit overnight in order to get rid of any moisture that accumulates.
Additionally, in order to make your bacterial plates, you’ll need to know how to pipette. Here is a nice video that shows you how to do so if you don’t already know.
Making LB broth is nearly the same as making a LB-agar plate. The only difference is that LB broth doesn’t have the agar, so it won’t ever transition from a liquid to a solid “jelly”. The LB broth is important for soaking the textile.
Where can I look for answers if I work with something different? Thankfully, there are many resources available at our fingertips. NCBI is always a fantastic and reputable source to use to look up research papers and articles on a particular topic. Some of these may be dense and can be more difficult to understand. Don’t be afraid to command+F and search keywords for particular things you are looking for.
Another great source is the FPbase (Fluorescent Protein Database). This has everything and anything you need to know about using fluorescent proteins.
- Petri dishes
- Pipette tips
- Glass bottles
- LB powder
- Agar powder
- Copper sulfate
- Textile of choice (silk is recommended)
- Autoclave tape
- Weigh boats
- Graduated cylinder
- Distilled water
Here is the step-by-step protocol:
- For a 250 mL bottle (melanin dyed textiles): 5 g LB, 2.28 g agar, 125 mg tyrosine, 400 uL CuSO4, 10 ul stock solution of chloramphenicol (34 ug/mL concentration)
- For a 250 mL bottle (fluorescent bacteria or cell-cell signaling textiles): 5 g LB, 2.28 g agar, 10 ul stock solution of chloramphenicol (34 ug/mL concentration)
- Add the LB, agar, and tyrosine (if using) into the autoclaved glass bottle first.
- Add the copper sulfate (if using). The antibiotic will be added after the liquid comes out of the autoclave.
- Fill bottle with distilled water until 250 mL. Preferably, add a stir bar to the bottle and mix on a stir plate. If not available, invert the bottle a few times until the solution is homogeneous.
- Place the lid on the bottle and a piece of autoclave tape on top. Make sure to label the bottle.
- Place the bottle in the autoclave on a liquid run.
- Once liquid run is done, immediately take the bottle out of the autoclave and let sit for just a couple of minutes. It needs to cool down enough so that the antibiotic isn’t denatured, but not too much so that is starts turning solid. After adding the antibiotic, pour the liquid into the plates. Make sure you use oven-safe gloves as the bottle will be very hot. Pour the liquid so that just the bottom of the plate is covered with liquid.
- Put the lid on the plate. Let sit for at least 45 minutes or overnight if time so that any additional moisture evaporates.
- Once plates are set, place desired textile that is autoclaved and cut to size on the plate. Soak the textile through on top of the LB-agar with LB broth using a pipette. Only use enough LB to coat the textile. (Note: in order to make LB broth, use the same ingredients in steps 1 and 2, just minus the agar. Make sure to autoclave.)
- Parafilm plates and let them chill in a 4 °C fridge until needed for further use.