Introduction: Bioluminescent Bacterial Lightbulb / Water Pollution Tester
Second Prize in the
Green Tech Contest
This instructable will show you how to grow and culture your own glowing bacteria and use it to reinvent the light bulb!
I"ll show you how to grow your very own Vibrio phosphoreum or Vibrio fischeri from fresh sea fish (squid work well) and culture it onto special agar gel that you can make at home or simply buy ready-to-pour. The gel can be formed inside all kinds of things that you want to make glow a cool greenish/blue color !!!!
The glowing vibrio bacteria are marine life forms that live alone or in a symbiotic relationships with fish and squid inside their light organs or as parastites. Alone the bacteria don't really glow but in groups of large enough number something amazing happens called quorum sensing ..... The bacteria upon realizing they have enough of each other around turn on genes that allow them to GLOW !!!! The light that is emitted is super efficient around 98 % meaning that only 2 % is lost through heat, thus the light is cool and that's very COOL.
Here is some good info on v. fischeri
It's these amazing little guys (1/1,000,000 of a meter long) that will power our lightbulb !!!! If properly cultured your lightbulb will glow for about a month using zero electricity and zero pollution!!!! Granted its not incredibly bright but you can save electricity costs if you replaced a night light , or you could use it to read maps or books while camping and not worry about changing batteries in your flashlight and disposing of the batteries causing more pollution !!!!
Water Pollution Tester
This instructable will also demonstrate how glowing bacteria can test how toxic water is. The methods used are much more Eco friendly than chemicals and processing ..... So not only does bioluminescent bacteria reduce pollution , they can also DETECT IT !!!! The glowing process is a direct result of the bacteria's metabolism. Fresh clean water does not affect its cellular metabolism or how bright it glows. However even slight toxins that we can't smell , see or taste can alter the bacteria's metabolism and will reduce the amount of light they can produce. By comparing the amount of light emmited from bacteria treated with clean water to that of the sample at hand ..... Say from a stream near a power plant we can assess the level of toxicity in the water !!!!
Here is a video of what the bacteria look like ALIVE under high magnification:
Step 1: What You'll Need
- Fresh (but dead) Sea fish, squid or shrimp (if you don't have access to fresh fish you can simply buy the bacteria from Carolina biological supply here: http://www.carolina.com/product/vibrio+anguillarum%2C+living%2C+tube.do?keyword=Vibrio&sortby=bestMatches)
- Aquarium salt (from pet store) or fresh sea water
- Agar growth medium ( dehyrated tryptic soy agar, or homemade, or ready to pour photobacterium agar...you get to choose if you want to make it or buy it.)
- Sterile Petri Dishes (or glass bowls that can withstand being boiled)
- Sterile Q tip or loop of solid wire
- Pressure Cooker (optional but highly recomended)
- Large Beaker or saucepan
- Med size Erlenmeyer flask (optional)
- Distilled Water
Step 2: Get Your Hands on Some Dead Sea Fish
If you live by the ocean, GREAT. Just go down to the local fish market and get some fresh ( never frozen) squid, shirmp or fish from the sea. I tend to think squid is the best, then shrimp then sea fish since vibrio fischeri etc live inside the light organ of the bobtail squid, but there are tons of these guys all over the ocean. With a little luck the seafood you pick up will have the strains on it, if not, just try again or buy online like the rest of the land locked people. If you are land locked but visiting the beach you may be able to put the fish in a jar, then pack the jar inside a cooler with ice untill you get home but its not a guarntee. Or you can take some sterile agar culture plates with you and grow some bacteria while your on vacation and take it back home to your lab....your family may think your a bit strange but it's worth it ( I did it on a recent trip to Cali muwahahaha).
Step 3: Make Some Bacteria Food Method # 1
Like you and me bacteria need food to grow so let's make some food that our bacteria will LOVE. Its full of vitamins, amino acids, carbon, minerals and of course salt, without salt they won't live (they live in the ocean after all).
TSA agar gel recipe:
Per Liter of deionized water: (you can always reduce it by 1/2 or 1/4 if you don't need the whole liter)
- 40g TSA agar (or directions per liter depending on which company makes it)
- 30g Salt
Mix together in a large beaker or saucepan on a heating plate or stove.
If you are going to use a pressure cooker you just have to heat it up until it mixes because using the pressure cooker to autoclave it will sterilize it...if you don't have a pressure cooker bring the mix to a steady controlled rolling boil to sterilize it.
Pressure cooker instructions (wannabe autoclave)
- Pour dissolved mixture of TSA, and Salt in a flask with a rag SLIGHTLY in the mouth and place in cooker.
- Place glass petri dishes (not needed if you bought some sterile plastic sealed ones) in as well.
- Fill cooker with about 1-2 inches of water.
- Seal pressure cooker and set heat to high
- When the top valve starts to dance and hiss start a timer for 15 min
- When time is up turn off heat and let cool down until pressure valve retracts
Your agar gel and petri dishes are now completely sterilized !!! ( the steam molecules created under pressure bounces around and kills any organisms that were present)
P.S. you may want to research how to use pressure cookers if you haven't used one before as they can be a little scary.
No pressure cooker instructions:
I HIGHLY recommend buying some sterile petri dishes from the net (around $7 U.S.) because it will ensure your bacteria don't get contaminated with other bacteria that are competing for the same space. But if not you can try boiling glass bowls or dishes that can withstand high temps....like pyrex for example for about 15 min.
Now that you have sterilized agar solution AND sterilized petri dishes or bowls and while both are just cool enough to handle (use a oven mit) pour the solution into the dishes and cover with lid or plastic wrap if using bowls.
Let cool until agar solution cools and hardens into a gel, when it hardens flip the dishes upside down...this ensures no bacteria fall on the gel from the lid.
They are now ready for your bacteria.
Step 4: Bacteria Food Method # 2
Pancreatin can be found at your local vitamin store...it's used by people that have trouble digesting food due to problems with thier pancreas. The pancreas releases digestive enzymes. The pancreatin pills contain porcine (PIG!!!) amylase, lipase and protease that disolve protien, fat and carbs. OINK OINK.
You will need:
- Pancreatin (I used 325mg capsules)
- Skim/Lowfat milk 7g
- 100ml Distilled water
- Aquarium salt 3g
- Agar-Agar seaweed flakes 2g
- Pour 7g of skim milk into a beaker.
- Add 100ml of distilled water and mix. Then add the contents of 1 pancreatin capsule and mix.
- Allow to sit for 8 hours with occasional stirring.
- Next add 3g of salt and 2g agar-agar flakes.
- Heat up mixture until salt and agar-agar disolves (maybe a SLOW boil, you don't want to burn your agar).
- If you have an autoclave er uh.... I mean pressure cooker, sterilize it for 15 min. as per previous instructions.
- Pour still hot agar into sterile plates.
- Alow to cool into a gel and flip the plates over so gel is on the top.
Step 5: Bacteria Food Method # 3
OR.....you can buy ready-to-pour "photobacterium" agar from Carolina Biological for about 7 bucks for 125ml....it's pricey but is the best for luminescent bacteria.
Step 6: Let the Fish Glow
- Remove the squid from the bag and allow the ink to remain.
- Heat up your loop until it glows, allow it to cool for min, or use your sterile q-tip.
- Dip the loop/q-tip into the ink.
- Next streak the petri dish of bacteria food that you made with the loop or q-tip. (if you did good sterile technique, your dishes should not have any bacterial colonies growing on them when you streak it.)
- Streak the plates into 3-4 zones using an interrupted pattern as shown in the pic, this will help isolated colonies grow. Separate each streak with heating up your loop or using a new sterile q-tip. (The bacteria don't like to glow unless they are in isolated colonies, they are kinda shellfish uh I mean selfish)
- Place the plate in a cool dark (18-25 C) place and watch it grow and glow! I wrapped mine in newspaper to make sure they are in the dark.
Note: Depending on temperature of the room and other factors, a newly streaked colony will take about 18-48 hours to grow large enought to turn on their glowing genes (lux genes).
Step 7: Culture More Bacteria
Now that you have a starter culture of luminescent bacteria (either from your fish or your culture from carolina) you can grow more and get a healthy stock for your light bulbs and pollution tester!
You'll notice that the bacteria like to grow in "colonies" or little circles...pick the brightest one and swab it or even "fish it out" with the loop, then transfer it to a fresh agar plate and streak it many times across the plate. Again let it grow in a cool dark place. Incubate for 18-24 hours.
Your plate should be aglow with bacteria. Muwhahahahha! BACTERIA!!!!
P.S. Interestingly a cousin to vibro fischeri and phosphoreum is vibrio cholerea which is the deadly cholera bug (well deadly if you don't have acess to basic medicine like IV fluids) so just in case, I would always wash your hands and wear gloves and goggles when messing with bacteria.
P.P.S You may notice that your bacteria seem to streak across the plate as they grow, this is becasue your glowing buddies have tails called flagella that spin like a propeller on a boat and can swarm around!!!
Step 8: Make Your Bulb
- Pick any container that you can boil or autoclave in a pressure cooker to sterilize it. Be creative!!! What's cool is they make the perfect night light since their glowing follows a circadian rhythm...in other words they glow brighter at night than during the day! You can even try using something that is not sterile and hope that other bacteria can't grow in the salty agar (most household bacteria don't like salt).
- Make up some hot liquid agar and pour it into your container.
- Position the container so that it has what ever slant you think is cool to show off the bacteria.
- Make sure that your container is not completely sealed because these bacteria need at least SOME oxygen
I made mine by taking the back off of a lightbulb and pouring the gel in such a way that I made a nice slant of agar medium to display the bacteria.
Step 9: Inoculate Your Bulb
- Take a swab from your pure bacteria culture and inoculate (fancy for streak) your bulb's gel covering as much surface as you can and spreading the bacteria as best you can.
- Wrap your bulb in news paper and again place it in a cool dark room for 18-48 hours!
- Let it grow and GLOW!!
Step 10: Water Pollution Tester
It's simple to assess the quality of water with bioluminescent bacteria!
Just add a drop of distilled water to one half of a glowig agar plate and a drop the suspect water on the other half and observe the change in bioluminescence over a few days compared to the distilled water.
PS. This is a demonstration only.....although if the water I was testing made my bacteria go dim...I would for sure reconsider drinking it haha.
Step 11: How to Care for and Use Your Bacterial Buddies
Some IMPORTANT things to remember if you want all your hard work to pay off and keep your bacteria glowing......
- UV light damages their DNA...so keep them out of harsh light if you can..esp flourescent lighting.
- Keep them comphy at room temp. Too cold and they won't grow and too hot they just dry up. Try to keep them in the 18-25 deg C range.
- Typically the best glow is going to be 18-48 hours after you transfer the cultures to a fresh medium, then they glow less and less each day until they don't glow at all...they aren't dead just need to be re-cultured.
- Its a good idea to keep a rotating stock culture by transfering bacteria from your latest batch to new medium every 3 days or so, this will ensure you always have some living bacteria on hand....this is why it's good to know how to make homemade cheap agar medium! (see step 4).
Make Frozen stocks:
I've been experimenting with making frozen batches that can be stored then used...I'm testing how long the samples stay viable.
- Take 8ml of glycerine (99% USP) and add 2 ml of distiled water.
- Add about 0.5ml of your glycerine solution to a small test tube or vial and add 0.5ml of bacterial culture and mix.
- Immediatly freeze vial by placing it in a block of dry ice and keep it in the feezer wrapped in newspaper.
To use samples take a needle and heat it up till its red hot...let cool then scrape the frozen sample then streak it on a fresh agar plate.
Still experimenting on how well this works.
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