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Bioluminescent Bacterial Lightbulb / Water Pollution Tester

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Bioluminescent Bacterial Lightbulb

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
http://www.bionity.com/en/encyclopedia/Vibrio_fischeri.html

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:
 
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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
If making your own agar gel see step 4 for supplies

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) 

  1. Pour dissolved mixture of TSA, and Salt in a flask with a rag SLIGHTLY in the mouth and place in cooker.
  2.  Place glass petri dishes (not needed if you bought some sterile plastic sealed ones) in as well.
  3.  Fill cooker with about 1-2 inches of water. 
  4.  Seal pressure cooker and set heat to high
  5.  When the top valve starts to dance and hiss start a timer for 15 min
  6.  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

A second method if you want to make bacteria food is to make your own nurtient agar from scratch (funner and cheaper). We will use PANCREATIN to digest the casein in milk into nutrients the bacteria can use and add some salt for minerals and agar-agar flakes to thicken it into a gel.

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
  1. Pour 7g of skim milk into a beaker.
  2. Add 100ml of distilled water and mix. Then add the contents of 1 pancreatin capsule and mix.
  3. Allow to sit for 8 hours with occasional stirring.
  4. Next add 3g of salt and 2g agar-agar flakes.
  5. Heat up mixture until salt and agar-agar disolves (maybe a SLOW boil, you don't want to burn your agar).
  6. If you have an autoclave er uh.... I mean pressure cooker, sterilize it for 15 min. as per previous instructions.
  7. Pour still hot agar into sterile plates.
  8. Alow to cool into a gel and flip the plates over so gel is on the top.

Step 5: Bacteria Food Method # 3

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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. 

http://www.carolina.com/product/nutrient+agar%2C+prepared+media+bottle%2C+125+ml.do?keyword=photobacterium+agar&sortby=bestMatches

Step 6: Let the fish glow

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  1. Remove the squid from the bag and allow the ink to remain.
  2. Heat up your loop until it glows, allow it to cool for min, or use your sterile q-tip.
  3. Dip the loop/q-tip into the ink.
  4. 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.)
  5. 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)
  6. 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

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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

  1. 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). 
  2. Make up some hot liquid agar and pour it into your container.
  3. Position the container so that it has what ever slant you think is cool to show off the bacteria.
  4. 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

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  1. 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.
  2. Wrap your bulb in news paper and again place it in a cool dark room for 18-48 hours!
  3. Let it grow and GLOW!! 

Step 10: Water Pollution Tester

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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

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Your bacteria has been good to you so be good to them. 

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.
 

  1. Take 8ml of glycerine (99% USP) and add 2 ml of distiled water.
  2. Add about 0.5ml of your glycerine solution to a small test tube or vial and add 0.5ml of  bacterial culture and mix.
  3. 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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drdan152 (author) 2 years ago
I've been doing some experimenting on adding things to the agar to make it glow more...it seems so far that by adding a crushed calcium vitamin (as calcium carbonate, CaCO3) AND some glycerol it glows brighter!!! I'll add the new recipe as soon as testing is done.
VijethaK4 months ago

Hi,

I am vijetha from Bangalore, India. I wanted to buy some bio-luminescent algae to grow in my lab. But I am not finding it anywhere here. Ca anyone please help me regarding this.

Thanks

VijethaK4 months ago

hi,

Can someone please help in finding the algae culture dealers in India. Preferable in Bangalore.

Thanks

theegghead10 months ago

A-W-E-S-O-M-E!!! would this work for any kind of ocean-fish residue?

gkpsanchez1 year ago
can you help me out? i just want to ask if the mediums that you have used (LA) is ready made, or you have mixed ever single chemical to make a Luminescent agar. hope you could help me out on this information.
hermes11 year ago
Dear Dan, I'm looking at the solar bottle bulb and thinking: http://www.instructables.com/id/How-to-build-a-SOLAR-BOTTLE-BULB/
I'm Thinking is there any cheap/free way of producing a living medium for bio luminescent algae for the poor people of the world to give free lighting at night from the solar bottle bulb idea... Any other links or even cheap/free bio luminescant chemicals that may help in the same situation.
mrg02d1 year ago
Any chance we might accidentally get ahold of the flesh eating cousin?
mrg02d1 year ago
How bright is this? Can you compare it to another light source?
ksail1 year ago
A 'lightbulb' that you can also use to test water quality - very creative.
I was wondering if it is possible to grow Vibrio fischeri in a liquid environment (saltwater) with little or no agar. I would appreciate your response.
jmapscience2 years ago
I want to do this expiriment. I'm just getting into biology and I'm 13 . If any one has tips and tricks on this expiriment ( like agar and equitment) please contact me at jmap2112@gmail.com Looks like a great expiriment thanks for posting it!
drdan152 (author)  jmapscience2 years ago
Hey! So happy to hear your gonna make a bio bulb! How's it going?
ksmith892 years ago
earlier you said "Just FYI I'm experimenting with a new agar to increase bioluminescence ..... It has more nutrients that should support more glowing ..... Calcium carbonate , yeast extract , and glycerol !!!!" so did you ever figure out your new agar? what amounts of the Calcium carbonate , yeast extract , and glycerol do you add to the ager recipe for the homeade ager method #2
qharding2 years ago
Hi. I know its been a long time since anyone has commented but I really need your help I'm currently growing Vibrio Ficheri and I have so many question. Mainly how did you get them to show up in pictures? Please help
drdan152 (author)  qharding2 years ago
HEY! Glad to see people growing bacteria ....haha. Well the best way to take a picture is in a completely dark room and get an SLR camera or any camera that you can adjust the shutter speed. Mount the camera on a stable surface or tripod and set the shutter to stay open for as long as possible...maybe even 30 seconds. I set the timmer so it counts down 5...4...3...2....1 then take the picture so i dont bump the camera and get a blurry shot. BIggest thing is the shutter speed though .....think LONG or SLOW shutter speed and keep the camera steady!
ozzyfast2 years ago
Hi friends,
where can i buy v. fischeri's culture from Europe?

regards
ryan88162 years ago
For anyone who would like to see the Tryptic Soy Agar label on the bottle
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nano nite2 years ago
Do u have any idea about biological shops in India where i can find theese bioluminescent bacterias ? Be soon buddy its for my science exibition.
nano nite2 years ago
hey can u give a clear comparison between bioluminescent light bulb and an electrical light bulb ? including all aspects.
dragonito2 years ago
This might be a bit of a stretch... but could a concentrated, sterilized dirt/compost extract combined with digested skim milk (and perhaps some yeast extract) be used to make a suitable photobacterium broth? The digested milk and yeast would amino acids and vitamins, while the dirt extract would provide (trace) minerals. And keeping the bacteria in a continuously agitated medium would also increase the oxygen content, possibly maximizing glow.
drdan152 (author)  dragonito2 years ago
Dude that sounds really cool
..... Not sure it of will work but I think it would be an awesome experiment to see if it does ..... As Bill Nye the Science guy says "TRY IT" !!!!
pchubb2 years ago
Any reason to use tryptic soy agar? I just mixed up agar into lamb broth made by boiling an old bone with a bit of meat on it. I added salt to make it 3% w/w, and I have the bacteria growing well.
drdan152 (author)  pchubb2 years ago
Well to be honest I'm not sure if boiled bone has all the nutrients that photo bacteria need..they are kinda picky eaters...I've noticed that the bacteria will readily GROW in different agar mediums (even polluted ones) but they will only GLOW if they have juuuuust the right conditions and nutrients to support their glowing reactions....thats why it makes such a good "water tester" cause they amount they glow is directly related to how good thier environment is. TSA has tryptones (short chains of amino acids) and other good stuff that fastidious (picky) bacteria like.
pchubb drdan1522 years ago
The colonies are growing and glowing well. It's my experience that most bacteria are not fussy eaters (unlike my ten y-o).
drdan152 (author)  pchubb2 years ago
Oh wow !!!! I'm sorry I thought you meant they were growing but not GLOWING !!!! Excellent !!!! Isn't amazing how glowing bacteria can come from a dead squid !!!! Good job culturing from different areas I only used the left over ink from the bag the squid was in. Can u post a picture of your glowin microbes ???? Would make a cool post for the instructable :-)
pchubb pchubb2 years ago
Oh, and I tried culturing from different parts of the squid. I got nothing from the ink sac, but plenty from the body cavity (inside the `tube' of the squid. Our local squid don't glow.
drdan152 (author)  pchubb2 years ago
Try adding a pinch of powdered calcium carbonate (from a vitamin crushed into a powder...it shoul say on the back "calcium as calcium carbonate") to the medium you made...or maybe a few ml of glycerol for a carbon source?? also try letting your eyes adjust in PURE darkness for 5 min...if you find a glowing colonie , fish it out and reculture it in fresh agar......BUT it may be that the squid was previously frozeon OR you just got a bad egg...or uh squid. DO you have acess to a microscope? If so you can see if the bacteria you grew is indeed the a vibrio species.
pchubb drdan1522 years ago
As I said before, the bacteria cultured from the head and body cavities (under the mantle) are glowing well. It's just that I found no colonies growing from the ink sac.
yellowcatt2 years ago
Great instructable.
It got a mention on the This Week in Microbiology podcast:

http://www.microbeworld.org/index.php?option=com_content&view=category&layout=blog&id=107&Itemid=275
drdan152 (author)  yellowcatt2 years ago
REALLY !! thats so awesome i love microbe world hahaha thanks for the info!
Your instructable was mentioned right at the end of podcast 39, when they were reading out the listeners emails, (at 1hour 11minutes and forty seconds)

Vincent Racaniello described it as "really quite amazing".

Aw dude this is awesome i've been trying to grow this for so long and this just simplified it sorry i'm new so I don't know how images work
drdan152 (author)  Mushroom man2 years ago
Sweeeet! Do you have acess to fresh non frozen fish or squid???!
techno guy2 years ago
did the idea for testing water pollution with this come from the idea that the 15 year old who made the cancer test strips had
drdan152 (author)  techno guy2 years ago
No, it's a pretty common experiment with v. fisheri.....i wish i came up with the idea but its been around :-)
FuzzeeDee3 years ago
Can this culture be grown in a liquid culture without going to the final gel step. Was thinking of using long glass tubes bent around and around to create rectangular lighting panels that could circulate fresh medium liquid periodically. Seriously curious. • • • Fuzzee Dee OUT • • • }~{ <> }~{ ^~^ :->
drdan152 (author)  FuzzeeDee3 years ago
Why yes it can ..... You need "photobacterium broth" you can buy it online. But I'm experimenting on how to make it ..... For some reason it's considerably more complex to make a broth than a solid medium. This really only the Begining of this instructable do stay tuned as I add more pictures and more cool stuff to do with the bacteria !!!! Also I know that you can make a bacterial suspension in normal saline for a cool liquid effect but the microbes don't last very long that way
They do market a "tryptic soy broth" which is a liquid mix without the agar, you can use that with the rest of the mix I believe to make a liquid medium.
Kewl, tnx for the info - that will be helpful
f• • • Fuzzee Dee OUT • • • }~{ <> }~{ ^~^ :-> 
You need a shaker if you want to grow a lot of bacteria in liquid broth. Even though they're tiny they'll settle to the bottom of the liquid if you leave them alone. They need to be shaken up to keep them happy.
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