Luminous Glowing Bacteria - Homemade!!

Upon searching some more:

The bacterium causing luminescence is Vibrio (Photobacterium) phosphoreum, it thrives between 20 and 30 deg C, and grows well at saline conditions. The bacterium requires oxygen to produce light, and the reaction roughly works as a luciferase (enzyme) - FADH2 (flavo-cofactor) complex, where light is emitted once it reacts with O2 and RCH2OH to form RCOOH and H2O. Light is emitted in the bluish green range (460–490 nm).

THen, at http://www.disknet.com/indiana_biolab/b203.htm it says:
It grows on glucose, mannose, fructose, or glycerol as a sole carbon source, and synthetic or real seawater as a growth medium.

ISOLATION :

Incubate fresh fish, squid, or octopus at 10-15 deg Celcius, half submerged in natural or synthetic seawater (I take it this shouldn't be sterile seawater! This will stink, so do it outside!). Examine at half-day or daily intervals for glowing colonies of bacteria (in total darkness after your eyes are dark adapted).

Streak these colonies on LM (Luminescence Medium). LM is synthetic or natural seawater containing per liter: 3 ml glycerol (glycerine), 5 gram yeast extract, 5 g tryptone, 1 g CaCO3 (powdered limestone or chalk) and 10 g of agar (per litre). For streaking, sterile toothpics can be used (i.e. soak them in ethanol and dry over flame, and use immediately after they cooled down)

I looked up agar, and it is a linear complex sugar made from beta-galactopyranose linked to 3,6-anhydro-L-galactopyranose. Basically, its just a polysaccharide/carbohydrate, or complex sugar. I should think any other complex sugar would do, such as starch, alginate (a linear complex sugar made from beta-D-mannuronic acid and alpha-L-guloronic acid) etc.

Regarding tryptone, this is a pancreatic digest of casein, a protein. I should think any protein extract should do, you will get that in Health shops/Fitness shops etc.
Yeast extract is similarly easy to get, it is basically a whole cell lysate of yeat, and thus contains vitamins, protein, DNA/RNA, lipids, minerals etc. Isn't Marmite a yeast extract?

Anyway, back to the isolation of Photobacterium phosphoreum!

So, once you have streaked luminous colonies onto the plates, you can either grow them at room temp. or at 4 deg C (fridge). With the latter, check every day for growth by looking at the plates in the dark!

For growth in liquid medium, just leave out the agar (whcih is the gelling agent).
So I would first grow the bacteria on the fish, and meanwhile prepare covered plates with the growth medium. Alternateively, use a small bottle that is sterile (Ethanol-rinsed) and poor in the hot growth medium. Let it cool down, and once at room temperature, add a bit of the luminous colonies from the fish. Only very very little is needed! Even if you dont see anything you have picked up on the tooth pick, there still will be zillions of bacteria on it!
Then grow the bacteria for a day or two, take out a small amount and bubble it with air! Luminescence will ensue! Apparently, luminescence can be so bright that you can read a newspaper with it in the dark!
How cool is that!!


Anyway, apparently it is better to use fresh fish from a fish market next shore lines, simply because transport wasn't so long and the fish are fresh. Water from a salt water aquarium gives varying, but usually poor results. If you live inland and happen to be on a seacoast, place a fresh squid in a plastic bag with trapped air and place it in crushed ice for storage for the trip home. For better results place fish or part of a fish in a jar that is pushed into crushed ice in a cooler. The purpose of the jar is to protect any growing colonies from smearing. By the time you get home glowing colonies may be present. After one or two days at 4 to 18C, luminous colonies may be present. Examine the squid or fish daily. Incubation at 15 to 20C may be the best temperature. Or use your home refrigerator which is about 4 C or above.

By the way, Photobacterium phosphoreum is the strongest light emitting bacterium known!
Whoever tries this gets the Mad Biochemist award!


PS An alternative, but similar growth medium is
30 g NaCl
1 g Glycerol
10 g Peptone (Bacto-Peptone)
3 g Beef Extract
(to obtain solid medium you should add 15 g of Bacto-Agar)
Dilute the ingredients in water (pH should be about 7,3)
Fill with H2O up till 1000 ml

PS2 At AEM Publications, I found a paper 'Isolation and Identification of Photobacterium phosphoreum from an Unexpected Niche: Migrating Salmon' by Budsberg et al. they use 0.38 M NaCl, 0.02 M MgCl2 · 6H2O, 0.25 M MgSO4 · 7H2O, 8 mM KCl, 0.5% peptone, 0.3% yeast extract, 0.3% glycerol as a growth medium. The first few are artificial seawater, which by itself is 0.4 M NaCl, 0.1 M MgSO4 · 7H2O, 0.02 M KCl, 0.02 M CaCl2 · 2H2O.

[Edited on 7-2-2004 by chemoleo]

More info :)

Ok I had a better look around in the net. There is heaps of information!

Firstly, as to the mechanism of luminescence , it appears that there are many different types of luciferins (see http://www.lifesci.ucsb.edu/~biolum/chem/detail1.html for structures of diff. organisms). Luciferins are mostly non-proteinaceous organic molecules.
Also, there are a number of different luciferases, which are the enzymes catalysing the light-emitting reactions of the luciferins. For a layman eplanation of a mechanism, see http://www.lifesci.ucsb.edu/~biolum/chem/index.html


Obviously, luciferases and luciferins have been researched extensively, and a large list of research publications is available at http://www.lifesci.ucsb.edu/~biolum/research.html#papers

Here is a link collection for natural photoluminescence sites . http://siobiolum.ucsd.edu/Biolum_web.html
which covers more you can read in a lifetime


For a quick peek (=picture) of some streaked out turquoise glowing Photobacterium phosphoreum, see

http://www.luxgene.com/
http://www.biology.pl/bakterie_sw/bac_mf_en.html



I think it's quite intriguing that photoluminescence is so widely spread in the animal/plant/bacterial kingdoms, it seems it has evolved very early, and many times over. A classic example of independent evolution of a certain phenotype to yield the same outcome!

[Edited on 16-3-2004 by chemoleo]

From the info I have read, you should use fish AS FRESH AS POSSIBLE. Markets next to shorelines are best (or fish stores, if they can guarantee you the fish was caught on the day u purchase it).
Processed fish, i.e. fish you can find in a supermarket (even if it's fresh) often has been treated with chemicals , such as bleach...so they wont have much bacteria on it.

Tap water? Now that's a no no! these bacteria are adapted to sea water conditions, thus they will lyse (burst open) due to osmotic forces.

Just use artifical seawater, the info for it is in the first post. NaCl, CaCl2, MgSO4 and KCl shouldn't be hard to obtain anywhere.
If in doubt, KCl is in low salt substitutes, and MgSO4 is a certain fertiliser that can be found pure in garden stores.

No luck

Ad Vibrio phosphoreum: I like the Idea of a homemade bacterial lamp. For the garden, a transgenic tabacco plant with luciferase inserted into the genome, would be a nice accessoir too:
http://www.mun.ca/biology/scarr/Fig15_transgenic_tobacco.gif


ad ATP synthase: basically, you are right, but you are mixing up a few things.
During the electron transport chain in bacteria, protons are translocated from the cytoplasm to the periplasm (which lies between the inner membrane and the peptidoglycane mentioned above), thus creating a proton gradient between the two compartments. H+-Ions then go down the gradient through the ATP-synthase (which phosphorylates ADP to ATP) and reduce O2 to H2O (though in anaerobic bacteria there can be many other electron acceptors). Therefore, if the pH at the outside of the bacterium would be - as you said - slightly alcaline, it would be unfavourable for the bacterium, because until reaching equilibrium, it will have to invest energy, before even being able to drive the ATP-synthase.
There are alkaliphilic (opposite to acidophilic) bacteria, which have optimum growth rates at pH-values >7, but the reason for this rather lies in the corresponding metabolic routes.

Ad mesosomes: their function is so far not determined. They are present in some bacteria and might be involved in ATP synthesis. If I remember correctly they are possibly involved in the septum formation during cell division.

@chemoleo: I didn't know lysis of bacteria works with osmotic pressure alone. If I am out of lysozyme, I will remember that. But so far we haven't done very much practical work yet and so my knowledge is rather academical.


[Edited on 30-3-2004 by ungebetenergast]

I don't want to get off topic, but I have to say, people are very nice in this Forum. Usually, as a newcomer, I expect to get flamed immediatly.


Well, in order not to be entirely off topic:
I mentioned previously, that mesosomes might be involved in cell division, but after I searched some articles and web pages about the topic, I found out, that about half of the articles say, that mesosomes are artifacts of fixation:

http://www.pubmedcentral.gov/articlerender.fcgi?tool=pmcentr...
(1964 June: "We confirm that " mesosomes ," hitherto quite often considered to be essential organelles in all procaryotes, are artifacts ."



http://mmbr.asm.org/cgi/content/full/62/4/1371
(1998 December: states, that "the close similarities in structure, function, and genesis between murosomes and mesosomes have led to speculations that mesosomes must be considered as being nothing but enlarged and multiplied murosomes induced by external factors like sucrose-mediated compression"



And the rest writes about mesosomes, as if there would be no doubt in their existence; the last article claims, that " mesosomes clearly are not artifacts of chemical fixation".

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=27...
(1964 June)


http://www.pubmedcentral.gov/articlerender.fcgi?tool=pmcentr...
(1976 September)



http://www.pubmedcentral.gov/articlerender.fcgi?tool=pmcentr...
(1973 January)

[Edited on 31-3-2004 by ungebetenergast]

And, are there any colonies? I wonder whether these bacteria can be found in all major oceans, in warm and in cold waters.

Anyway, as to your questions:
I am not sure whether boiled milk is a good idea. It contains a lot of fat, and may be detrimental to bact. growth.
More importantly, a yeast extract contains all the soluble components of lysed yeast - which includes vitamins, minerals, DNA, RNA, proteins, amino acids and so on. Milk doesn't contain some of the above at least.
Lysing yeast cant be done with water alone, unfortunately. The cell wall is stronger than with bacteria, and already with bacteria lysis with water alone is not efficient.
The text books I have quote lysing yeast with liquid N2 and such, which is probably not available to you. However, on the internet I found this:
http://www.mbb.yale.edu/fl/m_hochstrasser/protocols/EthanolL...

Essentially you can lyse cells with ethanol (100%), in the addition with mechanical shearing (in this case glass beads). A mixer should though just as well, particularly as you will probably need larger amounts than 500 microliter.
It's a very simple protocol - you mix the yeast with ethanol, vortex (mixer), always keeping the temp. down.
The ethanol has to be removed of course, in this case its done at RT/vacuum. This is unfortunately necessary as you cant just boil off the EtOH, as it would destroy many valuable vitamins etc. So thats your bottleneck

But then... using an electrical mixer, I am sure that an aqueous suspension of yeast can be lysed efficiently, provided you do it long enough. The danger is obviously that some life yeast will remain, which would mess up your culture plates. So almost certainly you wont get around a heating step.

But first of all... did you get glowing colonies? If you can see them with your bare eyes, use your digicam and do a long exposure in darkness onto the fish. Maybe you can pick up a few bright colonies then!

lightning bacteria

we want to produce lightining bacteria in the college environment but we fail.do you know how to do it?

You might want to read this very thread first. Chemoleo

[Edited on 21-12-2004 by chemoleo]

Help!

That's funny We did exactly the same experiment when I was a student. This was for a course in microbial ecology. We used "fresh fish" from the market nearby, but I've heard that fish from the market can be as old as two weeks

Maybe that was the reason that only one group actually got V. Phosphoreum. Our group wasn't able to spot any luminous colonies on the fish, so obtaining the right fish is the hard part I think.
We incubated the fish at 4 deg. C to deminish competition with other microorganisms. (V. phosphoreum can grow quite fast at 4 deg. C.) And looked for colonies twice a day...

Quite funny was that our professor told us that every now and then reports come in from terrified people that claim to have eaten "radiation contaminated" seafood. The bacterium however is of no harm IIRC.

I wonder what the reason is for it's dissapearance after the fish gets older. V. phosphoreum is facultative anaeroob and able to grow at a wide range in temperature. Maybe because of competition with other faster growing microorganisms?

Oh, and an idea for people that need yeast extract...There are these nutritional yeast pills that are told to be rich in Vitamin B. I believe they are mayde of pressed and dried cells of S. cerevisiae siccum

TCBS agar preparation

I don't think there are any phototrophic bacteria known that grow in fresh water...


Bereal - well next time make a glycerol stock, to conserve the bacteria! Shame you lost them
With a glycerol stock, you just dip a sterile needle into the first successful culture, which is then dipped into to sterile (heated prior to this) 50/50 glycerol/water.
Then you should be able to restart your growth as many times you wish!

Quote: Originally posted by chemoleo

I read something very interesting recently - I got a pdf but can't remember where I got it from. Anyway, here it goes: Quote: PASSENGERS on board cruise liners are sometimes startled by an eerie phosphorescent glow emitted by seawater flush lavatories. The explanation lies in luminous bacteria, which require oxygen for their light-generating reactions. Luminescent bacteria are present in all marine environments and develop abundantly on the surface of decaying fish. Their presence can be a problem — most of the research on marine bioluminescence has been funded by the US Navy, embarrassed by glowing trails behind surface vessels, and more recently by bacterial interference with the laser light used for submarine communications. Upon searching some more: The bacterium causing luminescence is Vibrio (Photobacterium) phosphoreum, it thrives between 20 and 30 deg C, and grows well at saline conditions. The bacterium requires oxygen to produce light, and the reaction roughly works as a luciferase (enzyme) - FADH2 (flavo-cofactor) complex, where light is emitted once it reacts with O2 and RCH2OH to form RCOOH and H2O. Light is emitted in the bluish green range (460–490 nm). THen, at http://www.disknet.com/indiana_biolab/b203.htm it says: It grows on glucose, mannose, fructose, or glycerol as a sole carbon source, and synthetic or real seawater as a growth medium. ISOLATION : Incubate fresh fish, squid, or octopus at 10-15 deg Celcius, half submerged in natural or synthetic seawater (I take it this shouldn't be sterile seawater! This will stink, so do it outside!). Examine at half-day or daily intervals for glowing colonies of bacteria (in total darkness after your eyes are dark adapted). Streak these colonies on LM (Luminescence Medium). LM is synthetic or natural seawater containing per liter: 3 ml glycerol (glycerine), 5 gram yeast extract, 5 g tryptone, 1 g CaCO3 (powdered limestone or chalk) and 10 g of agar (per litre). For streaking, sterile toothpics can be used (i.e. soak them in ethanol and dry over flame, and use immediately after they cooled down) I looked up agar, and it is a linear complex sugar made from beta-galactopyranose linked to 3,6-anhydro-L-galactopyranose. Basically, its just a polysaccharide/carbohydrate, or complex sugar. I should think any other complex sugar would do, such as starch, alginate (a linear complex sugar made from beta-D-mannuronic acid and alpha-L-guloronic acid) etc. Regarding tryptone, this is a pancreatic digest of casein, a protein. I should think any protein extract should do, you will get that in Health shops/Fitness shops etc. Yeast extract is similarly easy to get, it is basically a whole cell lysate of yeat, and thus contains vitamins, protein, DNA/RNA, lipids, minerals etc. Isn't Marmite a yeast extract? Anyway, back to the isolation of Photobacterium phosphoreum! So, once you have streaked luminous colonies onto the plates, you can either grow them at room temp. or at 4 deg C (fridge). With the latter, check every day for growth by looking at the plates in the dark! For growth in liquid medium, just leave out the agar (whcih is the gelling agent). So I would first grow the bacteria on the fish, and meanwhile prepare covered plates with the growth medium. Alternateively, use a small bottle that is sterile (Ethanol-rinsed) and poor in the hot growth medium. Let it cool down, and once at room temperature, add a bit of the luminous colonies from the fish. Only very very little is needed! Even if you dont see anything you have picked up on the tooth pick, there still will be zillions of bacteria on it! Then grow the bacteria for a day or two, take out a small amount and bubble it with air! Luminescence will ensue! Apparently, luminescence can be so bright that you can read a newspaper with it in the dark! How cool is that!! Anyway, apparently it is better to use fresh fish from a fish market next shore lines, simply because transport wasn't so long and the fish are fresh. Water from a salt water aquarium gives varying, but usually poor results. If you live inland and happen to be on a seacoast, place a fresh squid in a plastic bag with trapped air and place it in crushed ice for storage for the trip home. For better results place fish or part of a fish in a jar that is pushed into crushed ice in a cooler. The purpose of the jar is to protect any growing colonies from smearing. By the time you get home glowing colonies may be present. After one or two days at 4 to 18C, luminous colonies may be present. Examine the squid or fish daily. Incubation at 15 to 20C may be the best temperature. Or use your home refrigerator which is about 4 C or above. By the way, Photobacterium phosphoreum is the strongest light emitting bacterium known! Whoever tries this gets the Mad Biochemist award! PS An alternative, but similar growth medium is 30 g NaCl 1 g Glycerol 10 g Peptone (Bacto-Peptone) 3 g Beef Extract (to obtain solid medium you should add 15 g of Bacto-Agar) Dilute the ingredients in water (pH should be about 7,3) Fill with H2O up till 1000 ml PS2 At AEM Publications, I found a paper 'Isolation and Identification of Photobacterium phosphoreum from an Unexpected Niche: Migrating Salmon' by Budsberg et al. they use 0.38 M NaCl, 0.02 M MgCl2 · 6H2O, 0.25 M MgSO4 · 7H2O, 8 mM KCl, 0.5% peptone, 0.3% yeast extract, 0.3% glycerol as a growth medium. The first few are artificial seawater, which by itself is 0.4 M NaCl, 0.1 M MgSO4 · 7H2O, 0.02 M KCl, 0.02 M CaCl2 · 2H2O. [Edited on 7-2-2004 by chemoleo]

I know this thread is somewhat old, but I just wanted to recount an experiment we did in biology last year (14 yo). We inserted bacterial plasmids engineered to contain antibiotic resistance and a gene from jellyfish which produces a UV fluorescent protein into some bacteria, then killed off the bacteria which had not taken up the plasmid using an antibiotic solution (very clever IMHO). This required a very specific procedure involving cold shock and I didn't get any colonisation but it worked for some other groups. I remember my biology teacher saying that the required materials cost several hundred pounds.

[Edited on 15-4-2010 by 12332123]

Quote: Originally posted by The WiZard is In

Let me introduce a dirty word here — Book. People on la Net spit and scream when someone suggests a book and absolutely have sissy-fits if you suggest they to the (an even dirtier word) Library.

Not a bacteria, but here's Mycena luxaeterna from the mushroom kingdom doing it out in the wild.





And Mycena chlorophos.

Quote: Originally posted by peach

Not a bacteria, but here's Mycena luxaeterna from the mushroom kingdom doing it out in the wild.

Another easy way to achieve glowing is inserting the genetic code that produces GFP in E. coli

first disk: +pGLO/LB/amp
second disk: +pGLO/LB/amp/ara

The two others are blanco's
3. -pGLO/LB/amp
4. -pGLO/LB

LB is a specific medium for G- bacteria, like E.coli

Small question: is it possible to get luminous glowing bacteria that are glowing spontaneously during the day e.a. with luciferin ?

Not only fish but chicken also can work!

*carolina also sells cheap vibrio culture.

QUOTE pumukli:"Not only fish but chicken also can work!

anyone try growing a glowing chicken? that's a new one for me.
there is a bioluminescent form of cholera. so maybe it was that?

Another option (for people not located near the ocean) might be hornworm-caterpillars,
if you find a caterpillar that looks paralyzed, it may be infected with glowing nematodes. (Actually it's a bacteria in the nematodes that glows.)

The nematodes first infect a plant, the caterpillar eats the plant, and becomes infected,
inside the caterpillar they release a paralyzing toxin MCF (Makes Caterpillars Floppy)

interesting, in olde times this glowing bacteria sometimes infected wounded solders,
seen as a miracle, their bandages would glow, and it reportedly healed their wounds.

ii think it would be a fun project to grow the glowing nematodes bacteria.