Sciencemadness Discussion Board - Luminous Glowing Bacteria - Homemade!!

Luminous Glowing Bacteria - Homemade!!

chemoleo - 7-2-2004 at 10:23

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]

Blind Angel - 7-2-2004 at 19:01

Do you think that a growth medium made for other bactria like Streptocoque, Coli or some like that would do too?

chemoleo - 7-2-2004 at 19:42

Yah, except you have to bear in mind that this organism grows at saline conditions (sea water) - so in fact you could use seawater, sterilise it by boiling and add the nutrients (yeast extract, agar).
To be honest, from what I read, about any nutrient solution could be used, as long as there is plenty of salt. Seems to be a fairly robust organism otherwise.

Nick F - 14-3-2004 at 10:05

I've done it a while back, after a few very smelly failures. Please, do it outside if you value your nose!!

"I take it this shouldn't be sterile seawater!"

Sterile seawater will work (otherwise synthetic seawater could not be used) - the bacteria are on the fish.

ziqquratu - 14-3-2004 at 23:03

Sterile seawater should work - I think that the salinity is the key reason for the seawater, and as Nick mentioned the bacteria ARE on the fish!

As to agar... If you have access to pre-made lab agar, something designed for growing skin bacteria (particularly Staphylococcus spp.) should work, as these plates are often reasonably saline.

Secondly, if you're making your own media, simple gelatin works well as a solidifying agent. In fact, it used to be the most common medium for solid culture of bacteria, until agar came along... agar is preferred because many bacteria grow best at 37*C, and gelatin melts around 25*C (whereas agar doesnt melt until near 100*C... but solidifies at around 40*C... it's a very interesting substance, really!). But really, substituting gelatin for agar should work like a charm here because you dont want to incubate at the higher temperature! (not necessarily in a 1:1 ratio... I'm not sure how much you would use, as I've made gelatin plates but never agar, so I can't make a practical comparison).

The purpose of the agar (or gelatin) is not as a nutrient, but simply as a gelling agent. Quite frankly, there are surprisingly few bacteria that are capable of metabolising either of them.

Tryptone is just a protein extract that the bacteria can metabolise, and just about any protein should suffice as a substitute. And marmite/vegemite is a yeast extract, although I dont know if there are any other preservatives that could cause problems...

[EDIT]: One more thing... when you're doing this, make sure that you're working under sterile conditions. This means the media should be as sterile as possible (normally you'd autoclave, but in this case simply boiling the mixture for 15-20mins should suffice. Of course, you could always throw your plates into a pressure cooker with some water for a similar time, but it probably isn't necessary). Also, work underneath a spirit lamp/bunsen burner (ie. have one very close to the plate and so forth when you're working). For sterilising the toothpicks, you could boil then store them in a jar that has been washed out with 80% ethanol... you could throw the picks and the jar in the pressure cooker with your plates if you were that way inclinced! As an alternative to toothpicks, you could use a simple nail, which you can sterilise by holding in the flame of your burner for a few seconds... you must then let it cool before streaking your bacteria, or you'll kill them!! Practice a few times by heating the nail for say 3 seconds, then letting it cool - touch it to the back of your hand to work out how long it takes to cool to normal temperature. Then flame it again, let it cool and do your streaking. Also, make sure you do use 80% alcohol, not 100%, as the water is required for effective sterilisation. Metho would work fine, just dilute it to 80%. The reason for these seemingly slightly excessive precautions is that skin bacteria such as Staphylococcus epidermidis which is carried by almost everyone should be able to grow quite well under the given conditions and may out-compete the luminous bacteria, meaning you'd nd up with some extraordinarily boring off-white colonies of nothing remotely interesting... hence, go for the sterility - it's really not that difficult!!

[Edited on 15-3-2004 by ziqquratu]

More info :)

chemoleo - 15-3-2004 at 18:27

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]

Iv4 - 16-3-2004 at 05:30

About a year ago I was forced to gon on vacation to a place near the ocean and the markets all had fresh fish.

There were some old fish left in an area of the shore where nobody usually goes(because of the smell)but being with a solid 8.5 I ignored it.When we went there though there was a weirdish light liek you say on some of the fish.Not nearly bright enough to read a newspaper but still it was visible if you looked firectly at the fish.

I'm just saying this because it seems if it can live like that I'll have good luck for it as my first experiment when at my new place.I'm gonna try the two mediums youve sugested(if I can get agar) and out in the open and with only salt water and with normal tap water.

Blind Angel - 16-3-2004 at 14:25

Perfect, I'll get the Staphylococcus Spp. agar medium quickly (well went all the rest will be ready), and i was wondering if fresh fish from the fish store will do, since it's winter around here and that white fishing has endend recently, i'm looking for a fish source, also i'm wondering if i need to add a protein extract to the culture or it's already included in.

chemoleo - 16-3-2004 at 15:02

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.

Blind Angel - 16-3-2004 at 16:00

Tap water?
I plan to make them grow on blood agar that it's use in medical laboratory to grow the Streptococcus

chemoleo - 16-3-2004 at 16:34

Lol..
tap water refers to IV4's post.

ziqquratu - 16-3-2004 at 21:49

Blind Angel: NOT blood agar, and NOT Streptococcus!! Mannitol-Salt agar and Staphylococcus!! There's a significant difference. Staph are happy in a saline environment, whereas Strep don't like it so much... plus blood agar probably isnt saline enough. The only thing I thought of since my last post is that perhaps the mannitol would inhibit the growth of the desired bacteria... on re-reading the original post, I see that we're trying to grow Vibrio, so if you can get TCSB (Thiosulfate Citrate Sucrose Bile salt) agar, you have a selective medium on which only the desired bacteria (ie Vibrio species) will grow. They might not phosphoresce on this medium, but you can easily get a pure culture from the TCSB and transfer it to the medium mentioned in chemoleo's first post.

No luck

Iv4 - 24-3-2004 at 04:49

Maybe it's the fish(was alive-not cheap but myfortunatly my grandparents like that stuff)but I had no success.

The first sugested formula(without the peptone though) and I also used plain water and just a bit of the seawater in an open dish.SMelled prety bad but nothing.

Esplosivo - 24-3-2004 at 11:43

I often culture funghi on a mixture of around 70% jelly (the one that is eaten) / 20% gelatine (the powdered form) / 10% glycerol. For fungi I usually added some weak acid. This worked for me. I also cultured bacteria once, but this resulted in numerous colonies which couldn't be identified.

For culturing bacteria some weak base, such as some sodium carbonate should be added (I've read that most bacteria prefer a slightly alkaline environment). Add some sea water to the mixture before it solidifies. It should work fine, I suppose.

[Edited on 24-3-2004 by Esplosivo]

chemoleo - 29-3-2004 at 09:02

I do believe that you have to use seawater only, and no water dilutions thereof, to avoid the bacteria from lysing - which means bursting, due to osmotic pressures!
In fact, in biological laboratories, gentle lysis of bacteria IS done with distilled water.

On the note of using slightly basic media for optimum bacterial growth - isn't that because the bacteria are more acidic inside, and thus have a greater proton gradient and hence a greater rate of energy conversion (ATP production)?

Esplosivo - 29-3-2004 at 10:11

Quote:

On the note of using slightly basic media for optimum bacterial growth - isn't that because the bacteria are more acidic inside, and thus have a greater proton gradient and hence a greater rate of energy conversion (ATP production)?

As far as I know that should be the idea. Bacteria, having no mitochondria, produce their ATP on proteins found attached on mesosomes ('curly' parts of the cell membrane). The electron chain for the production of ATP is therefore carried out more efficiently in alkaline conditions(because of a lower H+ gradient outside the cell) and the bacteria divide more often forming larger colonies in a shorter period of time.

Don't bacteria have a cell wall?! Therefore cell lysis cannot occur right?

[Edited on 29-3-2004 by Esplosivo]

chemoleo - 29-3-2004 at 12:14

I am not sure whether a cell *wall* can prevent cells from lysing. Plant cells have a cell wall, and do swell up in low salt conditions. Similarly, they shrink in size when a lot of ions are around (i.e. see the salad effect, where salad shrinks once the dressing is added)...which of course is not the same as lysis.

Bacteria have a cell membrane, with a few proteins sticking out, or peptidoglycans etc.. To my knowledge I haven't seen it termed 'cell wall', only membrane. On the other hand, I know there are bacteria with a solid cell wall, such as the infectious strain of salmonella.
Nonetheless, E.coli can be lysed by osmotic shock. Of course this won't apply to all bacteria, but most certainly it will apply to Vibrio phosphoreum, which simply won't grow in de-salinated conditions. This is presumably because the ion gradients are messed up, and because the cells are likely to lyse.

ungebetenergast - 29-3-2004 at 12:24

Almost all bacteria have a cell wall. With the exception of archaebacteria and some rare species they can be classified as gram-negative or gram-positive (which refers to the gram stain). Basically, gram-positive bacteria have on the outside a peptidoglycan layer (consisting of N-acetylglucosamine-N-acetylmuraminic acid polymers, which are crosslinked via aminoacids) and the cytoplasmatic membrane. Gram-negative bacteria have an outermembrane with attached polysaccharides (the outer membrane is therefore called Lipopolysaccharide) and a thinner peptidoglycan layer than gram-positive bacteria without crosslinking (because of this the reagens of the gram stain stays within gram-positive bacteria and elutes from gram-negative bacteria after Iodine is added) and an inner membrane.

Osmotic lysis only works on bacteria after the destruction of the cell wall, e. g. by lysozyme. The peptidoglycan is rigid enough to withstand a (pretty huge) osmotic pressure, but as soon as it has a leak, the membrane ruptures.

[Edited on 29-3-2004 by ungebetenergast]

chemoleo - 29-3-2004 at 13:23

Thanks for clarifying this. Great to see there are increasingly more people knowledgeable in biochem/biology!
I use lysozyme in the lab myself, and it works great. However, I still got it to work without it, albeit slowlier...

ungebetenergast - 29-3-2004 at 15:49

Quote:

Originally posted by Esplosivo

As far as I know that should be the idea. Bacteria, having no mitochondria, produce their ATP on proteins found attached on mesosomes ('curly' parts of the cell membrane). The electron chain for the production of ATP is therefore carried out more efficiently in alkaline conditions(because of a lower H+ gradient outside the cell) and the bacteria divide more often forming larger colonies in a shorter period of time.
[Edited on 29-3-2004 by Esplosivo]

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: :cool:

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]

Esplosivo - 29-3-2004 at 21:26

Thanks for correcting me ungebetenergast. I've oversimplified the process. Thanks.

It seems there quite a large interest in Biochem around here and that is very very good.

Cell lysis is prevented by the action of the cell wall. Bacteria do have a cell wall which is made of murein (a peptidoglycan). This murein is formed from mucopolysaccharides and short polypeptide chains. Mucopolysaccharides are made up of alternating monomers of N-acetylmuramic acid with N-acetylglucosamine. The polypeptide chains join the long mucopolysaccharide chains together. Gram +ive bacteria have a thick murein cell wall, while gram -ive bacteria have a thin murein cell wall but with an additional layer of glycoprotein.

This bacterial cell wall is not as strong as a cellulose cell wall (as in plants) but is strong enough to prevent lysis under normal conditions. Except treatment with lysozyme I found that the bacteria can be treated with a detergent/alkali/alkaline salt(CH3COOK) mixture (the detergent used was Sodium dodecyl sulphate) Check out this site for more info.: http://www.marshall.edu/cellcentral/plasmidprep.html

[Edited on 30-3-2004 by Esplosivo]

ungebetenergast - 31-3-2004 at 09:57

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]

Esplosivo - 8-8-2004 at 04:13

Right now I am following the procedure described by chemoleo to grow such a bacteria. Three days ago I obtained a fish (just right out of the sea) and I placed the latter in natural sea water. All was placed in a fridge. The fish being used is of the species Scorpaena scrofa which was obtained from Mediterranean waters. Right today the fish was in a phase of degeneration, so I took out of the fridge and placed it outside. Tonight I will check for any luminous colonies.

Anyway, I have a couple of problems to culture such a bacteria. I have all the necessary chemicals (such as agar and the sort)to carry out the process, except the protein digest and the yeast extract.

As mentioned by chemoleo both tryptone and peptone are partially digested proteins. I was thinking of the possibility of using strongly boiled milk. The boiling is required to kill any microorganisms present in the milk, such as bacteria and fungi. Could this work? Can 'protein soups', the ones used by body builders and the type, be used instead?

The yeast extract is also another of my problems. I tried placing bakers' yeast in distilled water. After approx. a day the colour of the water turned brownish yellow, and a sort of 'debris' precipitated. I hope that this is some sort of indication that yeast cell lysis did occur. Can this solution be used as a yeast extract?

A long story made short, I can always buy what is needed but I would love to make all that is required myself. Another fact is that the supplier sells such chemicals in bulk, which is rather expensive.

Another question. Can the yeast extract, and peptone etc... be boiled? Just to make sure that denaturation of the proteins is not a problem. Thank you for any suggestions.

chemoleo - 8-8-2004 at 14:36

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!

Esplosivo - 8-8-2004 at 20:54

I couldn't see any of such colonies, but the reason could be that there was still some faint star light. I will try again tonight in total darkness. If I don't get any visible growing colonies I will still try innoculating a medium. If at least 1 luminiscent bacteria with all the bacteria living on the fish, then this would theoretically grow into a colony right? I would then innoculate another medium with such a colony. Lets just wait and see.

Thanks for the help chemoleo. If I get growing colonies I'm off to the supplier.

Lysing yeast with 100% ethanol is interesting, but it is a little tricky to remove all the yeast cells. Liquid N2 ... well its out of range for now.

Esplosivo - 9-8-2004 at 12:00

I checked the fish right now. I could not see any clear luminous colonies, but on the part of the fish which was left outside the water I could see a very faint light. I am not sure that these could be the bacteria. So what I will do is that tomorrow I will buy the necessary chemicals and try innoculating a medium with all the bacteria present on the surface of the fish as mentioned previously.

If I manage to collect just a couple of luminescent bacteria a colony will form and from this colony I could make millions of others.

Btw, I tried taking a picture with my cheap camera but nothing was visible. The camera has no aperture settings so the ammount of light entering the cam cannot be controled. Anyway, I will try enhancing the pic.

Edit: The growth of the bacteria will be postponed since I cannot obtain the yeast / beef extract till next month. Till then I will try acquiring two different species of fish, and probably even a squid or octopus.

[Edited on 11-8-2004 by Esplosivo]

lightning bacteria

gorkem - 21-12-2004 at 13:00

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]

vulture - 21-12-2004 at 13:35

Read the thread first and DO NOT crossposts three times!!

One more stunt like this and your posting ability will be gone with the wind.

Madandcrazy - 10-7-2005 at 07:20

Vulture you meant the crosspost to chemolos metabolic pathway topic ?

I don`t know the method isolating lightning substances from insects would walk ?
(wings of chafers or some tropic insects)

[Edited on 10-7-2005 by Madandcrazy]

Help!

Ravenstein - 9-8-2005 at 00:30

Hi all, I am from Singapore and and trying to do this ... I bought some octopus and and letting them rot in my fridgerator half submerged in sea water. Its been nearly 18 hrs since i started. At first i did'nt read the things well and left my octopus outside for about 12 hrs and earlier this morning, i placed them in my fridgerator after i realised what happened. Will this affect the photobacterium?

For the tryptone, i am in early high school and am not very sure how to obtain this, early in this thread someone said any protein extract should work... is it true? and if not, what should i use.

For the medium can i use the LM and just dont add agarose gel?

How fast does photobacterium grow?

How long will it take to get about 100ml of the bacteria?

How do i grow the bacteria in such a way i can get alot of it quickly?

In singapore, anyone know where i can buy petri dishes and the rest of the ingridents?

Bugger - 31-8-2005 at 14:43

I've got a question:

I live in an inland state in a town where there's no fresh sea-fish available; everything's either frozen or already cut up and probably washed and cleaned. Is there no other way I could get a sample of this glowing bacteria from nature then? Would aquarium water from a tank w/ live rock contain the bacteria, for instance?

Thanks for any help!

sparkgap - 1-9-2005 at 02:57

OK, let's review what has been said thus far...

-P. phosphoreum is a *marine* organism.

-Freshly-caught marine animals *may* carry the microbe. Store-bought animals may have already been cleaned and as such may have little, if any, microbe present.

-Yeast lysate and tryptone are possible protein sources. (There should be bakeries nearby that have yeast, FCOL).

-Use gelatin or agar as a medium. Shouldn't be difficult if you live in a tropical country... they're extensively used in a good number of Oriental deserts.

sparky (~_~)

Xque - 17-11-2005 at 06:51

You might want to look into Pseudomonia ssp. too. There's several species which are flourescent when lacking iron, such as P. aeruginosa, P. flourescens , P. putida and P. syringiae. It seems Pseudomonia is pretty common.

I wouldn't mess too much with aeruginosa though, as it is a pathogen.

nitro-genes - 12-1-2006 at 08:52

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

Esplosivo - 12-1-2006 at 09:01

That's nice to hear. So you did manage to grow colonies on the fish. I have tried with two freshly caught fish (caught by a friend that very moment and kept in sea water - fresher than that is impossible) but with no apparent success, except on the second time where I think there were visible light emitting colonies which were very faint.

Could you please give us more detail regarding this experiment. You said you incubated the fish at 4degC - for how long? When did the first colonies become visible? Did you incubate the fish in sea water? Do you have any idea of what species of fish did the bacteria actually grow on (the common name is sufficient)? Thank you.

[Edited on 12-1-2006 by Esplosivo]

nitro-genes - 12-1-2006 at 13:01

I'm sorry if I made the impression that luminous bacteria are specific for some species of fish... What I meant was that you need a fish with the "right" fressnes. I/m saying "right" because I haven no clue why the colonies show up in some cases as in others it doesn't. Could be a lot of things, but In our experiment we used plain cod. The group that had succes found colonies after less than two days @ 4 deg C already.

The light is only emmited during the growth fase, so you should be able to see the luminescence quite quickly. That is probably also the reason why you must have very fresh fish.The bacteria will still be there, but not emmitting any light anymore!

Luminous bacteria are present in almost all seas at any depth and can be found in almost every marine organism. Of course there are more species of luminous bacteria, so I don't know if this is the case for V. phosphoreum. But seen it's large temperature range in which it can grow, and the commenly found niche it occupies, it is quite likely.

In the attachment is a pdf that is very simple, it contains almost every information you need, like media, growth conditions and background information.

[Edited on 12-1-2006 by nitro-genes]

Attachment: Luminous bacteria.pdf (186kB)
This file has been downloaded 2196 times

TCBS agar preparation

Vitus_Verdegast - 8-4-2006 at 09:30

Thiosulphate Citrate Bile Salts Agar (TCBS)

(Oxoid designation is Cholera Medium TCBS)

Basic ingredients

Peptone 10,0 g
Yeast extract 5,0 g
Sodium citrate 10,0 g
Sodium thiosulfate.5 H2O (Na2S2O3•5 H2O) 10,0 g
Sodium chloride (NaCl) 10,0 g
Ox gall powder 5,0 g
Iron (III) citrate (FeC6H5O7) 1,0 g
Sodium cholate 3,0 g
Sucrose 20,0 g
Thymol Blue solution (0,2%) 20,0 ml
Bromothymol Blue solution (0,2%) 20,0 ml
Agar 15,0 g

Distilled water 1,0 L
pH 8,6 ± 0,2

Preparation

Mix all ingredients and apply heat until all goes into solution. DO NOT STERILIZE USING AUTOCLAVE. Allow to cool to 45-50°C and distribute.

Notes:

from http://www.disknet.com/indiana_biolab/b041.htm:
Peptones are the most widely used source of nitrogen in microbial media. Some are made by cooking milk or meat products in acid, but most are made by incubating milk or meat with trypsin, pepsin, or other proteolytic enzymes to digest the protein to a mixture of amino acids, peptides, and polypeptides. Many microbes, called proteolytic, can digest proteins, but most can't. The choice of peptone is sometimes of importance.

Some water-based inks contain ox gall (bile) as a dispersant. Pure ox gall can be obtained from art supply stores (eg. Winsor & Newton brand).

Sodium cholate, a bile salt, is used as an anionic detergent.

kazaa81 - 9-6-2006 at 05:25

Maybe this is a little off-topic, but I would mention some dinoflagellates of the genre Noctiluca, that, when they're shaken, emits light.
Obviously, being monocellular organism of larger size, it would be more difficult to spot and grow as done with Photobacterium; it has been reported of some species that can reach 2mm of diameter...that's very big for a monocellular organism!

bioluminescent dinoflagellates at luxgene.com
http://www.bioart.co.uk/lux/dino.html

"Noctiluca" on wikipedia
http://en.wikipedia.org/wiki/Noctiluca

Noctiluca scintillans information pages
http://www.marbot.gu.se/SSS/dinoflagellates/Noctiluca_scinti...
http://www.nmnh.si.edu/botany/projects/dinoflag/Taxa/Nscinti...

Noctiluca scintillans photo
http://micro.magnet.fsu.edu/featuredmicroscopist/vanegmond/i...
http://micro.magnet.fsu.edu/featuredmicroscopist/vanegmond/i...
http://members.fortunecity.com/anemaw/noctiluca.jpg

The Subcellular Origin of Bioluminescence in Noctiluca miliaris
http://www.jgp.org/cgi/content/abstract/50/5/1429

[Edited on 9-6-2006 by kazaa81]

JohnWW - 28-3-2007 at 02:10

What enzymes, and substrate, are involved in the chemoluminescence reactions? In the cases of fireflies and cave glow-worms, I understand it is luciferase and luciferin.

bereal511 - 26-6-2007 at 20:58

Hmmm, I wonder if anyone was successful in growing and maintaining a culture of the luminous bacteria so far.

I just spent the past week conducting the procedure to allow growth of bacteria on fish, with great success. The fish I caught off the pier, a mackerel I believe, so it was as fresh as it was going to get. I immediately iced it to both kill and preserve the fish. I filled the container half way (about 250 mL) with ocean water and added about 5 grams of salt to ensure that only the luminous bacteria would be growing on the fish and to also make up for the addition of tap water from the ice. The container was left in another container which had ice water and was allowed to sit for 2 days.

I got a very strong glow in a completely dark room, and I just inoculated a 1000 mL solution of 20 grams sodium chloride, 5 grams of yeast extract that I prepared using a 50 mL of 5 molar sodium chloride lysing solution, 10 grams beef boullion (with delicious beef extract, yeast extract, salt, hydrolyzed proteins, and fatty acids), and 0.5 grams of magnesium sulfate. I hope by Thursday I can get some results up. I can't get a clear picture of the glowing fish on my digital camera, but I kept it outside to see if it'll have an even stronger glow. Updates soon!

chemoleo - 27-6-2007 at 14:54

Fantastic, that is good news!
Just make long exposures (>1 sec) and I'm sure you'll get good images, and do post with reference objects! Even cooler if you can grow it in some sort of dish!
Well done!

bereal511 - 28-6-2007 at 09:01

I don't think my camera has a long exposure function. Which is unfortunate, because the bacteria was out-competed, so no more glow. I still have the inoculated culture medium though, so I'm placing my bets on that. I just wish I had isolated it on an agar plate first, but I have no plates. Hopefully the conditions will be good enough that the luminous bacteria aren't killed off by other bacteria. Otherwise I'll start over with a culture plate.

Spargine - 6-12-2007 at 09:02

Has anyone had success with fairly inland fresh fish? I am thinking about attempting this and I am wondering if anyone else has had success.

chemoleo - 16-12-2007 at 16:10

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!

ChrisWhewell - 25-12-2009 at 11:23

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:

Awesome. they ought to coat the toilets with a triboluminescent zinc layer

12332123 - 15-4-2010 at 07:01

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]

The WiZard is In - 15-4-2010 at 10:27

The most common source of luminescence in da sea
dobe luminous alga e.g., Pyrocystis luna and Pyrocystis noctiluca.

This may take all the fun of self discovery out -

Wards sells a —

Fascinate Students with One of the Simplest Light-Producing Organisms Luminescent Bacteria Living Specimen Set

These glowing green and bluish-green examples of bioluminescence make a fascinating addition to microbiology studies. Cultures include: Azotobacter vinelandii and Vibrio fischeri. The set comes with photobacteria medium, instructions, and the Working with Bacteria manual.

http://wardsci.com/product.asp?pn=IG0007238

If the back of whats left of my mind was --- Difco sold photobacteria medium, however, I find not it listed.

Run Photobactium medium through Google or same such.

The WiZard is In - 15-4-2010 at 12:02

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.

I would mention in passing :—

E Newton Harvey's 692 page opus.

A History of Luminescence : From the Earliest Times Until 1900
American Philosophical Society
Philadelphia 1957

He discusses luminescence in all it variations, e.g.,

The Journal of industrial and Engineering Chemistry
2 [11] 478-9. November, 1910.

Tribo-Luminescence. [1]

The term “luminescence” has been applied to the light which is emitted by various substance at a temperature much lower than that which is necessary to show visible incandescence, as illustrated for example in the light of the firefly, decaying wood, and many natural and artificial mineral compounds.

The general phenomenon of luminescence has been divided into several classes by Dr. Sylvanus P. Thompson, as shown in the following tabulation which is copied from his book entitled "Light Visible and Invisible," page 175 (1893).

Phenomenon. Substance in which it occurs.

1. Chemi-luminescence………... Phosphorus oxidizing in moist air;
decaying wood; decaying fish; glow-worm;
firefly; marine organisms, etc.
2. Photo-luminescence
(a) Transient: Fluorescence............Fluor-spar; uranium-glass; quinine;
scheelite; platino-cyanides of vari-ous
bases; eosin and many coal-tar products.
(b) Persistent: Phosphorescence…. Bologna-stone; Canton's phosphorus
and other sulphides of alkaline earths;
some diamonds; etc.
3. Thermo-luminescence ……………. Fluor-spar; scheelite.
4. Tribo-luminescence……………. Diamonds; sugar, quartz; uranyl
nitrate; pentadecyl-paratolylketone.
5. Electro-luminescence
(a) Effuvio-luminescence Many rarefied gases; many of the
fluorescent and phosphorescent bodies.
(b) Kathodo-luminescence Rubies; glass; diamonds; many gents
and minerals.
6. Crystallo-luminescence………… Arsenious acid.
7. Lyo-luminescence…………… Sub-chlorides of alkali metals.
8. X-luminescence…………….… Platinoeyanides; scheelite; etc.'

Light Visible and Invisible can be DL'd from Google.com/books.

Sedit - 15-4-2010 at 12:06

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.

Well, Welcome to Science madness where we will spit and scream if your ass don't read a book before speaking.

peach - 6-10-2010 at 10:07

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

And Mycena chlorophos.

Morgan - 11-12-2012 at 08:14

I remember camping in the mountains near Mt. Hood zipping up my tent at night and happening to look down at a vivid green light coming out of a perhaps 3 inch wide hole in the ground. It really startled me and I froze for an instant trying to make sense of it. I had just pitched the tent that day with my girlfriend and was so close to the ground pulling the zipper the rest of the way to the floor and there was this beam of green light just a foot from the tent. The rotted wood was not very far from the surface of the soil a few inches down. I dug the dirt away and collected a few pieces of this wood. Even in the day you could cup your hands around a chunk and make out the green light. It lived for several days after I returned home, being kept moist in some damp paper towels.

scottjm - 29-6-2013 at 16:02

Quote: Originally posted by peach

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

Wow, those look crazy cool!
Are they legal to cultivate, or are the psychoactive?

scottjm - 30-6-2013 at 16:09

I found out that those mushrooms would be legal to cultivate as they have no psychoactive compounds in them, but they are very hard to grow.

dontasker - 10-8-2013 at 09:44

I purchased a culture of Panellus stipticus from SporeWorks a few years back. I was afraid to buy it at first due to their selling spores of magic mushrooms, but figured I wasn't really doing anything illegal.

I inoculated some wood chips (hickory, I think) and let it grow. It never fruited and I didn't see any signs of luminescence, so I put in in a cabinet to keep it away from contamination.

Completely forgot about it for a couple months. When I checked on it again the mycelium was colonizing most of the wood. It was also starting to produce a very faint, greenish glow.

There's something almost magical about it. Fireflies always fascinated me, but there was a logical reason for their using it for communication. A fungus. on the other hand, defies all explanation.

Morgan - 10-8-2013 at 16:39

Panellus stipticus
http://www.youtube.com/watch?v=Rezwma4MOn8
List of bioluminescent fungi
http://en.wikipedia.org/wiki/List_of_bioluminescent_fungi

[Edited on 11-8-2013 by Morgan]

JAVA - 20-5-2014 at 11:15

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!

Pumukli - 9-11-2017 at 06:29

A few days ago one of my friends had bought a few chicken drumsticks at a local store. (Tesco)
At home he forgot to put the bag containing the chicken parts into the fridge but put them on a table instead in a cold building where his fridge is.
After say 24 hours incubation in the cold (12-15 Celsius) he entered that storage room in the following evening and to his surprise he found the chicken bag emitting eerie green light on the top of the table! According to his storytelling the light was visible enough to catch his attention so he did not switched on the light.
He was frightened and shocked and everything you can imagine a layman would do who never heard about luminescent bacteria and such oddities. :-)
He even called his family to check the "thing" and they tried to make photos but never heard about the "B" setting on the camera and they failed to capture any meaningful detail on those pictures.
Next day though they boiled and ate the drumsticks as they had no substitute and anyway, they were destined for the Sunday lunch. :-)

Morgan - 9-11-2017 at 07:05

I saw a curious effect out fishing one night anchored about 20 miles off the coast of Florida. The heavy monofilament fishing line weighted to the bottom glowed as the current passed over it. You could see it for quite some depth in the darkness.

[Edited on 9-11-2017 by Morgan]

NEMO-Chemistry - 14-11-2017 at 06:48

Looking for something else, i came across a paper that seems to suggest this bacteria can grow in fresh water. While does say a 50% diluted marine solution is optimal, the paper deals with some of the bacteria being isolated from migrating salmon, these were 1200KM from a marine environment.

I thought i would post it incase someone finds it useful at some point.
https://doi.org/10.1016/S0167-7799(98)01223-2

Similarly other papers i came across, seem to suggest the organism is a spoilage one, it is somewhat sensitive to CO2 and O2. I didnt read the entire paper so I may have slightly misread the point of the paper.

However it does tally with the bacteria being found in vac packed fish that have spoiled.

https://link.springer.com/article/10.1007/s00217-002-0575-1

I have also attached a paper that gives optimal culture conditions (apparently).

https://www.jstage.jst.go.jp/article/jgam1955/26/2/26_2_75/_...

[Edited on 14-11-2017 by NEMO-Chemistry]

[Edited on 14-11-2017 by NEMO-Chemistry]

mayko - 9-7-2019 at 17:14

I ran across this protocol for isolating luminous bacteria, leafing though "Experimental Microbial Ecology", by Sheldon Aaronson

Quote:

Examine surface of agar in a darkened area with red illumination reduced to the point that nonluminescent colonies are barely visible.The luminescing colonies will have a bluish tinge. These may be marked and subsequently transferred to fresh nutrient agar medium for purification and subculture.

"Gelysate" is a gelatin-based peptone

Attachment: aaronson_luminous.pdf (279kB)
This file has been downloaded 573 times

TriiodideFrog - 22-12-2020 at 21:24

A few years back, I got some dinoflagellates online.

Here is the website: https://www.carolina.com/algae/bioluminescent-dinoflagellate...

manomanom - 9-5-2021 at 12:54

*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.