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chemoleo
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Luminous Glowing Bacteria - Homemade!!
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.
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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]
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Blind Angel
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Do you think that a growth medium made for other bactria like Streptocoque, Coli or some like that would do too?
/}/_//|//) /-\\/|//¬/=/_
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chemoleo
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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.
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Nick F
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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.
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ziqquratu
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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]
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chemoleo
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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]
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Iv4
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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.
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Blind Angel
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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.
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chemoleo
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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.
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Blind Angel
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Tap water?
I plan to make them grow on blood agar that it's use in medical laboratory to grow the Streptococcus
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chemoleo
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Lol..
tap water refers to IV4's post.
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ziqquratu
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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.
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Iv4
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No luck
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.
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Esplosivo
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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]
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chemoleo
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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)?
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Esplosivo
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| 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)?
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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]
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chemoleo
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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.
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ungebetenergast
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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]
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chemoleo
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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...
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ungebetenergast
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| 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: 
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]
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Esplosivo
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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]
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ungebetenergast
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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]
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Esplosivo
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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.
Theory guides, experiment decides.
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chemoleo
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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!
Never Stop to Begin, and Never Begin to Stop...
Tolerance is good. But not with the intolerant! (Wilhelm Busch)
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Esplosivo
Hazard to Others
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Registered: 7-2-2004
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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.
Theory guides, experiment decides.
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