About microscopic forms of life, including Bacteria, Archea, protozoans, algae and fungi. Topics relating to viruses, viroids and prions also belong here.
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Hello there i am new to this forum and i really need some help. My lab partner and i are performing an experiment and we would like to know if anybody would know what types of results to expect (to make sure everything was done well). Our school gave us some e. coli in triptic soy agar to grow and we were going to examine the effect of bleach, handsoap, and dishwashing liquid on the colony growth of e. coli. We diluted the concentrated ecoli 100 fold and put 1 ml of the solution on separate agar dishes. Can anybody please help us by telling us (approximately) the amount of colonies that should be found on each dish before and after the separate application of these antiseptics. Any help would be greatly appreciated!!! thank you.
Yep studiying E. coli resistance to antibiotics is one of my favourite past-time.
Different protocols could be used to perform the kind of experiment you are planning to do.
A good one would be to just prepare a batch of tubes with broth with increasing concentration of whatever antiseptics you want to study. Then inoculate each tube with the same amount of E. coli (a drop of your 100 fold dilution is OK) and see at which concentration you do not see any growth. Prepare a tube with the same broth as a positive control to show that the inoculum was good enugh to start a culture when there are no antiseptic.
But reading your post you seem to have chosen to use a plating method. You could also prepare plates with different concentrations of your antibiotics and plate your culture onto them. The only way to know how much bacteria you had in the beginning is to plate on a control plate without antiseptic and count the bacteria.
E. coli in rich media like TS, or LB grows to a concentration of 10^9 per ml overnight at 37C. If you want to be able to count, you should then dilute your suspension 10^7 or 10^8 times (by serial dilutions in water or better 0.8% saline) and plate both dilutions.
Counting 5 plates will give you a good average of the real number of bacteria in your initial solutions. Plating different concentrations of bacteria on different concentrations of antiseptic could then give you an idea of the resistance and the percentage of bacteria with a resistance to the antibiotic in your suspension. But that does not constitue an easy experience.
A simpler way would simply be to prepare a set of plates with increasing concentration of antiseptic, from 0 to whatever you want. Prepare a bacterial suspension of 1 isolated colony in about 5 ml of water (prepare one per plate). Flood the plate with 2 ml of the suspension, kelet it soake about 30 seconds, discard the bacterial suspension and see which concentration of antiseptic inhibits growth. The bacteria could not be enumerated (they will form a "lawn", but it will give you a rough idea of the inhibitory concentration.
Any way in my eyes, the first solution is probbaly the better and the easier to give the result you want.
If you have any other questions, the resident canadian can help. E. coli is my favourite pet.
It is also one of my all time favorites, too, although I have a special place in my heart for O157 in particular.
I do have a question, please.
Why or what about soy broth, do the little darlings love so much? Certainly with the genetically engineered soy, the bT is a killer.
TSB, like LB is rich in all sort of nutrients and vitamins but also contains added sugar (Dextrose or Glucose), so E. coli really find here all it needs for fast growth: everything is provided, so it is faster to use what is provided in the medium rather thant to build it from scratch.
As for bT bacteria do not see this as a killer, it is a toxin for insects. Just one more protein from the bacterial point of view
Patrick, just another protein, yes, but not necessarily a rose by any other name. bT is a bacterium itself, I wonder about excitation.
I also wonder about peer review, are the results you are getting with the new SB the same as with the older studies? Within a certain boundary? Was there a turnover when the new soy hit? In other words, were there inconsistent results that we may have turned some work away because it didn't jibe with the older studies? The new soy has been around for a while, but not that long that it wouldn't be a wash yet with some studies built on the older conclusions. If my observation is credible, how was this explained in the community? Perhaps the colonies adapted?
Thank you, Stew
Bacillus thuringensis is indeed a bacterium. But only one protein as been engineered from it and inserted in soy. So Bt soy differs from normal soy for only one protein. Moreover Soy peptone is the product of protein digestion, so the Bt protein is probably not much more of a concern than any other proteins, and I would bet that there is so much difference in the real protein content from 2 different strains of soy in 2 lots of soy tryptone than is induced by just the insertion of an insect toxin that does not impair bacterial growth anyway.
You have to realize that, SB as LB and many other media are not "chemically defined" as M9 (or some other) is. And As far as I know there have never been any study on the effect of lot to lot variation of media. And they are probably irrelevant in my opinion, because if you really want to keep chemically stable composition then you switch to synthetic media and avoid the others.
Thank you, Patrick.
I work in health care and am seeing a great deal of celiac Sprue-like reactions to the genetically engineered soy and wheat. In some cases, people are being diagnosed with Crohn's even. The celiac Sprue cases are odd because they are happening with alarming frequency in people who are not of Irish descent. I take these people off soy and wheat and they're fine.
I was investigating what was bothering the normal flora of the intestinal tract and wondering if the bT was irritating the e. coli. At first I thought the molecule was too big with the bT insertion and it was sitting out, undigested, and being attacked by the immune system. But recently I heard about a studiy where this was examined using mice and the molecules were absorbed as with any other nutritient. Although, I did not look at the study myself to determine the conditions.
I thought perhaps the toxin was killing a certain amount of e. coli or at least bothering them enough to cause a Herxheimer reaction.
Also, I rely heavily on established works for data, a contamination problem would shoot holes in my logic and conclustions.
I guess I'll just have to look at something else.
Thanks again for all of your attention to this matter. Stew
Ah, Canalon, you are holding up the entire board on this issue.
I understand your dichotemy.
I hit this board/forum on a search when I was lookiing at the composition of cell walls. I had already noted that some pathogens have hydrophobhic qualities. I was researching a case of Hyperhidrosis.
I noticed MiT's Brain Teaser. I like that stuff.
I noticed Kyle's deference to you. Good, I thought, let's talk at agar level. Here was my chance to take my theory out for a spin. I'm a throwback to BacT. You love e. coli, Kyle loves amoebae, but me, although I have a place in my heart for O157, II truly love the elegance of Ebola.
Now can we take a look at this?
Square One, Allergy.
It didn't seem to be an allergy to me because we could not seem to adapt, consequently my question, do the colonies adapt? Our children did not.
As a toxin, could there be another entity that is affected in the intestinal tract that could be reacting to this? If it is a bug/animal toxin, could that body have been infected with a virus, a protazoan, or worm? How could we tell that on a plate? What could we say to the supervisor regarding several days of incubation? When a request for a 24 hour prelimiary report came in, what would be our response? What shall we do when our mycotoxin tech says, "I think you better have a look at this?/" Should we have a better synthesized array of agars that addresses the new diseases?
I notice you did not address my observation of excitation. Have you not read about the contamination reports of PCR's?
Stew, I am sorry to answer so late, but I am quite busy in the lab these days, and haven't that much time to answer.
But I am puzzled by your questions, and do not really understand them. Please forgive me, english is not not my native language. But I will do my best to answer
I understand 3 points in your questions:
- Could Bt soy in TSA have an effect on E. coli growth?
The answer is probably no. First because non chemically defined media are tested by the manufacturer to sustain bacterial growth of defined strains in the same limits as a part of quqlity control. And second because the soy used in this preparation is digested by proteases, meaning that the Bt toxin is probably completely degraded.
-Could undigested Bt toxin affect E. coli growth?
I do not know if it has ever been tested, but basically it would just require to add Bt soy protein extract to chemically defined medium (M9 for example) and compare bacterial growth to Media with non Bt soy extract or with protease degraded Bt soy extract. However I do not think that a toxin specifically targeted to insect cells could affect E. coli, but I'd be happy to be proven wrong.
-Are your patient affected by Bt toxin?
Maybe so, but as you told us you put them off soy and wheat. And no Bt wheat is yet commercially available (and it would be better if it could stay like this, even though I think it is already created) so your patients may suffer from something completely unrelated.
Feel free to ask any more question though, I'd be happy to answer if I can get out of the petri plates tsunami that is menacing me these days
hi i am also working on the same project(effects on antiseptics on ecoli)
can anyone give me a protocol of the same?????????????
i am just interested in finding out the estimated number of ecoli b4 & after d exp.
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