Bill's Comments:
What did everyone see on their D29 plates today? Did you guy's autoclave your media? Who is Louis Pasteur? What did he do? Student: glassware and probably looking for spontaenous generation. Bill: swan neck flask drawings. Asked what media was? It was actually Koch's wife that came up with idea for agar. If came up with idea to grow bacteria, what would you grow it on? Student: Milk or sugar water. Bill: good idea. Bacteria sometimes in decomposing matter. Company called Difco. Won't forget Difco after this. What might DIFCO mean? At time found that piece of meat digested could break down AA and could make brain-heart infusion. Company started at Yale over 100 years ago, learned how to isolate enzymes even though didn't know what they were, and named them digestive ferments. "Digestive Ferment Company." Most microbiologists don't know this; but it makes perfrect sense...and made different infusions. Brain Heart infusion becomes part of the media. Bacteria like having rich AA's and proteins around to eat on and it is not uncomon. Louis PAsteur liked fermentation alot. He solved a big problem. French wine is a serious commodity. Batches of wine. Grape juice + bacteria --> alchohol. Bad bacteria will make it all sour. Couple years of wine saved because bad bacteria tracked down and known how to get rid of. Could innoculate with correct yeast and bacteria to make good wine. At time, people thought could get bacteria spontaneously. Pasteur said no, he took glass of swan neck and sterile media...how do we make something sterile? We cook it. It turns out that if we just boil media it is not hot enough to kill all bacteria. Difference between an autoclave and just boiling something is that the autoclave combines heat with pressure to kill all bacteria, and unless you live at the bottom of the ocean, in the hydrothermal vents, there are archaebacteria with enormous pressure and heat...it is almost like finding life on mars. You cannot imagine something as funky as living there. Absolutely amazing. But we don't have these on the mainland. All the bacteria we are familiar with will die in the autoclave.
Bill asked for a plate we poured last week and left out. Do you see anything? It is pretty obvious that nothing arose from the media. We autoclaved this media and still nothing is growing on it. Students then examined intentionally contaminated plates in the back and noted environmental organisms. Bill asked if everyone saw colonies on their 10-3 D29 plates from last week. Students discussed colony morphologies of the environmental organisms with Bill. Bill: okay whose Charles Darwin? STudent: evolution. Bill: who opposed him? what did they say? Lemark. Student: Giraffe necks stretched. Bill: so in fact evolution could occur in the presence of a selective pressure. What did Darwin say? Student: natural selection; Bill: mutations prexist in one case Lamark says pressure then mutations; Darwin says mutations spontaneously occur. Now you guys have done a very important experiment. It is often designated as the beginning of modern molecular biology, let me tell you what you have done.
When you plated your M. smeg last week, first of all, would it be hard for you to count the # of colonies on your 10-3 plate? You used different cultures of M. smeg, didn't you? Please count the # of colonies that you see. If there are many colonies, just count a quarter of the plate and multiply times 4. Demi, when you look at your plates over there, do you see isolated colonies coming up on your plates? Demi: I ihave some from dilutions from library or smeg. Bill: when done counting, let me know. Bill to Demi: controls first?
Bill, okay, we've heard about L. Pasteure, who has heard of R. Koch? Student: heard of him. Bill: he discovered causative agent of Tb. Next, Max Delbrook and Salvadore Lurie. Okay, all these guys got noble prizes for different experiments. The last two got the noble prize for proving Darwin was right. Let me tell you their experiment. If you take D29, a lytic phage, and add it to mc2155, ...do you know why you are using mc2155? My mentor neighbored his strain collection the chi collection because he started it at the university of chicago. When I came to AECOM, I thought long and hard what I would name all my strains, and in honor of Albert Einstein, I decided to name my mycobacteria culture collection the mc2 collection. So I started with mc21, we are now at mc2-6000. 155 was an isolate gotten by my first graduate student, and is very genetically manipulable. If you take these phage and cells, theh phage attaches, shoots in it's DNA, that DNA replicates, starts making a bunch of head and tail proteins, and the next thing you know is that you start making lots of new phages, and then the cell lyses and phages release. When you see a plaque, you had 1 cell that was infected with 1 plaque forming unit and then if you blow up this region here, you initially started with 1 phage producing up to a hundred phages...and the reason that you get this clearing is because you the bacteria are dying...stop making plaques because bacteria stop growing and non-growing bacteria don't make phage. D29 is almost as good as an antibiotic in killing. If we titered the amount of bacteria that we put down the other day, it was at least 10 million bacteria, and on any one of your plates 600 or 700 bacteria are surviving on your D29 plates. (See data above). So it is 1 in 100,000 that are survivors. Remember, we talked about the pennies...if you had to go through a mutant hunt like this, trying to get 2500 pennies...trying to go back and fill up the bank. The problem is that rare mutants...these are m. smeg mutants that confer resistance to the phage. they have a mutation in the chromosome of m. smegmatis so that they no longer become susceptible to the phage, and they become resistant. On half a plate, each of you streak for isolated colonies from your mutants...add colony to 1 mL media...10x6 organisms, isolate a colony, and later in the week we will show you that you have isolated colonies. Each of you pick 10.
What is the selecting agent in this case? That is only 1 in 100,000. You are telling me right now that these mutants exist and have a frequency of ... the beauty of mixing the D29 with the cells is that it killed everything except those mutants that preexisted and they had a selective advantage. If you happened to have a mutations so that you could no longer be infected with m. smeg, you survived the weekend. You actually selected for mutants over the weekend. Those colonies seen over the weekend are phage resistant mutants.
If Lamark was right, if each of you go back and start an independent smeg culture, and lamark is correct, how many mutants will you have on the plate? Probably the same number, but if you all used independent cultures and you started with the same number of cells, you will get the same number of mutants. But if Darwin was right and each of you started a culture independently, then you would expect to see variation in the number of mutants that arise. Start M. smeg cultures with about 1000 organisms, mutations are 1x10^5, so no D29 resistant phage will be in these cultures. So we don't have resistant culures before, but once we grow them up, we might. If Lamark is right, we will get mutants at 10-5. However, if Darwin is right, then we will have 1 bacteria go into 2. And then they will go into 2 more. It will take 2 or 3 days to get the smeg to grow up. So if we get a mutation early, you will have mutations at the frequency of 1 in 1000. You are going to get more because they will grow up spontaneously. You will have no D29 around...and we want to grow up everyone's culture. If Darwin is correct, each of you should get very different numbers of D29 resistant mutants. If Lamarck is right, you should get the same number of resistant mutants. This is a very key experiment, and they did this experiment with T4 phage. We will show you their data in their paper and you will get exactly the same sort of data. We are going to see if Darwin is right for mycobacteria.
Today, you want to go in ... today plate our D29 for titer. Meanwhile, pick a colony and put in a mL of media. Don't fret that you won't see anything...you have to go by faith that you will get plenty on...and with your loop, 1 mL = 1000 uL. With your loop, take 1 uL...if in a colony expect 10^6 cells. If you grab a colony, expect 10^6 in that mL, and when you grab a sterile loop of this you will have 1000 bacteria and anywhere from 100 colonies. Go by faith they are still there. Isolate them out, and test to see if they are phage resistant. Bill to Demi: D29 for you may not be titered enough. In Demi's case, we will use a very special library that already have mutations caused by transposons. In your case, you haev spontaneous and prexisting mutants. You have won at Lotto...1x10^6...meanwhile..
Here's what we will end up finding by the end of the course: not all phages use the same receptors, and phages use different receptors. Just because you are resistant to D29, you can be susceptible to another phage...
REgarding Stephen's phage:
Bill smelled his smegmatis; there are 2 possibilities. One is that this is very resistant to lysogenize. What do the individual plaques look like? Do the plaques become turbid? Look at independent plaques at D1,D2,D3...and let's record what we see. Could become resistant because of phage. Isolate a plaque, look at D1, D2, D3...look at all week long.
Regarding Kristen's plaque:
Concern about how well smeg was grown up. The smeg doesn't look totally normal. I don't think the smeg culture was dense enough...we have fresh phage grown up, repeat this.
Sample XX...same smeg, and this
We seemed to have a problem with our picking of plaques. Stuff just happens...just back yourselves up, pick 2 or 3 plaques.
Thursday zoo trip planned. Planned to get samples from a globe trotting
Pseudo-lysogeny, becoming in the bulseye, it is like D29, where there is pseudo-lysogeny. Chiara: why the clear ring? People have seen the ring with pseudomonas and same thing. People have tried to look at this and tried to study...but if you go and pick these cells they are not phage resistant mutants. It is really not well understood at all. Science is like a black-box; techniques to go in and probe science but to go in and probe it we don't always get to the full understanding. lot's of people describe these at phage meetings and not yet described. We'll think about this later in the week, and will think about