Day 6 thoughts.  Phagefinders. 

Planned:  Dilute a colony of our M. smegmatis streaked plate into 10 mL 7h9 culture media.  Culture soil samples that have incubated, shaking in MP buffer over the weekend.  Look at plates to determine pfu/mL of D-29.  Dr. Jacobs should be meeting with us today, also.  Need to make 7h9 liquid media w/Tween for Smeg culture; plate 2 7h9 inkwell smeg solutions-1 from a colony, another from Demi's culture (when I use her media and culture, my culture clumps) which seems to be clumping on me; also make another top agar since when I autoclaved the agar twice it had a yellow precipitate.  Blake recommends 10-5 to 10-8 for phage sample plating. 

3 pm: make 7h9 and top agar for autoclaving.  Start autoclave.  While waiting for the autoclave to finish, make 20% tween and determine how to do the calculations.  As an alternative, just add the tween to the media and filter the media and tween without the use of an autoclave.  0.05% Tween = 

We Did:  Note that our BXZ-1 virus made very small plaques for us.  The sample given to us generated lacy plates at the 10-1 and 10-5 dilutions, I'm not sure if a student made a mistake with the calculations or not.  The dilutions of the phage seemed to be pretty consistent, otherwise, with the expected proportionate decline in the number of plaque/plate as the solutions were diluted.  Note that the 7h9 liquid media that we made today only contains 2, vrs. 5 mL glycerol/L (as the bottle recommends instead of the lab protocol), this could be a problem later if not noted. 

Accomplishments:

1)    Harvested BXZ-1 phage from plates.  We gently rocked the plates by hand every few minutes. 

2)    Counted and determined D29 PFU/mL in our high titer phage prep from last week.  Reminder: this phage had been used for practice dilution plating, in order to determine whether

3)    Plated our soil samples that incubated with M. smegmatis over the weekend (1 plate per sample).

4)    Rocorded how well our plate streaking method went. 

5)    Collected paragraphs regarding the questions that were given last week, and told students that new questions would be given tomorrow. 

6)    Blake (our returnee student from last year) diluted 2 phage samples that were prepared at high titer last year; at 10-5, 10-6, 10-7, 10-8.  Tomorrow, these samples will be plated.  

Students decided that the mass of a single bacteria would be about 1 femtogram. 

Students will need to make media on their own, soon. 

Dr. Bill Jacob's Visit:

Bills recommendation: check out HHMI.org  .  Asked class who Howard Hughes was.  woman chasing bachelor made his money; dad invented the oil well drill.  countless movie star affairs, set up hollywood and made his own films...famous flying film where he was the pilot, and he bequeathed his billions to medical research institutes.  Students introduced themselves to Bill, Bill read an Einstein quote:  "the important thing is to not to stop questioning...one cannot help but to be in awe when one questions the marvels...it is enough to comprehend the myseteries of everyday...never, never lose the holy curiosity."  Bill says the mark of the good scientist is the scientist that asks good questions.  The sin is to remain ignorant.  Don't be afraid of asking questions this whole summer...never stop asking questions...if you want to be a good scientist.  Bill explained his math major background.  Bill admits his own shortcomings of not knowing but his strength of ....Dr. Sherer got Amanda involved, Steven spoke to Steinman who put his email on the website.  Kristen ...with Dr. Sagathy, ;  Chris heard through Dr. Sherer, also.  50% of our students were becoming juniors, 50% becoming seniors. 

Bills questions:

Why would we look in soil for phages?  How many bacteria are on earth?  Explained prokaryote; no bacteria has a nucleus.  "Karyote means nucleus, eu means good, pro means before...before a nucleus.  Can you name me other bacteria?  tuberculosis was mentioned...mycobacterium tuberculosis...vrs. mycobacterium smegmatis that we have been working with.  "Myco means fungi-like."  Do you know what syphillis is caused by?  Treponema pallidum.  Bacteria that often cause infections.  We are covered with hundreds of different bacteria outside our skin; in our colon we have E. coli.  In fact, this is the most widely studied bacteria.  Of course we are going to replace that because we are going to make M. smegmatis more user friendly around the room.  And by the way, this whole project of you coming over the summer is an experiment.  So if you were goint to look for E. coli, if E. coli lives in your gut, where might you look for virus that lives on E. coli.  Chiara: exrement.  Bill: sewer...this is where we used to isolate E. coli from.  Turns out that we know when we look at dirt, we might think dirt is really boring but it in actuality a dynamic environment.  Degraded plant, are a bunch of different bacteria that degrade the plant.  There is a whole set of bacteria that lives in the soil, actinomycetes, are responsible for this...they are the name of a big family of bacteria, and include the mycobacterium, streptomyces.  has anybody heard of the antibiotic streptomyces?  Who discovered penicillin?  Alexander Fleming.  You know in the dirt, prokaryotic organisms...we have mycobacterium, the 2 pathogens are leprosy bacillus...have you guys seen anybody with the leprosy bacillus?  Did you know there are 2 lepor colonies in the US?  I took a microbiology course as a math major...have you prepared media yet?  The M. leprae bacillus cannot be cultured on media.  M. smegmatis grows if it basically has sugar and salt.  That's pretty amazing, you can't grow on just sugar and salt.  You cannot, do you know why?  Chiara: we need to make protein.  How many amino acids are there in nature?  About 20.  It's amazing that in all of life it is basically the same 20 AA's...that's the same for cockroaches, slime molds, bacteria, cows.  If we have to eat certain amino acids, if you have to eat something with Lysine and Tryptophan, you're dead.  A bacteria with just glucose will grow fine.  Student: chemical conversion.  Bill: great! and humans cannot make all twenty amino acids; in fact, there seems to be essential and nonessential amino acids.  Mycobacterium can make all 20 amino acids from glucose.  Let's see...what did we talk about...we're still going back to this dirt.  When I was in 4th grade or about then...we used to see this movie about life that said all life came from the sun.  In fact, it does.  The sun is a tremendous source of energy.  There are ways to convert that energy into glucose.  How? Photosynthesis.  There is a constant recycling of glucose, plants are consumed.  Does anybody have any cockroaches, ants at home?  Can you go home and get some ants?  I mean, we are experimentors, we are not afraid to try anything...science should be fun.  The first time we had chemistry...let me tell you about my high school experimentation.  In chemistry, they kept saying sodium is really dangerous...and our Navy man kept trying to hit on the girls in class.  I kept asking if I could put Na in water.  It was the day before XMAS break, and he said I could put Na in water.  I had this big glass battery jar, and the Na was under oil, and I cut a big hunk he made me make smaller...and I had these tongues, and he had enough forsight to have a couple guys with water nearby...and i got on some sort of gown and I drop the sodium into the water and it makes this ton of purple smoke and I'm wipping off my mask and the NaOH flowed and the ceiling was on fire...now we don't want to try dangerous things here....and the other experiment that I did when I was in 9th grade...I looked at an Algae, and we can see these grow in your swimming pool, pond, etc...and these are single celled organisms that can be seen like bacteria...and they have growth hormones that stimulate the plant to grow.  One of these hormones is Gibrellic Acid...then we didn't know if plants had these, and took GA from a Germanium and asked if it could increase the growth of this single celled Algae.  And I showed that it made the Algae grow faster compared to the control...I could see that by the density they were growing more rapidly.  It looked under the microscope that they were multiplying faster.  I only got an honorable mention.  I grew up in the pittsburg area, and I grew up less than 2 miles from the largest steel mill in the world where they would dump slag everynight and there would always be this orange glow.  I couldn't understand why the air smelled so funny when I went to college...first time I smelled clean air.  Everybody in my neighborhood worked in the steel mill.  One day I was playing baseball out in the street, and the ball went into the mean neighbor yard, and he said I told you before that you can't play on the street and he said he was keeping the ball.  I was mad.  I thought i was going to get even, and I got my bottle of gibrellic acid, dumped into an atomizer bottle, climbed the fence over the night and he had planted tomato and corn plants coming up and for the next week, every one of his plants grew a foot in a really thin manner and then they just fell over dead.  Had I done the control experiment I could have probably written up a really nice paper.  Isolate DNA from your ants, from a tomatoe, etc., the phages that we are going to isolate have DNA.  Student: Do all mycobacteriaphages have DNA?  You know some of the E. coli phages are RNA viruses.  To date, nobody has isolated a mycobacteriaphage that has had anything but DNA.  If we could isolate an RNA phage, that would be a great publication.  None of the actinomyces have yet had an RNA phage discovered.  Many of the viruses that infect us are actually RNA.  What are some viruses that infect us?  ...back to bacteria, flesh eating streptococcus...Jim Henson...the muppets.  Steptococcus pneumoniae is a big killer of developing world children.  Ebola is a DNA virus, Herpes is a DNA virus...rhinovirus causes colds, RNA.  Student: flu...is an RNA virus.  Smallpox is a DNA virus...okay that's enough for me talking today.  We are still going to be talking the rest of the week about soil and why actinomyces.  What I can guarantee is that anyplace that you find bacteria you will find viruses that infect those bacteria.  Student: if you didn't have viruses limiting the bacteria, bacteria would just keep reproducing.  Bill: not the case.  Another quote to remember is that the goal of bacteria (quote from Stanley Falkow) is to be bacterium.  E. coli doubles every 20 minutes.  Here is your homework assignment from me for tomorrow: how many bacteria would you have, starting with 1 bacteria, if it doubles every 20 minutes for ... look up on the web and find the weight of a single bacteria, and determine what the mass of that number of bacteria is.  Chiara: where did you go to school?  Edinburough university, etc.  I'll tell the story tomorrow...write out any questions you have for me. 

I think it is safe to say that the students collaborated and formed a group where, for

Additional:  I met with Dr. Nosanchuk today, and having conversed with bill, we decided to begin  a Candida screen.  The idea is to hunt for a phage that will infect a fungi.