The lab so far.

[gemfyre]

I was actually in the mood to do work so I wrote out what I know of my experiment so far. Any suggestions, ideas, help would be great!! I seemed to come up with more questions than answers.


The story so far...

Ingredients

20 petri dishes
400mL nutrient agar
1 McCartney bottle
Streaking loop
Bunsen burner (always gotta have them)
Laminar flow hood (ooer)

Method

Pour agar into 20 petri dishes.

1st plating - 2 dishes
2nd plating - 8 dishes (2 for each colony)
3rd plating - 8 dishes (2 for each colony)

Collect sample from soil at contaminated site in Bellevue (find info about Bellevue fire).

Plate onto two dishes.
Incubate 1 week.

Take sample from four different colonies, plate on 2 dishes each. Incubate (how long??)
Repeat next week.

Now we (hopefully) have nice pure cultures.

Gram stain cultures. Find the gram negatives.

Test for growth on heavy metals. Maybe choose metal most likely to have contaminated original site (mercury?) What concentration??
Nutrient agar with metal included. Not exactly sure how to do this. Diluted dots on the plate? No idea what the original concentration of cells will be so no idea how much to dilute.

For the stuff that does grow. Grow a nice sample in nutrient broth. (perhaps keep metal concentration in the broth the same, yeah, I think that's a good idea.)

Conjugate with non-resistant bacteria (which one? E. coli?)

Test if conjugated bacteria grows (how will I know which cells are the originals and which are the receivers??)

THEN, get the original culture. Start growing it in plain broth. Take samples at time intervals (how long interval? 10 minutes? 20 minutes? What's the replication time of my original sample? I have no way of knowing.) Grow on plates with metal.

See what grows, how long does it take to lose resistance? Does it lose resistance at all?

After all that crap

Write the smegging lab report.

Comments

[marzipan9.livejournal.com]

heh, E. coli is like.....the wonder bacteria with all the stuff it can do with its plasmids.

Can you figure out exactly what you have and look it up and see if it does, um, crap, what's that called? when it picks up DNA from the environment? I just had a test on that.
But anyway, if you could locate some of your heavy metal bacteria that is not heavy metal dependant, then degrade some of your heavy metal bacteria, stick in some of the non heavy metal dependant variety, then see if the non heavy metal dependant bacteria can then live in the presense of heavy metals.....kind of like the encasulated/nonencapsulated pneumonia bacteria experiment.

Transformation? Is that what it's called? Or Transduction? Too many trans!!!!!

[gemfyre.livejournal.com]

What's it called when it's plasmids? That's what I'm looking for.

My big problem is, how do I know that what's growing isn't my original resistant bacteria and not the new transformed bacteria?

[gemfyre.livejournal.com]

!
I remember now, I DID figure that out.
You use a bacteria that grows on another particular medium (and hope the one from the environment CAN'T grow on that media - it really sucks that we'll know nothing about whatever we culture, all I'll know is the shape and whether it's gram negative or positive, and we have to find a gram negative.) then grow the mix of bacteria on that medium and see what happens.

But once again, more questions than answers. ARGH!

[marzipan9.livejournal.com]

plasmid transfer is conjugation.
what i'm talking about is transformation

and how, with transformation, that you know it's not your original heavy metal resistant bacteria growing because you use dead heavy metat resistant bacteria and allow the non-resistant bacteria to pick up the resistant gene from the environment w/ the dead bacteria in it.

but for plasmid transfer? you pray? ;)