micro lab studyguide 2

micro lab studyguide 2 - Review the procedures...

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Review the procedures for: -Selecting a single colony from a streak plate for further growth in a broth or plate -Bacteriophage titration - serial dilutions pipette 1.0 mL phage from stock tube = 10-1 dilution mL from 10-1 tube into the 10-2 tube mL from 10-2 tube into the 10-3 tube transfer 1.0 mL of 10-1 suspension to soft agar tube and mix 1.0 mL of E. coli to the agar tubes pour into the agar plates, let dry and then invert and incubate -Transformation chemically competent bacteria must stay on ice during the majority of the experiment add 10microliters, using a pipette, of DNA prep from the tube marked “D” to your competent cells on ice. Flick the tube to mix DNA and bacteria; immediately return the tube to the ice bath for 30 min Place the tube in the 42 degree C water bath for 20 seconds then return it to the ice bath for 2 minutes using the micropipette, pull up LB broth to the base of the bulb (about 0.5 mL) and add drop by drop to the competent bacteria/DNA. Flick to mix then place the tube in a 35 degree celcius heat block for 30 min prior to the end of the heat bath, transfer one tube of X-gal onto one plate of agar (using a micropipette) containing 50 micrograms of ampicillin. Open the bent glass rod (from the handle) spread the X-gal over the surface of the plate. Let dry and repeat using the second tube of X-gal and the second ampicillin-containing plate. Put the rod back into the package label the bottom of one amp plate “2 drops” and the other “10 drops”; label the plate without amp “control” remove bacteria/DNA from the heat block and using the micropipette add the respective amounts of bacteria/DNA to the agar plates spread the bacteria using the bent glass rod let plates air dry briefly; invert and incubate at 35 C overnight -Conjugation Divide one of each of the following plates (LB, LB+str, LB+amp) into halves (labeling half I and half II) Divide the LB+str+amp plate into thirds (I, II, I+II) Mix strain I and using a sterile loop, remove a loopful, and streak; sterilize the loop repeat for the remaining plate sections labeled I repeat using strain II Place another loopful of strain I on the ede of the remaining third of the LB+str+amp plate; sterilize loop and place a drop of strain II next to it and mix them together, streaking the area into a dime invert and incubate at 35 degree C -Taking a nasal swab
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insert sterile cotton swab 2-3 cm into one nostril; swab the surface and remove roll the swab over one quadrant of a mannitol salt agar plate; disregard the swab in the BacDown beaker using a sterile loop, streak the plat to obtain isolated colonies. Do NOT sterilize your loop between quadrants -Testing bacterial susceptibility to antibiotics/antiseptics/disinfectants label the bottom of the plates with the numbers 1-5 where the disinfectant discs will go (not too close to the edge) dip a sterile swab in a well-suspended broth culture. Streak the agar completely
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micro lab studyguide 2 - Review the procedures...

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