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Data Summary Table

Media Type


Culture Type LBA LBA+A LBA+T
E. coli + - -
E. coli pBR322 + + +

Each Student Should Answer the Following Questions:

Why were the plates incubated upside down?
Plates are stored upside down to prevent condensation that might form on the top of the plate from dripping back onto the surface of the medium and causing the colonies to run together.

Were the results seen in the Table as expected?
Elaborate on sources of error.
Answers will vary.

Describe the physical appearance of a single colony of E. coli as it was found on your plates.
Colonies from "Unknown Type A" and "Unknown Type B" have identical appearance. Both are circular, off-white colonies that are slightly raised. Students should also understand that there can be no inference from the phenotypic appearance of colonies as to which strain carries the pBR322 plasmid.

What reasons have you to assume that any individual colony is genetically pure?
Each colony results from the growth of a single isolated cell.

E. coli strains that contain the plasmid pBR322 are resistant to ampicillin and tetracycline. How might this plasmid function to bring about resistance to antibiotics?
The strain of E. coli containing the plasmid (pBR322) possesses genes that encode enzymes that confer antibiotic resistance to ampicillin and tetracycline. The gene Ap that confers resistance to ampicillin encodes for the protein B-Lactamase which hydrolyzes four-member rings of penicillin-like molecules, rendering them ineffective against the cell. The Tc gene that confers resistance to tetracycline, encodes for Tet proteins which function to actively transport the drug out of the cell.

BIO SAFETY

All materials that have come in contact with bacteria cultures must be decontaminated prior to disposal. Students should be directed to place all used (contaminated) applicator swabs, slant, and plate cultures into biohazard bags for disposal. This kit provides a biohazard bag.

Decontamination may be accomplished by autoclaving (if available) at 121deg.C, at 15 psi for 15 minutes. Alternatives are to use a steam pressure cooker, or to treat contaminated materials by immersion in Chloroxreg. for 30 to 60 minutes.


Materials List

Kit Materials:

Cultures:
1 E. coli (wild)
TO ORDER --> [each]
85W1860
1 E. coli (with plasmid pBR322)
TO ORDER --> [each]
85W1868
Reagents:
3 Bottles, Luria agar (200 mL/btl)
TO ORDER --> [pkg/6]
88W1205
20 Tryptic Soy agar tubes
TO ORDER --> [pkg/12]
88W0912
1 Sterile water (2 mL)
TO ORDER --> [pkg/4 vials]
88W0502
1 Bottle, ethanol (denatured, anhydrous)
TO ORDER --> [500 g]
39W0273
1 Capsule, tetracycline (250 mg)
TO ORDER --> [pkg/100]
21W2816
1 Vial, ampicillin (14 mg)
Other Materials:
2 Pipet, sterile (1 mL)
TO ORDER --> [pkg/500]
18W2974
60 Applicator swabs (sterile)
TO ORDER --> [pkg/100]
14W5502
1 Microfuge tube
TO ORDER --> [pkg/500]
18W1361
40 Petri dishes (sterile)
TO ORDER --> [pkg/20]
18W7101
1 Biohazard bag
TO ORDER --> [pkg/100]
18W6905
1 Teacher's Manual
1 "Student Study Sheet" copymaster
1 "Student Analysis Sheet" copymaster

Materials Needed But Not Supplied:

1-3 Waterbath (or microwave) Hotplate
TO ORDER --> [each]
15W8010
1-3 Beaker (1,000 mL)
TO ORDER --> [pkg/6]
17W4080
30 Safety goggles
TO ORDER --> [each]
15W3119
30 Apron (disposable)
TO ORDER --> [pkg/100]
15W1050
1 Microburner
TO ORDER --> [each]
15W0602
1 Pair, gloves (thermal protection)
TO ORDER --> [each pair]
15W1095
Index Page
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Copyright © 1996
   


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