Antibiotic resistance in Escherichia coli
A practical investigation of bacterial conjugation
In this practical protocol students investigate one way in which bacteria acquire antibiotic resistance through conjugation. The recipient strain Escherichia coli J-53R carries on its chromosome a gene conferring resistance to the antibiotic rifampicin. The donor strain, E. coli HT-99, harbours a plasmid that includes a gene conferring resistance to a second antibiotic, chloramphenicol. Liquid cultures of the two strains of bacteria are 'mated'. The recipient, donor and 'mated' cells are then plated on three different types of media: one containing rifampicin, one containing chloramphenicol and a one containing both antibiotics. After incubation, students interpret the results and they should find that antibiotic resistance has been transferred from one strain of E. coli to the other.
Students will learn about the process of horizontal gene transfer through bacterial conjugation and acquire the skills of good microbiological practice.
Pre-knowledge and skills required by students
Familiarity with aseptic techniques would be useful.
Time taken in lesson
50 minutes x 3
120 minutes x 2
Specialist equipment and materials required
Slope culture of Escherichia coli HT-99
Slope culture of Escherichia coli J53-R
Chloramphenicol powder, 25 mg
Rifampicin powder, 100 mg
Plus numerous other items for practical microbiology (see protocol). Bacterial cultures are available from the NCBE, University of Reading.
Context where originally used
Because of the manipulative skills required and the scientific concepts involved, this practical work is in general best suited to students over the age of 16 years.
Good microbiology laboratory practice is essential to the success and safe execution of this practical activity. Teachers carrying out the work should therefore be trained in the use of aseptic techniques, and should teach these to the students. Non-specialist teachers (e.g., those without a biological background who may be teaching science) should not carry out or supervise this work. Also bear in mind that without specialist knowledge, non-biology teachers may not be able to adequately evaluate or explain the hazards associated with the use of E. coli and antibiotics to students and others who may be concerned about these aspects of the work.
Laboratory technicians must also have appropriate microbiology training to prepare the microbiological media and cultures and to dispose of them safely.
This protocol was originally devised for the Wellcome Trust's 'Survival Rivals' project. 'Survival Rivals' is the Wellcome Trust's Darwin 200 offering for secondary schools in the UK (www.survivalrivals.org). The Wellcome Trust is an independent charity that funds research to improve human and animal health (Registered charity No 210183).
Dean Madden for the Wellcome Trust
University of Reading
2 Earley Gate
Text copyright © The Wellcome Trust, 2009
This protocol is covered by a Creative Commons 'attributed, non-commercial, share alike' licence. This means that you may use it, adapt it, translate it and so on. You may not use it for commercial purposes however, and you must mention the source of the original work (The Wellcome Trust). Any derivative works must be distributed on the same terms.