Kate Andrews Tricia Geraghty

1. Aim: To investigate the spread of antibiotic resistance between bacteria. Kate Andrews Tricia Geraghty

2. Conjugation takes place between two strains • of bacteria resistant to different antibiotics • Result some cells resistant to both • antibiotics • So pupils will: • practise using aseptic techniques • Investigate this method of horizontal gene transfer • www.survivalrivals.org

3. Curriculum links • Int . 2 Biology Unit 2. Environmental Biology and Genetics • Factors affecting variation in a species (xii) Natural Selection • Higher Biology Unit 2. Genetics and Adaptation b) Selection and speciation (v) High speed evolution of organisms such as antibiotic resistant bacteria and the melanic peppered moth.

4. Int. 2 Biotechnology Unit 1. The Biology of Microorganisms • Structure and reproduction of microorganisms Unit 2. Working with Microorganisms • Microbiological procedures • Higher Biotechnology Unit 1. Microbiology • The structure of microorganisms Unit 2. Microbiological techniques • Growth limitation and sterilisation techniques Culturing techniques

5. Revised Higher Biology Unit 1: DNA and the Genome 3. The genome (c) Evolution (i) Inheritance Genetic sequences are inherited vertically from parent to offspring as a result of sexual or asexual reproduction. Prokaryotes can exchange genetic material horizontally, resulting in rapid evolutionary change. Prokaryotes and viruses can transfer sequences horizontally into the genomes of eukaryotes.

6. Unit 2. Metabolism and Survival • Investigating metabolism in microorganisms • Genetic control of metabolism • (i) Genetic variation Some bacteria can transfer plasmids or pieces of chromosomal DNA to each other or take up DNA from their environment to produce new strains.

7. Antibiotic resistance in bacteria Darwin thought that hereditary characteristics were always passed from one generation to the next, i.e. vertically. This does happen with microorganisms and some genes e.g. With MRSA Genes conferring resistance held on bacterial chromosome http://chsweb.lr.k12.nj.us/mstanley/outlines/bacteria/bacteria.htm

8. Horizontal gene transfer Often microbial genes are transferred horizontally, from one bacterial strain to another. • Transformation • Transduction • Conjugation – via ‘sex pilus’

9. www.survivalrivals.org

10. The procedure E coli J-53R carries a gene on its chromosome conferring resistance to antibiotic rifampicin. E coli HT-99 (the donor) has a plasmid which has the gene conferring resistance to chloramphenicol. Once they have conjugated, the three cultures are grown up on three types of media • Containing rifampicin • Containing chloramphenicol • Containing both antibiotics Top ups to kits – Philip Harris www.philipharris.co.uk

11. Our Procedure • Preparation of cultures – getting the bacteria into active state of growth (could do with pupils) • Mating the bacteria (Lesson 1) • Plating out the cultures (Lesson 2) • Results and interpretation (Lesson 3) N.B. Aseptic technique throughout - GMLP

12. Good microbiological laboratory practice (GMLP) Someone trained to level 3 – to prepare the microbiological media and to dispose of them safely. “Teachers should be trained in the use of aseptic techniques, and should teach these to students.” http://www.sserc.org.uk/members/SafetyNet/Microbio2/Main_Menu.HTM

13. General points: • Wear a lab coat • Wash hands before and after each ‘step’ • Cover cuts or scratches on hands • Wear safety glasses when diluting Virkon™ and using • bleach

15. At each stage: • Wipe the bench surface with disinfectant before and at the • end of the procedure • Work within a 20cm radius of a lit Bunsen flame (blue) - to • create an up-flow of warm air which will carry away any • potentially contaminating organisms • Label bottles and plates carefully (plates on underside) • Handle sterile syringes, pipettes and loops carefully – • dispose of these into Virkon™ • Flame necks of bottles after removal and prior to • replacement of lids • Don’t put lids of plate or bottles on bench….. • Inoculated plates should be sealed diametrically with small • pieces of sticky tape and incubated upside down.

16. Ensuring bacteria are in an active state of growth Bacteria from the slopes have been streaked onto nutrient agar plates and incubated at 300C for 24hrs to make stock plates of each strain. Single colonies of each strain of bacteria are then transferred into separate nutrient broths, and incubated at 300C overnight.

17. Conjugation - ‘mating’ • Using sterile syringe aseptically transfer 1.8cm3E. coli J-53R into labelled (‘mating’) nutrient broth • Discard syringe into disinfectant • Use a new syringe to add 0.2 cm3 E. coli HT-99 into the ‘mating’ nutrient broth • Discard syringe into disinfectant • Place all three cultures into an incubator or water bath at 30oC for 4 – 24 hrs.

18. Plating out the bacteria

19. Plating out the bacteria Now label the plates Inoculating the plates

20. Look for the bubble

22. Make sure you streak bacteria right onto the agar

24. Plating out the bacteria Inoculation plan

25. Invert the plates and incubate at 30oC for 24 hours. Seal the plates.

26. Predictions Chloramphenicol Chloramphenicol + Rifampicin Rifampicin

27. Results Rifampicin Chloramphenicol Rifampicin and chloramphenicol

28. X- bacteria overview

29. http://evolution.berkeley.edu/evosite/evohome.html

30. Further activities/ resources • BBC Learning Zone includes clips on evolution www.bbc.co.uk/learningzone • Tree of Life An interactive fly-through of evolution on Earth, narrated by Sir David Attenborough.www.wellcometreeoflife.org • DNA to Darwin free resources for 16-19 year olds around molecular evidence of evolution www.dnadarwin.org • Evolution Megalab Did you know that thanks to a common little snail that you can find in your garden, in the park or under a hedge, you can see evolution in your own back yard? www.evolutionmegalab.org • CREST Awards to accredit young people’s work in science and technology www.britishscienceassociation.org/crest • Royal Society for the Protection of Birds Information about birds, including education pages www.rspb.org.uk • Society for General Microbiology Information about microbiology, including educational resources www.sgm.ac.uk • Singtastic Featuring the Mr Darwin song www.singtastic.com • Your Genome Educational information from the Sanger Centre www.yourgenome.org