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LMS Web site: http://www.lms.unimelb.edu.au/
[enter your email uname & pwd, then go to the 526321 page]
Coordinator: Dr M. Dyall-Smith () Tel: 8344-5693
lab 3.07, third floor of dept. (Haloarchaea Genetics Lab)
Demonstrators: Louise Adams, Lara Grollo, Nicole Kountouri, Claudie Thia,
Raju Mantena, Brendan Russ, Jan Stent
Prep room: Eleanora Puglia, Justine Hines
UNIVERSITY OF MELBOURNE
Department of Microbiology
2007
Prepared by the staff of the department of Microbiology and Immunology
Molecular Microbiology Techniques
Contents
PAGE
Laboratory rules 3
Care of Microscopes 4
Safety summary 5
Class times, Lectures, Objectives, Assessment, Reports 6
Reference material, Library resources 7
Introduction, Background, Aims, (to practical) 8
Course Outline 9
WEEK 1 - details 10
METHODS for WEEK 1 12-27
Weeks 2 - details 28
METHODS for WEEK 2 30-36
Weeks 3 - details 37
Weeks 4 - details 38
Weeks 5-6 - details 39
Methods
M-protein extn, SDS-PAGE and Western Blot methods 40-45
Final reports and Workbook - marking outline 46
Discussion questions for week 1 47-48
Discussion questions for weeks 2-6 (to come)
LABORATORY RULES
1. You must wear protective clothing (lab coat, gown etc.) during practical work in order to protect normal clothing against possible contamination and chemical splashes. Remove this clothing when leaving the laboratory and store in a plastic bag. Students who do not bring a lab coat will be required to purchase a disposable plastic apron before commencing practical work.
2. You must wear some protective footwear - bare feet and thongs are forbidden.
3. Do not bring food or drink into the laboratory.
4. Do not place on the laboratory bench any object which may be transferred to the mouth, consciously or unconsciously, e.g. fingers, pens, pencils, labels, handkerchiefs, etc.
5. Do not sit on the bench.
6. Do not bring bags to the work bench. Leave them in the designated areas.
7. Should any culture be spilt or broken inform your instructor or persons in charge of the practical class immediately. Do not attempt to clean the bench or floor without permission.
During Classes
8. When in doubt as to a laboratory procedure, seek the advice of an Instructor.
9. Every precaution must be taken in handling cultures whether they are pathogenic or non-pathogenic. Develop sound technique.
10. No mouth pipetting is permitted.
11. All instruments used for subculturing must be sterilized after use by either (a) heating in the bunsen flame; (b) transferring to a chemical disinfectant; or (c) placing in a discard tin for autoclaving.
12.. Cotton wool plugs from sterile tubes or from culture tubes must not be placed on the bench.
13. Requests for extra materials or equipment must be made in the first instance to demonstrators or class supervisors not directly to technical staff.
14. All culture tubes must be labeled clearly with grease pencil/felt pen or with a gummed label with group number, the name or initials of each student and the name of the organism or specimen. Labels should be moistened under the tap or on a wet filter paper; not with the tongue.
15. Cultures for incubation or storage should be placed in the labeled tins or baskets provided. It is your responsibility to ensure you use the correct receptacle.
Cleaning Up
16. Benches must be left clear and tidy before you leave the laboratory. Each student is responsible for their own bench. Anything left on the bench will be discarded.
17. After tidying benches students should swab their bench area with the 80:20 v/v alcohol and cotton wool provided at the end of each bench.
18. After tidying benches and swabbing with disinfectant, wash hands using the recommended procedure before leaving classroom.
Discarding materials
See the demonstrator for details about discarding contaminated and other wastes.
Accidents Should Be Avoided
Details of hazards associated with the handling of microbiological materials are in the Techniques Manual on page 3a, and a table with details of recommended precautions on pages 3b, 3c, and 3d. There is a table on page 3e giving advice on what to do in case of an accident.
OLYMPUS BINOCULAR MICROSCOPES: CLEANING INSTRUCTIONS
Always use the microscope that corresponds to your bench number
• Leave covers in cupboards
After Use
• Remove and discard slides from the stage.
• Return the voltage control dial to its lowest setting.
• Turn off the microscope light
Cleaning
• Fold a ‘Kimwipe’ cleaning tissue in half and moisten with microscope cleaning fluid (found at the end of the bench)
• Wipe the 10x objective first
• Do not wipe the 40x objective if it has not been used (Never use oil on a 40 x objective)
• Wipe the 100x objective lens once, turn tissue to the other side, then wipe the lens again.
This ensures you do not re-wipe the oil back onto the objective
• Wipe the stage
• Return the microscope to the correct numbered position in the cupboard
Eye protection
Some form of eye protection must be worn. Contact lenses are not eye protection. The options are:
1. Safety glasses to AS 1337
2. Normal prescription glasses (if the lenses are not too small*)
3. Safety goggles as issued by the Chemistry Department stores
* If the lenses on prescription glasses are too small, safety glasses or goggles must be worn over the top.
Eye protection must be worn by all people during the time in which practical work in being carried out in the laboratory. Remember that others around you may be carrying out hazardous procedures, so even if you are not actually working, others may be. Frames, lenses, and ear-pieces must be decontaminated at the end of the session, using 70 to 80% ethanol and lens tissues. This is also sufficient for most contamination problems that may arise during the practicals.
Some safety glasses will be available from the preparation room, but this is not to be considered a permanent supply. You will have a pair from your chemistry practicals anyway, and will probably not want to wear a pair that is not yours.
Gloves
Remember that you wear gloves to protect you from something that is potentially harmful. Do not touch pens, pencils, books, door handles, lift buttons, computer keyboards and other objects that other people may touch with their bare hands, as you may transfer the hazard to unprotected and unsuspecting people.
Latex or synthetic gloves must NOT be worn when working with Bunsen or other gas burners.
PLEASE REMEMBER - SAFETY FIRST !
1.DISCARDS: Students must discard all their own materials at the end of the practical session into the CORRECT discard containers.
Contaminated Plastic disposable bins (plastic bins with yellow biohazard bag)
· DISCARD: Plastic centrifuge tubes, petri dishes, disposable filters etc.
· Found at end of each bench
Glass bottle discard (drawer at end of every 2nd bench)
· DISCARD: Contaminated glass bottles including macartneys, bijous, 200 ml bottles.
Plastic jugs with autoclave bag
· DISCARD: plastic 1ml, 10ml & pasteur pipettes, gilsen tips, toothpicks.
Sharps discards (Yellow 2.8L plastic container on bench)
· DISCARD: Slides, coverslips, needles, syringes, broken glass
Waste paper bins
· Discard all pipette wrappings
· Paper towels etc.
· Disposable plastic aprons
3. MICROSCOPES: Students must switch off and clean their own microscopes after every Practical class. Use the Microscope cleaning fluid and Kimwipes provided at the end of each benchtop.
Microscope cleaning instructions are posted on Classroom NOTICEBOARDS.
4. INCUBATIONS:
· Materials for incubation (plates, bottles, tubes) must be placed in the correct containers. These are labelled with the Group and Experiment number and are found at the front of the Classroom.
· Prep. staff will not take responsibility for materials not placed in the correct containers.
5. “DO NOT DISCARD”
· Materials eg, plates, slides etc. with the label “DO NOT DISCARD” should be left at the group site bench.
6. DISINFECTING BENCHES & HANDWASHING
· At end of session Students are to use the 80:20 ALCOHOL squeeze bottles and toilet tissue rolls provided at the end of each bench to wipe their benches. These can also be used to mop up any spillage.
· Students must remove their labcoats before washing their hands with chlorhexidine handwash at the washbasins.
7. Pipettes , pasteurs & micropipettes
· Disposable plastic 2ml , 10 ml and pasteur pipettes will be provided in boxes at the front of each classroom or at group site.
· Micropipettes and tips will be provided as required . The micropipettes should always be hung on the hooks above each bench when not in use.
· All plastic pipettes and tips should be discarded into the jug provided on each student site bench.
526-321 MOLECULAR MICROBIOLOGY TECHNIQUES
Held in the 3rd year practical laboratories (2nd floor, western end, Microbiology and Immunology dept.)
Class times:
Mon 11-12 pm Lecture Small Russell-Grimwade Thtr (Biochem Dept)
Tues 10am – 1pm Prac Class, Rm 213
Wed 11 am - 5 pm Prac Class, Rm 213
Thur 9 am - 10 am Prac OR Lecture (Med Ctr, Thtr 3) (to be arranged)
Lectures: Mon 10-11 pm (Small Russell-Grimwade Thtr); Some at Thur 9-10am (Med Ctr Thtr 3).
TOPIC SPEAKER
Mon Wk 1: Streptococci; identification, characteristics, diseases MDS
Mon Wk 2: Sequencing emm genes of GAS: practice and theory MDS
Mon Wk 3: M protein: from western blots to vaccines MDS
Mon Wk 4: Typing 40,000 GAS isolates: the CDC experience MDS
Mon Wk 5: Genomics, bioinformatics and epidemiology of GAS MDS
Mon Wk 6: Review of course and results (and bring your questions along) MDS
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MDS - Dr Mike Dyall-Smith; NC - Dr Nigel Curtis, Royal Childrens Hospital
Objectives : By the end of the course you should:
· gained some understanding of the principles and procedures involved in the culture, isolation and identification of bacteria (particularly those of medical and environmental importance) based on principles of microbial physiology;
· used molecular microbiological techniques (e.g. PCR, DNA sequencing, western blot probing) to identify important characteristics of bacteria (e.g. virulence factors);
· used common bioinformatics methods to analyse DNA and protein sequence data (e.g. BLAST searches, translation of DNA sequences, emm virulence types of streptococci); and
· gained expertise in retrieving published scientific data related to the project using computer searches and library facilities (e.g. Medline).
General description of the course:
This subject covers various aspects of practical and molecular microbiology including conventional isolation and identification methods, PCR and sequencing, antigen detection using western blots. Each student analyses an uncharacterized isolate of a group A streptococcus, and writes up their results for assessment.
Assessment:
1. Regular written reports of laboratory work completed. Normally workbooks are taken up at the end of week 2 and week 4 for assessment by the demonstrator. You are expected to summarize your results so far and present them in an easily readable form (eg. tables, conclusions). There is also a final report, bringing together your results with those of the class. Workbook marks are worth 10% for each of 2 assessments (total of 20%) and the final report is worth 30% (total of 50% for these two components). Details of the requirements and format of workbook summaries and final reports are on the final pages of these course notes.
2. A two hour written examination (usually in week 6), that is worth 50%. This will involve both knowledge of the methods and organism, and interpretation of experimental data.
Experimental Reports
Students must use a notebook to record all their results and details of work done. You are expected to use the data in your notebook to write up your summary reports, and your answers to discussion questions. Using loose pieces of paper for this purpose is not acceptable, and notebooks will be taken up and used to assess particular experiments.
Final reports should be concise. The format and length will be given to you at the appropriate time during the course, but some generalizations can be made. Where methods are described in published material then the usual practice is to quote the source rather than writing out methods in detail. References should be given in a standard format (e.g. as in a standard journal, such as the Journal of Bacteriology), and data organized into figures (i.e. graphs, photos) or tables. Results must be described in writing (in the Results section), not just presented as tables/figures as being self-explanatory.
Lectures and tutorials
The lectures are meant to provide the necessary scientific framework for you to understand the practical exercises. This material is examinable. The provisional lecture schedule is given above. Any changes in dates, venues or titles will be given to you beforehand in class.
Specific times will be set aside by your demonstrator for tutorials. These will concentrate on specific topics (eg. answers to discussion questions) related to your experimental exercises. Take advantage of these to discuss problems relating to your practical work (experiments, reports, computer searches, etc.).
Textbooks and reference material
The Techniques Manual, produced by this department, is useful for techniques and some background information. You will need specialist texts to go into more detail, such as the texts described below. More up-to-date information is to be found in peer-reviewed studies published in scientific journals, and some of these will be discussed in the lecture course while others you will need to find yourself (or I will put them up on the LMS web site).
Shown below are some examples of useful texts:
Balows et al., The Prokaryotes vol. I-IV 2nd edn (Taxonomy/Classification of bacteria)
W&W Bergey’s Manual of Determinative Bacteriology, 9th edn. (Taxonomy)
ASM Methods for general and molecular bacteriology (1994) (Methods)
ASM Manual of Clinical Microbiology, 6th Ed., eds. P. Murray et al., 1995 (Methods)
Baron, S. (ed) Medical Microbiology, 4th ed.
Mims et al. Medical Microbiology, 2nd ed.
Greenwood et al. Medical Microbiology 15th ed.
Oral Microbiology Marsh and Martin, 3rd Ed. (1992)