Functional Genomics Research Stream Lecture: March 03, 2009 Aseptic Technique, Growth Media, Culturing & Microscopy
Agenda State of the Union Saccharomyces Review Short Term Research Plan Assignment Seven
Beginning of Phase II Phase I: Molecular Lab Skills & Communication Phase II: Cell Growth & Maintenance Phase III: Molecular Techniques & Diagnostics Phase IV: Biological Assays & Interpretation
Saccharomyces cerevisiae Our model organism of choice. First eukaryote fully sequenced. ~12 MB (1/200 Homo sapiens) ~6000 genes 16 chromosomes Haploid & diploid states. Model organism of cell-cycle elucidation (Hartwell, Nobel). Extensive whole genome studies. http://www.mrc-dunn.cam.ac.uk Wikipedia
Saccharomyces Strains Thousands of unique yeast strains. Differentiating: Wild type (S288C) Natural Mutants Mutants by DNA Damage (UV, agents) Mutants by Recombination Deletions & Fusions
Naming Conventions WT = Wild type rtg3 = Strain where gene rtg3 deleted aft1 = Strain where gene aft1 deleted bas1 = Strain where gene bas1 deleted
Work This Week
No more Chemistry!
The Plan (before Spring Break) Easier Assignments No Word, No Excel (this week) Phase II: Part A Clean Aseptic Growth, Media Production, Microscopy Phase II: Part B Differential Growth, Spectrophotometry
Assignment Seven A7 Assignment Eight SPRING BREAK Molecular Techniques MT
This Week
Assignment Seven Purpose: Aseptic Technique Media Production (Plates, Liquid) Solid & Liquid Growth of Cell Cultures Cell Density via Microscopy (Hemacytometer) Laboratory Notebooks (always) Due: Tuesday, March 10
Protocols Definition: Formalized method by which an experiment or procedure is performed. All protocols developed and tested by our research staff and posted regularly in online course notebook.
Section A Plate & Media Production Training Short Sessions - 20 Minutes Sessions Scheduled: Wednesday, March 4-10:00 AM Wednesday, March 4-2:00 PM Thursday, March 5-10:00 AM Thursday, March 5-12:00 PM Thursday, March 5-2:00 PM
Training Topics Basic Aseptic Technique Media Preparation: Liquid & Solid, Pouring Plates Sterilization by Autoclave: Media, Equipment, Glassware, Liquids
Aseptic Technique Prevents media, cultures, stock reagents from becoming contaminated. Also called sterile technique. Patterns of habit, movement, and prevention. Very difficult to know if you have good aseptic technique; confounding variables.
Section B Aseptic Technique Demonstration No - you, not me. Group work highly encouraged
Demonstration 1. General cleanliness; cleaning of bench space. 2. Required equipment: Bunsen burner, gloves, anything else? 3. Bunsen burner lighting, components, general use. 4. Bench-top aseptic work using a Bunsen burner. Flaming of bottles, technique and rationale. 5. Manipulating vessels, liquids using aseptic technique. 6. Sterilizing and using an inoculating loop to transfer from one liquid culture to another. 7. Making a culture spreader from a Pasteur pipet. 8. Handling of alcohol fires in beakers (occasionally incurred during use of culture spreader). 9. Each of the following: a. Use of inoculating loop to streak a culture on a plate. b. Use of toothpick to streak a clone on a plate. c. Use of a culture spreader to spread a culture on a plate.
Expectations Completeness: You will cover the material that is stated above. Participation: All members of a group must participate and/or have a defined section. Preparation: It should not appear that you read the material once and just rambled through some sub-part of it that you could remember. You should take notes (in your laboratory notebook), come up with a plan, and then present it when you are sure of what you wish to say. It is allowed, even encouraged, that you have your lab notebook open to reference during your talk. Professionalism: We have lots of fun and conversation in the lab. This is not one of those times. This should be considered a serious topic and presented as such. Research staff and students around will be both active and passive audience. You should pretend that these are researchers you do not know but wish to impress. Duration: The total presentation should last approximately 10 minutes.
Section C Saccharomyces cerevisiae Growth Group work highly encouraged
Saccharomyces Growth Liquid Media (Cultures) Solid Media (Plates) Both made from YPD Yeast Extract Peptone (animal protein) Dextrose (carbohydrate) Wikipedia
Using Solid Media Streak One Streak Two Streak Three Streak Four http://www.madsci.org/~lynn/micro/techniques/streaking/
Other Representation From Course Textbook (Wiley)
Alternative Method From Online Course Notebook
Plating for Growth Defect (serial dilution left to right) Hu, Killion, Iyer Nature Genetics 2007
Section D Cell Culture Quantitation by Microscopy +
Hemacytometer Device used with microscope to estimate cell culture density. Not so commonly used with Saccharomyces and bacteria cell culture (more common is spectrophotometry). Very common in mammalian cell culture where culture volumes and density low.
Sectional Constraints Constraint 1: Section C must run overnight. Constraint 2: Section C cannot run over an entire weekend. Constraint 3: Section D must be done the day after Section C.
Questions?