Lab #5 Review for Practical #1 Colony morphology (pg. 19) Review for Practical #1 Preparing a smear (pg ) Same procedure for all staining methods except Acid fast staining (1% albumin) Review for Practical #1 Gram staining (pg. 34) 1 min sec 5-10 sec Review for Practical #1 Bacterial shape and arrangement Gram positive cocci Gram negative bacillus Review for Practical #1 Endospore staining (pg. 37 38) Prepare and heat fix a smear Flood slide with Malachite green for 1 min Steam for 3 minutes Use Bunsen burner like a blowtorch, intermittently Make sure the stain doesnt dry up Rinse with water for minute Cover smear with 0.5% Safranin stain Rinse and blot dry Observe under the microscope (+) Green = spores (inside cells and outside) Pink = vegetative bacterial cell Acid fast staining (Pg ): Prepare smear Add 2 loopfuls of 1% albumin (helps hold bacteria onto slide) Mix in bacteria aseptically Air dry and heat fix Flood slide with Carbolfushin Blow torch for 5 minutes, intermittently DONT LET STAIN DRY Rinse with water Decolorize with Acid Alcohol until color stops to run Rinse with water Cover smear with Methylene blue for 1 minute Rinse with water Blot dry and observe under microscope Review for Practical #1 Acid fast positive = Red cells Acid fast negative = Blue cells Only Mycobacterium species will be Acid fast positive resist de-colorization and retain primary stain (Carbolfushin) Review for Practical #1 Hanging drop method to determine motility (pg ) Use a broth culture Need a depression slide, coverslip, and Vaseline Smear Vaseline on palm scrape onto the four edges of coverslip (Dont get vaseline mixed w/ culture) Transfer loopful of bacterial culture onto coverslip Place depression slide, concave side down, onto coverslip DONT PRESS CAREFULLY invert slide see the hanging drop Observe under microscope for motility Review for Practical #1 Microscope observation of hanging drop: Start at 10X objective look for the edge of the drop Center the edge of drop in field of view Switch to 40X objective FINE focus Bacteria will appear ghost like within the drop (no stain!) Motility is distinct movement from point A to point B Review for Practical #1 Streak plate method (pg. 26 29): Sample is only take once only for quadrant 1 Streak from top to bottom dont go up and down Flame loop in between each quadrant!! Work aseptically! Review for Practical #1 Capsule staining Some bacteria produce this external structure Capsules composed of polysaccharides Encapsulated bacteria is more virulent Procedure Negative staining (stain the background and the bacteria) Capsule appears as a halo around the bacterial cell Capsule staining