BIOD171 Microbiology Lab 6 Notebook Summer 2023 portage learning

Portage Learning BIO 171- Microbiology Lab Notebook Lab Notebook Bookmarks (click to navigate): Lab 1 Notebook Lab 2 Notebook Lab 3 Notebook Lab 4 Notebook Lab 5 Notebook Lab 6 Notebook Lab 7 Notebook Lab 8 Notebook Lab 9 Notebook Lab 1 Notebook Back to Home Page Lab 1 Title: Keeping a lab notebook Objective: To establish an organized template for keeping experimental records, procedures and results. Procedure: 1. This is where each step of the given protocol is recorded. 2. Be sure to clearly indicate any deviations that may occur during the experiment. Notes: Additional (helpful) information placed here. Results: A summary of the final outcome of the experiment should be described here. Lab 2 Notebook Back to Home Page 02 Title: Basics of microscopy Objective: To become familiar with the basic component of a light as well as how to load a sample for viewing. Procedure: 1. Review parts and components 2. Load sample slide onto microscope 3. Select magnification 4. Make necessary adjustments to optimize sample visualization Notes: basic parts of a microscope: eyepiece, arm/neck, objectives (different magnification lenses 4x, 10x, 40x, 100x), revolving nosepiece, stage (where samples are placed, using a clamp holder.), stage controls, coarse/fine focus, iris diaphragm, light source, base. Using a standard light microscope which uses a halogen light bulb. Only grab the microscope by the arm/neck to move it. A lot of eyepieces have a pointer. objectives (different magnification lenses 4x, 10x, 40x, 100x) two types-dry vs oil, intensity of light source: too bright-saturation, too dark-low visibility. [Power of objective] x [power of eyepiece]= Total magnification -if a cell is 15mm in diaqmeter, using a 40x objective and a 5x eyepiece the cell will now appear to the eye 200 times larger (200x) or 3000mm in diameter. Results: Magnification= objective x eyepiece Lab 3 Notebook Back to Home Page 03 Title: Mounting Techniques Objective: Microscopic examination of bacterial samples through various staining techniques. Identify color and shape of given samples. Procedure: Dry Mount 1. Clean glass slide (70% ethanol) 2. Circle area on slide for easy location of specimen(optional-with a sharpie on the b bottom side of slide so you don’t contaminate sample) 3. Apply organism to slide -if from culture, use sterile loop to spread onto slide -If from plate, use sterile loop to pick colonies and mix with a drop of distilled water 4. Air dry at room temperature until all moisture has evaporated Wet Mount 1. Clean glass slide (70% ethanol) 2. Circle area on slide with a wax/hydrophobic pen(keeps water inside the ring of wax) 3. Apply organism to the slide -if from culture, use sterile loop to spread onto slide -If from plate, use sterile loop to pick colonies and mix with a drop of distilled water 4. View under microscope -wet mount is ideal for viewing the motility of an organism. Do not dry out. Gram Staining 1. Clean the glass slide (70% ethanol) 2. Apply organism to slide: -Use sterile loop to spread ~1-3 drops onto slide -Spread into a thin film 3. Allow to air dry 4. Fix organism to slide by passing 3 times through a flame. Do NOT overheat slide! 5. Flood the slide with crystal violet for 30-60 seconds –sit for 1 minute 6. Rinse slide with water 7. Cover with Gram’s iodine stain for 30-60 seconds-sit for 1 minute 8. Rinse with water 9. Decolorize with alcohol rinse 10. Rinse with water 11. Counter stain with Safranin (red/pink dye) for 30 seconds-sit for 30-45 seconds 12. Rinse with water 13. Blot dry (with paper towel-rest will air dry off. Then blot dry with a chem dry to get the remaining moisture off) and examine under microscope -anything used during this procedure goes into biohazard waste for proper disposal that then gets put in the autoclave and finally disposed of -things needed for gram staining: distilled water, chem wipes, clips (optional), timer Notes: mounting technique-how to get a bacterial or pathogen species onto a glass slide for viewing. Gram Positive Bacteria= Purple -thick peptidoglycan layer, retains crystal violet stain Gram Negative Bacteria= Pink - Thin (single) peptidoglycan layer, damaged by alcohol rinse step and crystal violet stain washed away. -Pink color derived from Safranin (secondary counterstain) Stains and Shapes: -Gram (-) Rods (bacilli) -Gram (+) Chains (cocci-spherical chain) -Gram (+) Clusters (cocci-spherical clusters) Acid Fast Stain: -Strong resistance to decolorization -Very few structures are acid-fast -Commonly used to identify mycobacterium -Carbolfuchsin dye retained (red dye) Negative Staining: -Commonly used to identify organisms with opaque structures -Dark background via nigrosin dye -Negatively charged, repelled from membrane - Negrosin dye is negatively charged and most bacteria membranes are negatively charged so the dye is repelled from the bacteria(not absorbed) and creates a dark background on the slide. -Disadvantages to heat fixing samples- Excessive heat, particularly if it is prolonged, can damage cells and cause substantial shrinkage and hardening of the specimen.