BIOD 171 Microbiology Lab Notebook | Exam Questions With Answers | Portage Learning

Objective: Microscopic examination of bacterial samples through various staining techniques. Identify color and shape of given samples. Procedure: Dry Mount 1. Clean Slide (70% ethanol) 2. Circle area on slide for easy location of specimen (optional) 3. Apply organism to slide: a. If from culture, use sterile loop to spread onto slide b. 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 Slide (70% ethanol) 2. Circle area on slide with a wax/hydrophobic pen 3. Apply organism to slide: a. If from culture, use sterile loop to spread onto slide b. If from plate, use sterile loop to pick colonies and mix with a drop of distilled water 4. View under microscope a. Wet mount is ideal for viewing the motility or an organism. Do not dry out. Gram Staining 1. Clean Slide (70% ethanol) 2. Apply organism to slide: a. Use sterile loop to spread 1-3 drops onto slide b. 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 6. Rinse slide with water 7. Cover with Gram’s iodine for 30-60 seconds 8. Rinse with water 9. Decolorize with alcohol 10. Rinse with water 11. Counter stain with Safranin for 30 seconds 12. Rinse with water 13. Blot dry and examine under microscope Notes:  Gram Positive bateria= Purple o Thick peptidoglycan layer, retains crystal violet stain  Gram Negative bateria= Pink o Thin (single) peptidoglycan layer, damaged by alcohol rinse step and crystal violet stain washed away o Pink color derived from Safranin (secondary counterstain) Stains and Shapes:  Gram (-) Rods  Gram (+) Chains  Gram (+) 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 nigrosine dye o Negatively charged, repelled from membrane Results: 1st Sample-Staphylococcus aureus 2 nd Sample-Escherichia coli 3 rd Sample-Bacillus subtilis 4 th Sample-Pseudomonas aeruginosa 5 th Sample-Streptococcus Lab 4 Notebook Back to Home Page Title: SR04 Growth Media Objective: To understand the types and uses of growth media for the isolation and identification of unknown bacterial samples. Procedure: 4 Phase Dilution Streaking: Clonal Isolation 1. Use sterile loop spread culture in area #1 2. Using a NEW sterile loop drag through the end of area #1 ONCE 3. Using a NEW sterile loop drag through the end of area #2 ONCE 4. Using a NEW sterile loop drag through the end of area #3 ONCE -Use a back-and-forth pattern to dilute the culture in each zone. - Invert plate and incubate overnight at 37 degrees Celsius [Non-selective agar] Quadrant Growth: Rapid Test for Multiple Isolates 1. Using sterile loop spread unknown culture A in area #1 2. Using a NEW sterile loop spread unknown culture B in area #2 3. Using a NEW sterile loop spread unknown culture C in area #3 4. Using a NEW sterile loop spread unknown culture D in area #4 -Use a back-and-forth pattern to dilute the culture in each zone. - Invert plate and incubate overnight at 37 degrees Celsius Notes:  Non-Selective Media: Important for the expansion of unknown bacteria.  Selective Media: Used to eliminate irrelevant bacteria from mixed cultures.  Differential Media: Used to distinguish between species of the same group. Results: 1 st Plate-Zone 1 growth, culture was not as concentrated as we believed 2 nd Plate-Overnight culture, Zone 1-e coli strain grew rapidly, Zone 2-staph long uniform, Zone 3- strep sample nicely spread, Zone 4-psuedemonas strain wasn’t growing as strong as the others 3 rd Plate-E coli and staph, changed color because of the red blood cells, clear and dark, staph beta, e coli appears dark 4 th Plate-top is e coli and grows and appears green, bottom is gram negative psuedemonas strain and does not have a color change or strong growth like e coli Lab 5 Notebook Back to Home Page Title: SR05 Testing bacteria for antibiotic sensitivity Objective: To determine the threshold of antibiotic sensitivity across bacterial strains using the Kirby-Bauer method. (AKA Standardized Disc Susceptibility Test) Procedure: 1. Streak bacteria A across an LB agar plate for confluent growth using a sterile L-spreader 2. Evenly place the paper antibiotic discs on the plate 3. Invert plate and incubate at 37 degrees Celsius 4. Measure zones of inhibition (diameter) 5. Compare results with sensitivity chart