Portage Learning BIO 171 Microbiology Lab Notebook, Exams of Microbiology

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Portage Learning BIO 171 Microbiology Lab

Notebook

Portage Learning

BIO 171- Microbiology

Lab Notebook

Lab 1 Notebook

Title: SK01 Intro to Medical Microbiology & Lab Notebook Guidelines Objective: Cultivation of samples: growth conditions/equipment used Identification of samples: biochemical assays & tests Evaluation of samples: microscopy: visualization and key characteristics of specimens Equipment: Cleaning: Sterility is a must. Autoclave (125°C): uses heat, pressure, & steam to sterilize within minutes. Ensure it is opened slowly to allow it to depressurize Growing: 1. Fixed incubator: (37°C) airtight, shelving for petri dishes

2. Shaker incubator: (37°C) growth media (liquid nutrient broth) used to inoculate bacteria.; Shaker swirls it around to aerate it & aid growth Visualizing: microscopy Storing: (4°C) inhibits growth and helps preserve samples; Petri dishes wrapped in parafilm: airtight Lab Safety: 1. Never eat or drink in the lab: contamination 2. Use PPE: gloves- latex, nitrile (P, B, G), thermal, or cold gloves; eyewear if needed; lab coat 3. Never leave lab while wearing PPE Lab Notebook: Objective: To establish an organized template for keeping experimental records, procedures, and results. Procedure: each step of protocol is recorded , must include any deviations that occurred Notes: Additional information Results: summary of the outcome of experiment

1. Place sample securely on the stage and turn on.
2. On L side you can dim/brighten the light. Too bright: high saturation, too dark= low visibility *Tip: Start midway
3. Need to know what kind of objective is present: dry vs oil (required on higher magnification); add a drop of oil onto slide with light refraction and lens will be embedded in the oil
4. Stage guides below stage 2 knobs: Top controls: forward/backward movement; Lower: moves left/right. (Specimen now in range of viewing)
5. If unsure of magnification: start on lowest power; Power of Objective x Power of Eyepiece = Total magnification: 15 mm diameter object & total magnification is 200x larger & diameter is 3000mm
6. Coarse adjustment will raise/lower the stage- sample should not be touched with the lenses. Look through eyepiece & slowly make necessary adjustments Results : The parts/basic functions of the microscope can now be identified, as well as correct methods to visualize slide samples

Lab 3 Notebook

Back to Home Page Title: SK03 Mounting Techniques and Intro to Gram Staining Objective: Identify/characterize different genus & species of various bacteria. Microscopic examination of bacteria through various staining techniques Procedure: Dry Mount:

1. Clean slide (70% ethanol & Kimwipe); dirty=difficult to determine what you’re visualizing
2. Optional—Circle around specimen on slide for easy location
3. Apply organism to slide: × Culture: use sterile loop to apply to slide × 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:
5. Clean slide (70% ethanol)
6. Circle area on slide using a wax hydrophobic pen—will keep water inside circle
7. Apply organism to slide: × Culture: use sterile loop to apply to slide × Plate: if from plate, use sterile loop to pick colonies and mix with a drop of distilled water
8. View under microscope ** wet mount is used to view motility of organism; Don’t let it dry out—distilled water should be applied to maintain organism viability. Gram Staining:

-used for organisms with opaque structures -dark background (Nigrosin); both dye and cell membrane - charged so dye is repelled

• will be able to see the clusters and shapes of bacteria WET LAB:

1. Don PPE
2. place samples in tube and flame top of tubes to sterilize
3. cleaned slides with chem wipe
4. draw circle on slide with wax pen to keep bacteria in circle
5. flame top of tube again
6. put loop in tube and swirl to collect sample
7. spread thin layer inside circle on slide (wax keeps liquid inside)
8. let air dry
9. heat fix by running slide over flame 3 times – should be no liquid lef
10. repeat with remaining 4 samples *sterile loop should be disposed of in biohazard waste bin as well as anything else and put in autoclave to be sterilized before disposal. APPLYING STAIN TO EACH SAMPLE: Supplies: specimens, tray, Kimwipes, distilled water, clothespins, timer, crystal violet, alcohol, iodine, and safranin
11. Flood samples with crystal violet dye and let dry for 1 min
12. rinse off with water until dye stops running
13. Using an eye dropper, flood samples with iodine-don’t touch specimen! Dry for 1 min
14. Rinse again thoroughly, but carefully

5. decolorization with alcohol; some cells will lose dye, while some retain
6. rinse again
7. counterstain with safranin and let dry for 30-45 secs
8. final rinse
9. Allow water to run off on paper towel and let air dry
10. blot dry with Kimwipe; may re-mark # with hydrophobic pen, if needed EXAMINE SLIDES UNDER MICROSCOPE:
11. begin on 40x magnification & ensure stage is lowered all the way
12. open clips and place slide on stage to secure
13. set illumination to 50% and open diaphragm completely -Focus microscope:
14. raise sample until in focus using the coarse adjustment
15. If dark image is visualized, object is in focal plane, slow down & use fine focus to make image clear Results: Organism 1: S. Aureus: Gram +/Purple color/round cluster shaped Organism 2: E. Coli: Gram -/Pink color/rod shaped Organism 3: B. Subtilis Gram +/Purple color/ rod shaped Organism 4: Pseudomonas Aeruginosa: Gram -/Pink color/Rod shaped Organism 5: Strep: Gram +/Purple color/Chain

Differential Media: differentiate between species of the same group DIFFERENT TYPES OF PLATING: (different nutrient selections) NON-SELECTIVE PLATES:

1. LB: non-selective; most common; multipurpose (invert plate & place of 37 --don’t want settling on bacteria) 2. Blood agar: RBCs: important nutrients for growth 3. TSAYE: removed RBCs; general purpose; yeast & tripticase soy SELECTIVE MEDIA Plates:

1. MacConkey : red (slightly cloudy); selects for GRAM NEG, inhibits gram +
2. SMAC: pink color; sorbitol instead of lactose; Selects for GRAM NEG ; differential because it distinguishes between pathogenic and non-pathogenic E.coli
3. EMB : dark purple; selects for GRAM NEG; has eosin/methylene blue; differential based on ability to ferment lactose: -ferments: colonies turn green -partially ferments: colonies turn pink -no fermentation: colonies stay original color WET LAB: (don PPE!)
4. 4 Phase dilution streaks -individual packed sterile loops each time streaked, or loop flamed with Bunsen burner -Mix sterile loop into tube with culture and complete 1 st^ streaking a few times -Repeat process by rotating, and passing through previous zone only once

• Mark sample, invert, & place in incubator @ 37 C (12-14hrs), examine for growth

2. Quadrant Growth (divide into 4 quad on bottom with Sharpie; label and date) -culture already growing in tubes

-sterile loop mixed in sample & streak quadrant; invert plate and # , dispose loop

• repeat with remaining samples ensuring to cap them (spillage)
• Invert, place in incubator @ 37 C (12- 14 hrs), & examine growth

3. Blood agar: non-selective & differential media—ability to visualize hemolysis state of bacteria -divide plate in ½; 2 cultures: marked (one lyses blood cells the other does not)
   • Repeat same process with sterile loops, streak each half back & forth, invert, and discardPlaced in incubator etc. and examine for hemolysis state
4. EMB agar: 2 gram neg bacteria; 1 ferments lactose the other does not -divide plate in ½; mark samples, using sterile loop streak each side with sample; invert and discard; placed in incubator 37°C ** Don’t cross the zones Results:
5. Phase 4 Lab: able to visualize individual colonies in between zone 2 & 3; can now re- inoculate the colony and grow large scale
6. Quadrant Growth: (zone 1) E. coli-rapid growth; (zone 2) S. aureus- uniform, long growth pattern; (zone 3) strep- not as much growth; (zone 4) pseudomonas- formed a colony like dilution plate
7. Blood agar: (zone 1) S. Aureus- exhibits beta hemolysis (reg agar becomes clear); (zone 2) E. coli- doesn’t have lyses properties
8. EMB agar: (1) E. coli able to ferment lactose & gives off a green metallic color (2) pseudomonas does not ferment lactose= no color change & not as much growth

2. Add drop wise steps around plate for equal distribution and discard the pipette tip
3. Use sterile wrapped L spreader to evenly disperse bacteria around plate (may feel resistance), ensuring the lid is kept above the plate—discard spreader
4. Use forceps to remove disk and place on agar a finger width apart from edge ensuring room between other discs (don’t break agar surface!) -Label each disc and ensure there is equal spacing to visualize zones of inhibition; Repeat for each disc
5. Invert and place in incubator on 37°C overnight -If there is overlap, go to an area where there isn’t overlap, measure the radius and double for diameter **Results:

• Looking for zone of inhibition (clearing) for each disc**
• Reference chart to determine sensitivity levelResistant > Intermediate > Susceptible Antibiotic Resistant Intermediate Susceptible Cefazolin ≤ 14 15-17 ≥ 18 Clindamycin ≤ 14 15-20 ≥ 21 Erythromycin ≤ 13 14-22 ≥ 23 Gentamicin ≤ 12 13-14 ≥ 15 Oxacillin ≤ 10 11-12 ≥ 13 Penicillin ≤ 28 -- ≥ 29 Tobramycin ≤ 12 13-14 ≥ 15 Vancomycin -- -- ≥ 15

WET LAB RESULTS: On the back of the plate mark diameter of each sample to measure (mm) Antibiotic Diameter (mm) Result Vancomycin 30 Susceptible Clindamycin 16 Intermediate Oxacillin 17 Susceptible Tobramycin 22 Susceptible Erythromycin 24 Susceptible Penicillin 36 Susceptible Gentamicin 10 Resistant Cefazolin 19 Susceptible

1. Use sterile loop to pick an isolated colony from plate.
2. Add colony to tube of rabbit plasma, incubate overnight at 37 C.
3. Examine next day for precipitants (cloudiness) -Fibrin (blood clots) good for bacteriadefense mechanism used against antibody recognition and helps them escape phagocytosis -S. Aureuscoagulase positive Procedure : Lipase test : Qualitative test to determine the presence/absence of lipase (enzyme that hydrolyzes triglycerides (fatty acids/lipids))
4. Use a sterile loop to inoculate the culture onto spirit blue plates
5. Streak colony across plate, incubate overnight at 37 ℃.
6. Next day, examine sample for precipitants -Look for zone of clearance: indicates lipase is present and is changing the pH of media

• B. subtillisLipase positive Notes: Test Positive Result Negative Result Oxidase Purple No color change Catalase Bubbles present No bubbles Coagulase Fibrin aggregates present No precipitant Lipase Reduction in color (zone of clearance) No change

Results: Oxidase Test: (differential test of 2 gram NEG bacteria) ▪ E.coli- oxidase negative (no change) ▪ Pseudomonas- oxidase positive (turned bluish, yellow, green) Catalase Test: (differential test of 2 gram POS bacteria) ▪ S.aureus: catalase positive (bubbles formed) ▪ Streptococcus: catalase negative (no bubbles formed) Coagulase Test: (differential test of 2 gram POS, Staph bacteria) ▪ S. aureus: coagulase positive: cloudiness, no movement (coagulated) ▪ S. epiderminus: coagulase negative: no change, no clotting

3. While removing loop, streak slanted side of agar with loop and discard
4. Grow at 37 ℃ overnight
5. Examine samples next day API Test (Analytical profile index): Broad multi-panel test used for rapid characterization of bacterial species (multiple assays)
6. Fill each tube with bacterial culture.

• Fill CIT, VP, and GEL wells entirely
• Add mineral oil to ADH, LDC, ODC, H2S, and URE wells (anaerobic)

2. Add 5 mL distilled water into tray, add API strip and cover with lid
3. Incubate at 37 ℃ overnight
4. Examine samples next day Notes: -Indole test (1): ▪ Indole negative = yellow color ▪ Indole positive = red/pink orange color -Indole test (2): -Cloudiness indicates the culture has grown ▪ Bright pink band indole was produced (strong +) ▪ Light pink band indole was produced but weaker (weak +) -aids us in knowing bacterial physiology spore formation/drug resistance: if they are able to breakdown tryptophan into indole more resistant to drugs (antibiotics) and survive harsh environments (spore formation) -TSI test ▪ pH indicator slants are red; stays red= negative

▪ if bacteria is able to ferment sugars (glucose, fructose, sucrose) the color will change; if hydrogen sulfide gas is produced: sample turns black ▪ Yellow to pink: positive test -API test: ▪ blue arrow indicates need for mineral oil to create an anaerobic environment Results: Indole test: ▪ E. Coli (gram NEG): positive for indole (pink) ▪ Pseudomonas (gram NEG): negative for indole (yellow) ▪ Salmonella (gram NEG): negative for indole (yellow) TSI test : ▪ E. Coli (gram NEG) yellow, solid slant, positive for fermenting glucose ( sugars); negative for hydrogen sulfide but did produce a gas ▪ Salmonella (gram NEG) yellow slant in bottom positive for fermenting glucose; turned black in top of slant positive for producing hydrogen sulfide (ferments all 3 & hydrogen sulfide gas) API test : Test Sample #1 Sample # ONPG Positive Negative ADH Negative Positive LDC Positive Negative ODC Negative Negative