Portage Learning
BIO 171- Microbiology
Lab Notebook
Lab Notebook Bookmarks (click to navigate):
Lab 1 Noteboo k
Lab 2 Notebook
Lab 3 Notebook
Lab 4
... [Show More] 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.
Results: 1. Staphylococcus aureus: Gram(+) clusters, 2. Escherichia coli: Gram(-) rods, 3. Bacillus subtilis: Gram(+) rods, 4. Pseudomonas aeruginosa: Gram(-) rods, 5. Streptococcus: Gram (+) cocci chains.
Lab 4 Notebook
Back to Home Page
04 Title: 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(each sample only goes through the next sample once)
**Invert plate in between each step!!**
1.Using sterile loop spread culture in area #1
2. Using NEW sterile loop drag through the end of area #1 ONCE
3. Using a NEW sterile loop drag through the end of #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 Celsius
[Non-Selective Agar]
Quadrant Growth: Rapid test for multiple isolates( none of the samples touch)
**Invert plate in between each step!!**
1. Using sterile loop spread unknown culture A in area #1
2.Using NEW sterile loop spread unknown culture B in area #2
3.Using NEW sterile loop spread unknown culture C in area #3
4.Using 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 Celsius
Notes:
• Non-Selective Media:Can be Gram(+) or Gram (-) Important for the expansion of unknown bacteria( ex: LB-most common, light yellow. Blood agar-red, contains red blood cells-important nutrient source for plate, derivative of blood agar plate that blood cells are removed which gives you TSAYE-Triptic soy agar yeast extract complex general purpose media that contains the triptic soy and yeast as the nutrient source for the plate which are the foundational components of a blood ager plate- TSAYE + Blood extract= Blood agger plate.)(ex: Ld agar: non-selective but can be used in ampicillin, this can then differentiate and select against different bacteria that have resistance to different drugs.)
• Selective-Media: Used to eliminate irrelevant bacteria from mixed cultures: Selective media gram(+/-) may inhibit the grow of one but may promote the growth of another
(ex: MacConkey media-red, looks a lot like the blood media but is very cloudy. Used for selective media because only gram(-) will grow on it and inhibits growth of gram(+).
SMac-Sorbitol MacCokney-pink and translucent, substitutes lactose in MacCokney agar with sorbitol, gram(-), used to determine between ecoli k12(standard) and ecoli 0157 (potent pathogen) can also be a differential media.
• Differential Media: Used to distinguish between species of the same group
Invert plate when not in use because your bacteria will stick to the agar but remain contamination free. (ex: SMac-Sorbitol MacCokney-pink and translucent, substitutes lactose in MacCokney agar with sorbitol, gram(-), used to determine between ecoli k12(standard) and ecoli 1057 (potent pathogen) can also be a selective media. Eosin methylene blue(EMB) gram (-) bacteria-dark red but bluish/green sheen, can be used as a selective and differential media. Strong lactose fermentation ability the colonies will turn green on the plate, if not a lot of lactose is present then the colonies turn pink, if the bacteria has no enzymes capable of breaking down lactose then there will be no color change in the bacteria. Ecoli on EMB give off a green metallic color)
Results: Plate #1 Non-Selective Agar- 4 Phase Dilution Streaking: Zone 1 growth into Zone 2, and Zone 2 into Zone 3 single colonies growths.
Plate #2 Non-Selective Agar Quadrant Growth: Zone 1 ecoli strain-grew very rapidly( very wide and thick), Zone 2 staph sample (uniform, long growth pattern, Zone 3 strep sample not as heavy of growth but still present( more opaque than other samples) Zone 4 pseudomonas strain- single colonies grew but not very many.
Plate #3 Non-Selective Agar on blood agar plate-Divided plate in equal quadrant Zone #1 staph appearance is a white film, large growth present, red part of agar has changed because of the lysis of the blood cells so the red agar appears clear. Staph demonstrates Beta hemolysis. Zone #2 ecoli dark appearance, large growth present, no lysis properties-non beta hemolysis Plate #4 Differential EMB plate Zone 1: gram (-) ecoli present, ecoli is able to ferment lactose, give green metallic color, Zone 2 gram(-) pseudomonas present, no color change, does not ferment lactose
Lab 5 Notebook
Back to Home Page
Title:
Objective:
Procedure:
Notes: [Show Less]