Portage Learning
BIO 171- Microbiology
Lab Notebook
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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
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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
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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. [Show Less]