Portage Learning
BIO 171- Microbiology
Lab Notebook
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Lab 1 Notebook
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II01
Title: Introduction to Medical Microbiology
Objective: Cultivation of samples (growth conditions) and the equipment used in cultivation
Identification of samples (biochemical assays) and the tests that are available for identification.
Evaluation of samples (microscopy) involves the visualization and recognition of key
characteristics of the pathogens.
Procedure: Basic equipment used in the laboratory.
Cleaning (essential for working in a sterile environment)- An autoclave uses heat, pressure and
steam to sterilize samples or tools used. Autoclave uses a temperature of at least 125 degrees
Celsius.
Growing (to grow the bacteria and pathogens)- A Fixed incubator (at 37 degrees Celsius), and a
Shaker incubator (at 37 degrees Celsius) which shakes and rotates the culture in a swirling fashion
are used.
Visualizing- microscopy to visualize the bacteria.
Storing- Refrigerator at 4 C which slows the growth of bacteria and keeps for a long time.
General Lab safety-
Never eat or drink in the lab
Use PPE (personal protective equipment) such as Latex/Nitrile gloves, Thermal gloves, Eyewear
protection, Lab coat.
Never leave the lab while wearing PPE
Notes: General outline on how to set up a lab notebook for the course.
Results: Identification of basic lab equipment, general lab safety and how to setup a lab note.
Lab 2 Notebook
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Title: II02 INTRODUCTION TO MICROSCOPY
Objective: To identify and understand the basic components of a light microscope.
How to load and adjust a sample for viewing.
Procedure: Identifying different parts and functions of the components
Load sample slide on the stage of the light microscope
Select and differentiate the appropriate magnification.
Make necessary adjustments to ensure optimal sample visualization.
Notes:
Identifying different parts of the microscope and their function.
1. Eyepiece (ocular lens): To view the object or sample. It can be pulled apart or
compressed to fit both eyes.
2. Arm/neck: To move/carry the microscope with the other hand under the base.
3. Objective lenses: provide magnification of the sample. It has four different lenses (4x,
10x, 40x, 100x)
4. Stage: this is a flat surface for placing the sample. It has a clamp holder and stage clips
(which lay on the glass coverslip) to hold the samples in place.
5. Stage controls: It has two knobs. The top knob moves slide forward and backwards and
the bottom knob moves slide left and right.
6. Focus knobs: located on the side of the arm. The coarse adjustment is the outside knob
while the fine focus is the inner knob.
7. Iris/diaphragm: located below the stage and it determines the amount of light let into
the microscope.
8. Base: this is where the microscope is set to prevent introducing vibrations into the
imaging. It should be placed on a flat surface.
How to load samples for viewing
1. Place the sample on the stage and firmly secure the slide.
2. Determine the type of objectives to use, dry (4x, 10x, 40x magnification) or oil. The
oil requires a higher magnification (100x) and adding a drop of oil which helps with
light refraction for better imaging quality. Start on the lowest magnification if unsure
of the power to use.
3. Determine the intensity of light source. When it’s too bright, it causes saturation and
when it’s too dark, it causes low visibility. Control the amount of light entering the
sample.
4. Place the sample in the range of viewing. The top stage knob controls the stage
forward/backwards and the lower knob controls the stage left and right.
To indicate region of interest when viewing sample, rotate the ocular which controls the
pointer to point directly to the region of interest.
To determine the total magnification to be used. This involves the power of the objective
used and the power of the eyepiece.
Total magnification= power of objective X power of eyepiece
Results: Identification of the various components and functions of each component of the light
microscope.
How to load, view and adjust the sample.
How to determine the magnification to use and ensure proper visibility.
Lab 3 Notebook
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Title: II03 Mounting Techniques
Objective:
Microscopic examination of bacterial samples through various staining techniques.
Identify color and shape of given samples.
Procedures: Ensure all safety lab precautions
A. How to mount bacteria to a glass slide
Dry Mount
1.Ensure the glass slide is clean by using 70% ethanol and a Kimwipe to prevent contamination
and proper viewing under microscope.
2. Draw a circle area underneath the slide for easy location of specimen under the light
3. Apply organism with a sterile loop to the slide. If from culture, use sterile loop to spread onto
slide and 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
Wet mount is ideal for viewing the motility of an organism. Do not allow to dry out.
1. Clean the glass slide using 70% ethanol and a Kimwipe
2. Circle the area with a wax or hydrophobic pen to keep the water inside the ring
3. Apply organism to slide and ensure it stays wet. If from culture, use sterile loop to spread onto
slide and if picking from colonies, mix with a drop of distilled water to get a nice thin uniform layer of
the pathogen.
4. View under microscope
B. Gram Staining: helps to identify an organism.
1. Ensure a clean slide using 70% ethanol
2. Apply the pathogens to the slide
3. Allow to air dry to prevent excessive water on the slide
4. Fix the organism by sliding it through an open flame (heat fixing). Do not overheat the slide.
5. Flood the slide with crystal violet (a dark purple dye) for 30-60 seconds. Ensure to coat the area inside
the circle. Allow to rest for about a minute.
6. Rinse out with water
7. Flood with Gram’s iodine stain for 30-60 seconds and let sit for one minute.
8. Rinse out with water
9. De-colorize with alcohol (stains washing off the slide can be seen)
10. Rinse out with water
11. Counter stain with Safranin (a red or pink dye) for 30-60 seconds
12. Rinse out with water
13. Blot dry to get excess water off and examine carefully under the microscope [Show Less]