Introduction:
Decontamination of Lab Coat: if contaminated with bacterial culture, spot disinfection or sterilization in autoclave
Discards:
1. Paper waste: garbage
2. Petriplates, disposable gloves: biohazard
3. Glass slides, broken glass, Pasteur pipettes, swabs, toothpicks: broken glass discard
4. Pipettes: large cylinder Tolley, tips down (do not place used on bench)
5. Glassware: discard tolleys
6. Culture: never in sink, place on tolley
Spilled Bacterial Cultures:
1. Broken glass: do not pick up, notify TA
2. No broken glass:
• cover spill with paper towel
• disinfect onto outer edges of spill: work towards the centre to minimize the amount of
aerosols produced (bacteria in air)
• allow to sit for at least 1 min
• gather up soaked towels: discard garbage
• disinfect again with fresh paper towels + discard in garbage
Contamination of Body:
• skin: wash with alcohol, then soap and water
• eyes: rinse with water from tap/eye wash station. Avoid rubbing eyes
• mouth: rinse copious amounts of water and report to TA
• cover cuts or wounds on hands or fingers: bandage and glove
• clothes: spot disinfected with alcohol, dried, then washed with detergent + bleach.
Fire Safety: Turn off Gas, always light burner with strikers
Bunsen Burner:
• Platform: allows you to turn off flame without turning off gas.
• DOWN: gas will flow through unimpeded, sterilization flame. UP: pilot flame is lit, never use pilot
flame for sterilization
Workplace Hazardous Materials Information System: WHMIS, a system for safe management of hazardous
materials. Labs are considered a workplace. Use of cautionary labels and MSDS.
1) Cautionary Labels: controlled products. Must be placed on containers obtained by suppliers and
dispensed in the workplace
a) Supplier Labels: on controlled products
▪ Product identifier: chemical name
▪ Supplier Identifier: name and address
▪ MSDS: material safety data sheets, controlled products, biohazardous materials. Provide
information on chemical hazard data, control measures, handling, storage, disposal and
emergency.
▪ Hazard symbol
2
, ▪ Risk phrases: nature of hazards
▪ Precautionary measures
▪ First aid measures
b) Workplace Labels: in a workplace container, product name, precautionary measure and reference to
MSDS availability
3
, Lab 1 – Compound Light Microscope
Compound brightfield microscopes:
▪ Compound: use two sets of lenses to magnify specimen: ocular and objective lens
▪ Total Magnification of Specimen = Ocular Magnification x objective magnification
▪ Brightfield: the image of the specimen appears against a bright, illuminated background. Important for
observing colored specimens, like stained slides. Con: not ideal for colorless specimens (yeast +
bacteria)
Phase Contrast Microscope: contains a special condenser to increase the contrast bw the specimen and
background. Allows you to observe live, unstained specimens and motile organisms and endospores
Electron Microscopes: examine cells and viruses at much higher magnifications. Electron beam has shorter
wavelengths than visible light, allows for higher resolution
Know Parts + Functions of the Microscope:
1. Oculars: lens magnifies 10x, clean with Kimwipe
▪ Binocular: has 2 oculars, one for each eye
2. Nosepiece: holds multiple objective lens
3. Objectives: lens magnifies 10 to 100x.
▪ Magnifications: 10x, 40x, 100x
▪ Black ring on the bottom: 100x
▪ Clean all, especially oil: Kimwipe
▪ End: 40x above the opening in the stage, nothing should be sticking out
4. Stage and Slide holder: hold specimen, ensure at the end slide holders are at centre, nothing sticking out
and stage should be as far down
5. Condenser:
▪ a lens that focuses the light from your light source to illuminate the specimen through a
concentrated beam.
▪ Should always be positioned in the up position using a lever, as close to the stage for
best illumination.
▪ Note: if you find yourself focusing on the condenser, blue grainy, move 1mm down. If you move
lower, not have optimal light to view specimen.
6. Iris Diaphragm:
▪ allows you to control the width of the beam of light passing through the condenser.
▪ If iris is open: the full bean is allowed to pass through, allowing you to see colors. Keep fully open
for colored specimen. Ex: stained slide, green algae
▪ If iris is closed: beam is narrowed, interfere with ability to see colors. But if closed can improve
contrast, between colorless cells and colorless background. For unstained, colorless specimen, it
should be almost completely closed.
▪ As you move lever to the left: light increases
Figure: On the left, baker’s yeast, viewed at 400x magnification with iris diaphragm wide open.
Light passing through the iris has washed out the field of view leaving the cells barely visible. On
the right is the same specimen, but this time the iris has been closed to increase contrast.
4
, 7. Fine and Course Focus Knobs: move stage closer/farther
▪ Course Focus: large knob, bring specimen into few on low power
▪ Fine focus: higher objectives
8. Light source and power cords: light source control knob control brightness of image
Oil Immersion Lens/Objective:
▪ Using 100x objective: need a drop of immersion oil on slide
▪ Immersion oil: has the same refractive index as glass, which makes sure that light passing through the
specimen enters the objective and prevents light from bending and is not refracted away = increases amount
of light that enters the objective = better resolution
▪ Purpose: increase resolving power of microscope by changing the refractive index of the space bw
the objective and specimen
▪ Refractive Index: measure of the way light travels through a medium. When light passes from a
material of one refractive index to material of another it bends = reducing resolution upon magnification
▪ Important for viewing: bacteria strands, since it’s so small, dead and immotile organisms.
1) Focus first at 400x magnification
2) Move the 40x objective out of place, add a small drop of immersion oil onto the slide/coverslip,
slide directly to the 100x objective.
3) Do not adjust the slide. Since the objectives are parfocal, the specimen should remain in focus at
the new magnification. You may only needed to adjust the fine focus slightly
Grease Pencil: draw a line beside the specimen, to allow you to easily focus on the marking.
5
Decontamination of Lab Coat: if contaminated with bacterial culture, spot disinfection or sterilization in autoclave
Discards:
1. Paper waste: garbage
2. Petriplates, disposable gloves: biohazard
3. Glass slides, broken glass, Pasteur pipettes, swabs, toothpicks: broken glass discard
4. Pipettes: large cylinder Tolley, tips down (do not place used on bench)
5. Glassware: discard tolleys
6. Culture: never in sink, place on tolley
Spilled Bacterial Cultures:
1. Broken glass: do not pick up, notify TA
2. No broken glass:
• cover spill with paper towel
• disinfect onto outer edges of spill: work towards the centre to minimize the amount of
aerosols produced (bacteria in air)
• allow to sit for at least 1 min
• gather up soaked towels: discard garbage
• disinfect again with fresh paper towels + discard in garbage
Contamination of Body:
• skin: wash with alcohol, then soap and water
• eyes: rinse with water from tap/eye wash station. Avoid rubbing eyes
• mouth: rinse copious amounts of water and report to TA
• cover cuts or wounds on hands or fingers: bandage and glove
• clothes: spot disinfected with alcohol, dried, then washed with detergent + bleach.
Fire Safety: Turn off Gas, always light burner with strikers
Bunsen Burner:
• Platform: allows you to turn off flame without turning off gas.
• DOWN: gas will flow through unimpeded, sterilization flame. UP: pilot flame is lit, never use pilot
flame for sterilization
Workplace Hazardous Materials Information System: WHMIS, a system for safe management of hazardous
materials. Labs are considered a workplace. Use of cautionary labels and MSDS.
1) Cautionary Labels: controlled products. Must be placed on containers obtained by suppliers and
dispensed in the workplace
a) Supplier Labels: on controlled products
▪ Product identifier: chemical name
▪ Supplier Identifier: name and address
▪ MSDS: material safety data sheets, controlled products, biohazardous materials. Provide
information on chemical hazard data, control measures, handling, storage, disposal and
emergency.
▪ Hazard symbol
2
, ▪ Risk phrases: nature of hazards
▪ Precautionary measures
▪ First aid measures
b) Workplace Labels: in a workplace container, product name, precautionary measure and reference to
MSDS availability
3
, Lab 1 – Compound Light Microscope
Compound brightfield microscopes:
▪ Compound: use two sets of lenses to magnify specimen: ocular and objective lens
▪ Total Magnification of Specimen = Ocular Magnification x objective magnification
▪ Brightfield: the image of the specimen appears against a bright, illuminated background. Important for
observing colored specimens, like stained slides. Con: not ideal for colorless specimens (yeast +
bacteria)
Phase Contrast Microscope: contains a special condenser to increase the contrast bw the specimen and
background. Allows you to observe live, unstained specimens and motile organisms and endospores
Electron Microscopes: examine cells and viruses at much higher magnifications. Electron beam has shorter
wavelengths than visible light, allows for higher resolution
Know Parts + Functions of the Microscope:
1. Oculars: lens magnifies 10x, clean with Kimwipe
▪ Binocular: has 2 oculars, one for each eye
2. Nosepiece: holds multiple objective lens
3. Objectives: lens magnifies 10 to 100x.
▪ Magnifications: 10x, 40x, 100x
▪ Black ring on the bottom: 100x
▪ Clean all, especially oil: Kimwipe
▪ End: 40x above the opening in the stage, nothing should be sticking out
4. Stage and Slide holder: hold specimen, ensure at the end slide holders are at centre, nothing sticking out
and stage should be as far down
5. Condenser:
▪ a lens that focuses the light from your light source to illuminate the specimen through a
concentrated beam.
▪ Should always be positioned in the up position using a lever, as close to the stage for
best illumination.
▪ Note: if you find yourself focusing on the condenser, blue grainy, move 1mm down. If you move
lower, not have optimal light to view specimen.
6. Iris Diaphragm:
▪ allows you to control the width of the beam of light passing through the condenser.
▪ If iris is open: the full bean is allowed to pass through, allowing you to see colors. Keep fully open
for colored specimen. Ex: stained slide, green algae
▪ If iris is closed: beam is narrowed, interfere with ability to see colors. But if closed can improve
contrast, between colorless cells and colorless background. For unstained, colorless specimen, it
should be almost completely closed.
▪ As you move lever to the left: light increases
Figure: On the left, baker’s yeast, viewed at 400x magnification with iris diaphragm wide open.
Light passing through the iris has washed out the field of view leaving the cells barely visible. On
the right is the same specimen, but this time the iris has been closed to increase contrast.
4
, 7. Fine and Course Focus Knobs: move stage closer/farther
▪ Course Focus: large knob, bring specimen into few on low power
▪ Fine focus: higher objectives
8. Light source and power cords: light source control knob control brightness of image
Oil Immersion Lens/Objective:
▪ Using 100x objective: need a drop of immersion oil on slide
▪ Immersion oil: has the same refractive index as glass, which makes sure that light passing through the
specimen enters the objective and prevents light from bending and is not refracted away = increases amount
of light that enters the objective = better resolution
▪ Purpose: increase resolving power of microscope by changing the refractive index of the space bw
the objective and specimen
▪ Refractive Index: measure of the way light travels through a medium. When light passes from a
material of one refractive index to material of another it bends = reducing resolution upon magnification
▪ Important for viewing: bacteria strands, since it’s so small, dead and immotile organisms.
1) Focus first at 400x magnification
2) Move the 40x objective out of place, add a small drop of immersion oil onto the slide/coverslip,
slide directly to the 100x objective.
3) Do not adjust the slide. Since the objectives are parfocal, the specimen should remain in focus at
the new magnification. You may only needed to adjust the fine focus slightly
Grease Pencil: draw a line beside the specimen, to allow you to easily focus on the marking.
5
