Lab 2 Flashcards
Identify parts of light microscope
a. Ocular Lens (Eyepiece). Magnification value of 10X. The right ocular has a fixed focus, the left ocular has adjustable focus to account for differences between the user’s eyes.
b. Specimen Pointer: Pointer is mounted within ocular tube. Use the stage clip adjustment knobs to place the object of interest at the end of the pointer.
c. Binocular Head: Two ocular lenses and tubes. The distance between the ocular lenses is adjustable. To use, first focus on a specimen with the 4X objective using the right ocular lens, then adjust the focus of the left ocular lens. The head can rotate, if needed.
d. Arm. Use the handle at the top of the arm when transporting the microscope.
e. Revolving Nosepiece. Fixture for the objective lenses. Rotation of the nosepiece moves the desired objective into place. Use the rubber grip ring on the nosepiece to move the objective lenses, do not use the objective lenses as a handle. The nosepiece will ‘click’ into position for each objective.
f. Objective Lenses. There are four objectives on the JH 216 scopes. A 4X objective (scanning objective) lens with a red ring; a 10X objective (low power objective) lens with a yellow ring; a 40X objective (high dry objective) lens with a blue ring; and a 100X objective (oil immersion lens) lens with two rings – one black ring and one white ring.
g. Power Switch. Used to turn the lamp (light source) on and off. When the rocker switch on the arm is pressed to ‘0’, the lamp is off; when the switch is pressed to ‘1’, the light is on.
h. Light Intensity Knob. This knob on the arm of the scope near the power switch is used to control the intensity of the light coming from the light source. Turn to the lowest setting before turning the microscope on/off. The light intensity should be set to ~3.5 when first viewing a slide.
i. Y-Axis Stage Clip Knob. This knob controls front to back slide movement.
j. X-Axis Stage Clip Knob. This knob controls side to side slide movement.
k Coarse Focus Adjustment Knob: This knob moves the stage up or down very quickly. Use this knob ONLY with the 4X and 10X objectives.
l. Fine Focus Adjustment Knob: Turning the knob causes the stage to move up or down slowly. Always use this knob with the 40X and 100X objectives.
m. Lamp. Provides light for slide. There are no adjustments made directly to the lamp.
Base. Handle in front of base for use when carrying scope.
n. Iris Diaphragm Lever. This lever controls a set of shutters that alters the amount of light transmitted to the condenser lens. Use a setting of 0.5 for most applications; use a setting of 1.25 when using the 100X objective.
p. Condenser Lens Focuses and concentrates light on the specimen. The condenser lens should be set at or near its highest position for most applications.
q. Stage. The flat metal structure on which the microscope slide is placed. Note the opening in the stage that allows the light through.
r. Stage Clips. The mechanism that holds the slide in place. Use the small knob on the curved arm of the clips to pull the arm back to open the clips. Place the slide into the clips, then slowly release the arm so it does not crack the slide.
s. Condenser Height Adjustment Knob. This knob moves condenser (and iris diaphragm) up or down. This knob is on the left, directly under the stage. Use this knob to set the condenser lens at or near its highest position.
How to calculate magnification
power of objective lens * magnification of ocular lense
at PCC ocular lens is 10x
Describe the steps to bring a slide into focus using the oil immersion lens (100X objective).
- Focus with the 4X objective and center the object in the field of view - rough focus knob. Start with Iris diaphragm at 0.5
- Switch to the 10X objective, refocus, and re-center the object - rough focus knob
- Switch to the 40X objective, refocus, and re-center the object - fine focus knob
- Rotate the nosepiece halfway between 40X and 100X (do not go all the way to 100X yet).
- Add one drop of immersion oil onto the slide directly above the area you’re viewing.
- Carefully rotate the 100X objective into place so it contacts the oil. Increase intensity of light. Open iris diaphragm to 1.25
- Use only the fine focus knob to sharpen the image (do not use the coarse focus knob).
Describe the steps used to remove a slide from the stage.
- Rotate the 4X objective into position to prevent damage to higher-power lenses.
- Lower the stage to create space for removing the slide.
- Carefully remove the slide from the stage.
- Clean the 100X objective (if oil was used) by gently blotting with lens paper and cleaning solution.
- Turn down the light intensity to minimum before turning off the microscope.
- Wrap the cord around the guides.
- Store the scope in the cabinet, placing the cord on the outside.
Describe the microscope cleaning and storage procedures.
Clean the microscope
- Routine cleaning is not required for the 4X, 10X, or 40X objectives.
- Before storage, clean the 100X objective: Gently blot it with dry lens paper to remove any immersion oil. Then use lens paper and a lens cleaning solution to remove any remaining residue.
Store the microscope
- Rotate the nosepiece so that the 4X objective is in position.
- Lower the stage.
- Turn down the light intensity to its minimum.
- Wrap the microscope cord around the guides.
4 cell shapes
Bacillus: Rod-shaped; cells are longer than they are wide.
Coccus: Spherical or round-shaped; appear like small berries.
Spirillum: Rigid, S-shaped or open spiral; appears as a loose helix.
Spirochete: Flexible, tightly coiled spiral; resembles a corkscrew.
List / Describe cellular arrangements
Arrangement determined by plane of cellular division - cells remain attached after division. Cellular arrangements are not common. Seen most commonly in Coccus and bacillus shapes
Diplo-: Seen in both cocci and bacilli. Cells arranged in pairs -> diplococcus (paired cocci) and diplobacillus (paired bacilli).
Strepto-: Seen in both cocci and bacilli. Cells arranged in chains. -> streptococcus (chain-forming cocci) and streptobacillus (chain-forming bacilli).
Staphylo-: Seen only in cocci. Cells arranged in grape-like clusters. -> staphylococcus.
Tetrad: Seen only in cocci. Cells arranged in groups of four in a square.
Sarcina: Seen only in cocci. Cells arranged in a cube of eight (2 by 2 by 2).
Palisades: Seen only in bacilli. Cells arranged side by side like a fence.
Describe and list flagellar arrangements
A Monotrichous: A single flagellum located at one end of the cell.
B. Lophotrichous: A tuft (cluster) of flagella at one end of the cell
C Amphitrichous: One flagellum at each end of the cell.
D Peritrichous: Multiple flagella distributed over the entire surface of the cell.
Describe the bacterial capsule.
capsule is a layer of complex carbohydrates that covers the cell. It is visualized using a capsule stain, which involves two stains:
- One stain is repelled by the capsule and stains the background.
- The second stain penetrates the capsule and stains the bacterial cell.
- The capsule appears as a clear halo surrounding the cell.
Describe making a smear
- Label slide with wax pencil - easiest to use first letters of genus and species of organism.
- Put on chemical safety goggles and gloves
- Use a loop to place a small drop of DI water on the slide. Use loop to catch a drop of water from the DI squeeze bottle
- Transfer bacteria: Aseptically add a small amount of the colony to the water (for solid media). Slowly and gently move the loop in the water to add cells - stop when the water becomes cloudy but remains translucent. Make sure to sterilize loop after
- Spread the mixture into a thin, even smear on the slide.
- Air dry completely. (can now remove gloves / goggles)
- Heat-fix the slide by holding the slide (with a clothespin) over the mouth of the incinerator, smear-side up, for 30 seconds
Store
1. place smear on piece of paper towel
2. gently fold the paper towel to cover the slide
3. use a piece of tape to secure the paper towel envelope
- place smears in designated drawer
Smear should be thin and even: Thick smears may not stain properly and can make it difficult to view individual cells.
Slide must be completely dry before heat-fixing: If not dry, heating can cause cells to rupture or create artifacts.
Proper heat-fixing: Kills bacteria, adheres them to the slide, and preserves cell shape. Overheating can distort or destroy cells.
Describe the purposes/functions of heat-fixing in smear preparation
- Kills the bacteria, making them safe to handle.
- Adheres the bacteria to the slide, so they don’t wash off during staining.
- preserves cell shape and arrangement by slightly denaturing proteins.
the general function of staining in microscopy
- Enhance contrast between the bacterial cells and the background.
- Allow visualization of cell morphology and arrangement, which would otherwise be difficult to see since most bacteria are transparent under brightfield microscopy.
Compare and contrast a simple stain and a complex stain
Simple stain
- Uses only one stain.
- All cells take up the same color.
- Purpose is to reveal cell shape, size, and arrangement.
Example: methylene blue stain.
Complex (differential) stain
- Uses two or more stains.
- Different cell types or structures stain differently.
- Purpose is to distinguish between types of bacteria or cell components.
Example: Gram stain.
