Microscopes & Micropipettes

Cell Biology Lab Week One Review

What information should you add to your lab notebook?

Protocols, Data, Observations

What should be recorded at the top of the page of the lab notebook?

Day, Month, & Year

Whenever possible in your lab notebook you should?

Name things, quantify thing, and ALWAYS specify units.

How do you know if your notebook has sufficient information?

Your notebook should contain detailed descriptions, clear data, and comprehensive observations that allow others to replicate your experiments with only your notebook and without further explanation. .

What is a compound light microscope?

A microscope with multiple lenses and relies on light for the magnification of the specimen.

What types of microscopes are used in Auburn’s Cell Biology Lab?

Brightfield and Epiflourescence microscopes are utilized.

Typically what is the magnifcation of the ocular lens?

10X

What is the arm of the microscope?

The arm of the microscope is the part that connects the base to the head and supports the body tube. It is used for carrying the microscope and often holds the focusing mechanism.

What is the revolving nosepiece of the microscope?

Rotating disc with ocular lenses attached

What is the Objective Lens in a microscope?

The objective lens is the primary lens that gathers light from the specimen and focuses it to create an image. It comes in different magnifications, allowing for detailed viewing of the sample.

What is the stage of a microscope?

The platform that supports a specimen and is moveable in three directions

What is the base of a microscope. ?

Connects to the microscope arm and contains the light source

What is the light source of a microscope?

Typically a halogen or tungsten bulb.

What is the condensor of a microscope?

A lens system that focuses light onto the specimen, improving illumination and contrast.

What is the iris diaphragm?

The regulator for the amount of light that goes through the conductor.

What are the adjustment knobs of the microscope?

Moves the stage up or down (z-axis)

What are the coaxial stage controls of a microscope?

Moves the stage along the x and y axis

What is the magnification of a microscope?

The numerical ration of the virtual image of an object to the real image; typically expressed in a fold magnification.

What is resolution of a microscope?

The ability of a lens or a collection of lenses to distinguish two objects as separate.

What is the resolution of a light microscope?

200 nm

If you adjust the eyepieces of a microscope what are you adjusitng?

the interpupillary distance, allowing for a more comfortable viewing experience.

When is the ONLY time to use coarse adjustment on a microscope?

When using the lowest power objective lens. (4X and 10X)

What must you watch when using a High Powered objective?

the working distance to avoid damage to slides or lenses.

What does it mean for a lens to be parfocal?

It means that the lens remains in focus when switching between different objective lenses, minimizing the need for refocusing.

As you increase the objective power, …

the lamp needs to be brighter

True or False: Most of the time the diaphragm is wide open and the condenser as close to the stage was possible.

True

When preparing to use a microscope what are the steps?

1. Place microscope on top of workbench, facing you

2. Remove protective cover

3. Unwind Power Cord, do not plug in

4. Using coarse adjustment knob, lower stage to bottom of range

5. Use lens paper and clean all lenses

6. Plug in power cord and turn on power.

Prior to ending a microscope session what should you do?

1. Turn off power

2. Unplug Cord

3. Clean all Lenses

4. Use Paper towel to clean microscope base of spills

5. Use coarse adjustment and lower stage

6. Snap lowest power objective into optical view

7. Neatly wrap cord around base

8. Cover microscope

9. Stow microscope on cabinet

What is a consumable?

An item that is used once and then dicarded

What is the first step to reusing a slide?

Dispose of the coverslip in the glass waste container

What is the second step to reusing a slide?

Use Kim-wipe and wope both sides of the slide

What is the third step to reusing a slide?

Spray slide with 70% alcohol

What is the fourth step to reusing a slide?

Wipe dry with new Kim-Wipe

What is the fifth step to reusing a slide?

Allow slide to drycompletely before storing.

What are the advantages of doing a live mount on a microscope?

allow observation of organisms in their natural state, enabling the study of movement, behavior, and physiological processes that are not visible in stained or preserved specimens.

What are the disadvantages of live mounts?

The specimen deteriorates quickly and there is a very low contrast. Additionally, live mounts may introduce artifacts due to movement, and it can be difficult to focus on smaller organisms.

What is used to fixate a specimen?

By freezing or using chemicals like alcohols, formaldehyde, or acetone, specimens can be preserved for observation.

Why do we add stains to fixated specimens?

It allows us to view specific macromolecules like proteins, polysaccharides, DNA, RNA, Lipids, or collagen.

What does the stain being used in todays lab bind to?

Chitin, a major component of fungal cell walls

What is Lactophenol Cotton Blue (LPCB)?

? It is a staining solution used in microscopy that helps visualize fungal structures by staining chitin in cell walls.

In LPCB what deos the phenol do?

Phenol acts as a preservative and helps to clear the tissue.

In LPCB what does lactic acid do?

It is the Fixative

In LPCB what does cotton blue do?

a dye that stains chitin, making fungal structures more visible under the microscope.

What is a micropipettor?

a laboratory instrument used to accurately measure and transfer small volumes of liquid, typically in the microliter range.

What is the volume of a P-10 micropipette?

It typically has a volume range of 0.5 to 10 microliters.

What is the volume of a P-100 micropipette?

It typically has a volume range of 10 to 100 microliters.

What is the volume of a P-1000 micropipette?

It typically has a volume range of 100 to 1000 microliters.

What is the rule of thumb of the micropipette range?

Do not move volumes thatvare less than 10% if max volume

How do you draw fluid into the tip of a micropipette?

1. using your thumb, press control button down, until first stop; hold control button in place

2. Submerge the tip under fluid

3. Let control button go slowly

4. Remove tip and scrap tip against the wall of the container to remove excess liquid.

How do you dispense the fluid from the tip of a micropipette?

1. Introduce fluid to new contianer

2. Press control button until firsat stop; DO NOT RELEASE BUTTON

3. WHILE HOLDING CONTROL BUTTON, remove tip and scrap lip of container

4. Slowly release control button.

After dispensing the fluid in a micropipette what do you do?

1. Eject tip into waste container with ejection button

2. Grab a new tip between reagents

What should you not do with a micropipette?

1. Never lay the micropipette sideways

2. never adjust the volume more than the max

3. never let the plunger “snap back”

4. never immerse the barrel in fluid, only the tip.

Wha tis a 96 well Plate?

A 96 well plate is a flat plate with 96 small wells used for various laboratory assays and experiments, allowing for multiple samples to be tested simultaneously. Make sure to always have a blank sample

What is a spectrophotometer?

A device that allows specific wave length of electromagnetic radiation through a liquid sample and precisely quantifies the proportion of the radiation that passed through the sample.

What is the first part of a Spectrophotometer?

The light source, which emits electromagnetic radiation at specific wavelengths.

What is the second part of a Spectrophotometer?

the collimetor which is the lens

What is the third part of a Spectrophotometer?

The monochromator, which selects specific wavelengths of light for analysis.

What is the fourth part of a Spectrophotometer?

Wavelength selector

What is the fifth part of a Spectrophotometer?

The solution sample

What is the sixth part of a Spectrophotometer?

The photodetector, which measures the intensity of light, and the display.

What is Absorbance?

Absorbance is a measure of the amount of light absorbed by a solution at a specific wavelength. It is calculated as the logarithm of the ratio of incident to transmitted light.

How does a spectrophotometer work?

A beam of light passes through a sample and a reading of the intensity of the light is taken before and after passing through. The difference determines the absorbance which can be used to determine the concentration.

What wave length of light do we use to find the concentration of S. Cerevisae in a solution?

Typically, 600 nm is used.

What are the specific characterisitcs shared by all model organisms?

• Easy to maintain a thriving population in a lab

• Small enough to not require much living space

• Inexpensive to maintain

• Has a short life cycle

• Easily manipulated genetically

• must be able to generate economically usefule results.

What is Saccharomyces cerevisiae

A single celled eukaryotic organism that is. amember of the Fungi kingdom and is a model organism due to its simple cell cule, mitosis and meiosis.

In S. cerevisiae how does Asexual reprduction work?

Asexual reproduction occurs primarily through budding, where a new cell develops from the parent cell and eventually detaches to form an independent yeast cell. Using mitosis and cytokinesis

What are the mating types for sexual reproduction of S. cerevisiae?

There are two mating types, MATa and MATα, which can fuse to undergo sexual reproduction and form a diploid cell.