Module 1 Practical Review Guide (Micropipettes, pH, Microscopy, Spectrophotometry, Protein Assay, Transmitted Light Microscopy)

Flashcards covering key concepts from the lecture notes on micropipettes, pH calibration, transmitted light microscopy, serial dilutions, spectrophotometry, protein concentration assays, and basic embryology.

Which micropipette size should you use for pipetting 14 μL, 184 μL, and 670 μL, respectively?

14 μL → P-20; 184 μL → P-200; 670 μL → P-1000.

What are the general steps to use a micropipette to pipette a liquid?

1) Ensure you have the right size; 2) Set the volume with the dial; 3) Attach the correct tip securely; 4) Depress the plunger to the first stop and withdraw liquid; 5) Move to the target and depress past both stops to dispense; 6) Eject the tip into a waste/sharps container using the white button.

If you pipette 10 μL of liquid onto a tared weigh boat, what should the measured weight be and why?

Approximately 0.010 g, because 10 μL of water has a mass of about 0.01 g (density of water is 1 g/mL, so 10 μL = 0.01 mL).

When would you use a pipet-aid/serological pipet versus a micropipette (Pipetman)?

Use micropipettes for volumes less than 1000 μL (1 mL); use pipet-aid/serological pipettes for volumes greater than 1 mL.

When should you switch tips during pipetting?

Whenever you are dispensing a new fluid into a common mixture.

How do you use the analytical balance to weigh a liquid sample?

Open the balance door, place the weigh boat on the pan, close the door, press tare; pipette the desired amount into the boat, close the door, wait for the value to stabilize, and record.

What is the difference between accuracy and precision?

Accuracy is how close a measurement is to the true value; precision is how reproducible or consistent repeated measurements are.

How is percent error calculated?

% error = |observed value − true value| / true value × 100%.

How do you calculate pH?

pH = −log[H+].

How do you calibrate a pH meter?

Place the sensor in pH 4 solution and standardize (STD); when the display is stable, press STD again; rinse; repeat with pH 7 and pH 10 solutions; rinse before reading unknown sample.

What pH does most human/animal physiology typically stay at?

About 7.4.

How does blood help maintain pH?

Blood uses buffers such as bicarbonate and phosphate, along with charged molecules like serum albumin, to resist pH changes.

What are the different parts of the microscope slide orientation question answered in the notes?

The frosted side is the top of a microscope slide; the non-frosted side is the bottom.

What is Diff-Quik and how does it work (in general)?

Diff-Quik is a cytochemical stain that involves three steps: a fixative reagent, eosinophilic staining, and basophilic staining.

Which objective should you start with when viewing something under the microscope?

Begin with the lowest objective, typically 4x.

What is Permount and what is it used for?

Permount is a mounting fluid (resin) used to affix the coverslip to the slide and reduce optical imperfections.

What is the size difference between epithelial cells and bacteria?

Epithelial cells are much larger; bacteria are about 10x smaller than typical plant/animal cells.

What is the difference between swabbing only your cheek and swabbing both your cheek and your teeth?

Cheek only yields mainly epithelial cells; cheek and teeth yields cells plus particulates like food, saliva, and many more bacteria.

What is the role of the cells on the surface of the oral cavity in preventing infection, and how do they help prevent bacteria from entering the body?

Bacteria adhere to sugars on the surface of the oral cavity, cells containing these sugars shed off and are swallowed; bacteria are then destroyed by stomach acid.

What are serial dilutions and what are they used for?

Serial dilutions are stepwise dilutions used to establish the range in which an instrument can reliably operate and to determine concentrations of unknowns.

What are the step-by-step basic procedures to calibrate and use a spectrophotometer?

Turn on and set to 562 nm; zero transmittance with a blank; place the blank cuvette and adjust transmittance to 100; remove the blank, insert sample, and read absorbance.

What is a spectrophotometer used for?

Measuring the intensity of light that passes through (transmittance) or is absorbed by (absorbance) a sample.

What is the relationship between transmittance and absorbance?

They are inversely related: as transmittance increases, absorbance decreases, and vice versa (A = −log(T); T = 10^(−A)).

Why do we use absorbance instead of transmittance in many analyses?

Absorbance vs concentration is linear, making it easier to analyze data and create a line of best fit.

What does BCA stand for and how does the BCA assay work, including why we incubate?

BCA stands for Bicinchoninic acid. It binds Cu+ ions, causing a color change proportional to protein amount; incubation allows complete chelation and color development.

What does BSA stand for, and what is its role relative to BCA?

BSA stands for Bovine Serum Albumin. It is the protein used to create standards for the assay; BCA is the reagent that generates color with protein.

What wavelength should be set on the spectrophotometer for the BCA assay, and why?

562 nm, because the BCA-Cu+ complex absorbs strongly at this wavelength.

How can you use the standard curve in a protein concentration assay?

Plot concentration versus absorbance to create a standard curve; use the unknown absorbance to interpolate its concentration from the curve.

What is the difference between in vivo and in vitro?

In vivo means the study is conducted in a living organism; in vitro means the study is conducted outside a living organism, in a test tube or dish.

How are tissues/embryos prepared for microscopic examination (general workflow mentioned in pre-lab)?

Harvest from animals; chemically fix to preserve; embed in paraffin wax; cut into thin sections; float on water; mount on slides; melt/remove wax; stain; air-dry; coverslip.

What are the sagittal, frontal/coronal, and transverse planes?

Sagittal: plane from the forehead to the chin (z-axis). Frontal/Coronal: plane from one ear to the other (y-axis). Transverse: horizontal plane around the stomach/gut (x-axis).

How do early-stage and late-stage mouse embryos differ?

Early-stage embryos are smaller with less complex structures; late-stage embryos are larger and more complex.

Why might some parts of a mouse embryo undergo apoptosis during development?

To remove cells and sculpt structures, enabling the formation of more intricate anatomy (e.g., shaping digits).