1
Q
What is the dispensable range of a P20 micropipette in uL and mL?
A
2 - 20 uL, 0.002 - 0.02 mL
2
Q
What is the dispensable range of a P200 micropipette in uL and mL?
A
20 - 200 uL, 0.02 - 0.2 mL
3
Q
What is the dispensable range of a P1000 micropipette in uL and mL?
A
200 - 1000 uL, 0.2 - 1 mL
4
Q
How man sig figs do we use in Bio 225?
A
2; ex. 5.3
5
Q
How do we pick the precision of sig figs if we are using multiple devices?
A
What ever device is least accurate is our deciding factor.
6
Q
Define a standard curve.
A
A standard curve is a generated graph that depicts the linear relationship between an independent (x-axis) and dependent variable. In Bio 225, this is usually the concentration of a substance (x-axis, independent) plotted against the absorbance (y-axis, dependent) using a set of standard solutions compared against a blank to find an unknown value.
7
Q
What is the role of the field iris diaphragm?
A
It controls the size of the visible field
8
Q
What is the role of the condenser?
A
It focuses light onto the specimen and slide
9
Q
What parts make up the condenser?
A
The condenser focus knob, the centering screws, and the condenser iris diaphragm.
10
Q
What is the role of the condenser focus knob?
A
Gives the optimal light at kohler illumination
11
Q
What is the role of the centering screws?
A
Centers the condenser light
12
Q
What is the role of the condenser iris diaphragm?
A
Reduces glare and increases contrast
13
Q
How does the depth of field of a microscope change as you increase your objective?
A
As you increase in objective power, you decrease in depth, so the closer you are to a sample the harder it is to make out the different layers there are to a sample.
14
Q
How does the working distance of a microscope change as you increase your objective?
A
As you increase your objective power, you decrease your working distance, which is the distance between the lens and the sample/slide.
15
Q
How does the field of view of a microscope change as you increase your objective?
A
As you increase your objective power, you decrease your field of view. (Field diameter)
16
Q
List the steps of Kohler Illumination in Bio 225.
A
- Turn on the light and place specimen on the stage.
- Use the X, Y, coarse and fine focus to focus the specimen.
- Close the condenser iris diaphragm completely by sliding the lever to the right.
- Close the field iris diaphragm until a small circle of light forms
- Centre the circle with the centering screws
- Sharpen the edges with the condenser focus knob
- Open the field iris diaphragm until the edges just disappear
- Open the condenser iris diaphragm until the light intensity is comfortable
17
Q
Define kohler illumination
A
How to achieve optimal illumination throughout the sample to provide the best resolution, contrast and image quality possible.
18
Q
What are some benefits to fluorescent microscopy?
A
It can be used to visualize parts of the cell too small to see on a normal light microscope by dying them; it does NOT increase resolution.
19
Q
How does a fluorescent microscope work?
A
Fluorescent dyes are applied to a sample, chosen due to their ability to bind to different parts of the cell. Some can take advantage of the negative charge of nucleic acids and bind to DNA, the nucleus, etc, or the ECM. They absorb a light that is then emitted and refracted off the dichromatic mirror at a longer wavelength.
20
Q
DAPI is a fluorescent dye; what part of the cell does it bind to and what sort of wavelength does it absorb/emit?
A
DAPI binds to A-T rich regions in DNA (nucleus) and is excited by UV light and emits a blue light.
21
Q
Rhodamine-Phallodin is a fluorescent dye; what part of the cell does it bind to and what sort of wavelength does it absorb/emit?
A
Rhodamine-Phallodin binds to actin (cytoskeleton) and is excited by 540 nm and emits a red light.
22
Q
What is the role of Rhodamine in Rhodamine-Phallodin?
A
Rhodamine binds to other chemicals and is excited by 540 nm and emits a red light.
23
Q
What is the role of Phallodin in Rhodamine-Phallodin?
A
Phallodin binds to actin and Rhodamine.
24
Q
What was the role of Paraformaldehyde (PFA) when it comes to visualizing cells on a fluorescent microscope?
A
PFA fixes cells and permeates the membrane so dyes can enter the cells to bind to different components.
25
How were the cauliflower mitochondria extracted?
Mechanical distruption, cell fractionation.
26
How was mechanical distruption performed?
Sand, cauliflower, motar and pestle
27
How was cell fractionation performed?
Differential centrifugation
28
What is the goal of differential centrifugation?
To separate each subcellular component by size, shape and density to extract the mitochondria.
29
How does differential centrifugation work?
Each component is separated by changing the effect of gravity on the sample by varying and increasing speeds to increase the centrifugal force. The particles will separate based on density, shape and size.
30
Provide the order of isolation of subcellular components during differential centrifugation
Nuclei > mitochondria, chloroplasts, peroxisomes, lysosomes > ER, PM, large ribosomes > small ribosomes > cytoplasm
31
How is succinate produced in the mitochondria
The oxidation of pyruvate during the TCA cycle.
32
SDH oxidizes succinate to...?
Fumarate
33
What is released when succinate is oxidized to fumarate?
electrons and 2 protons
34
Where does FAD get its electrons from to become reduced to FADH2
When succinate is oxidized to fumarate
35
What does the e carrier FADH transfer its electrons and protons to to become oxidized to FAD?
Ubiquinone
36
What is the terminal electron acceptor in the ETC
Oxygen
37
Sodium azide is an inhibitor of the ETC, what does it inhibit?
The final transfer of e to oxygen
38
How does DCIP become reduced to DCIPH2?
It accepts the electrons avaliable for uptake since sodium azide is inhibiting the final transfer to electrons to oxygen that usually takes place.
39
What type of inhibitor is malonate?
Malonate is a competitive, reversible inhibitor of SDH
40
How does malonate inhbit SDH
It blocks the active site of SDH, preventing the oxidation of succinate to fumarate and the subsequent reduction of FAD to FADH2
41
The MF-M could not be used to analyze SDH activity. Why?
Malonate has been thought to cause apoptic stimuli by increasing the concentration of ROS, which are detrimental to a cell's health. They can cause the mitochondria and cell to collapse, release the free e into space for DCIP to accept and reduce to DCIPH2 which is not a measure of SDH activity.
42
How can we indirectly measure SDH activity?
We indirectly measured SDH activity with DCIP. It is a blue dye that turns colourless when reduced to DCIPH2, and a colourmetric test can be performed on a spectrophotometer. It was reduced with the excess electrons produced by the inhibition of the ETC by sodium azide. The rate of SDH activity increases as a rate of decrease in DCIP conc over time.
43
Describe the function of the 'A" valve in a blowout pipette
A = air valve, press it and squeeze to expel any air in the peleus bulb.
44
Describe the function of the S value in a blowout pipette.
S = suction valve, press to suck up the required volume
45
Describe the function of the E value in a blowout pipette.
E = exit valve, press and squeeze to expel any excess liquid
46
How do you blowout a blowout pipette?
Cover the E end firmly and push on the center bulb while squeezing E
47
What are pasteur pipettes used for?
Mixing or non-measuring transferring of solutions
48
What is spectrophotometry used for?
Used to quantitatively determine the concentration of an unknown from a set of known standards and a standard curve by measuring the absorbance of light in a solution.
49
After centrifugation, why must samples be placed on ice?
Cells and organelles under go autolysis quickly following isolation and ice helps prevent this (or at least slow it).
50
What is a hemocytometer used for?
It allows you to count cells and find the concentration of the original cell stock culture prior to dilution.
51
What is the known volume of 5 hemocytometer squares?
0.05445 mm^3, or 5.445x10^-5 mL (/by 1000mm^3/mL)
52
What objective do you focus on for a hemocytometer, and what do you start counting on?
4x and then 40x
53
What is the acceptable range of cells counted in 5 squares?
50 - 200
54
What cells are not counted in a hemocytometer?
Cells outside the grid, on the border, or small daughter cells
55
List the basic steps for using a hemocytometer
1. Dilute original cell culture to desired concentration (usually 1/10 or 1/20 to get 50 - 200 cells)
2. Get hemocytometer, clean with kimwipe, vortex culture, and load 15 uL into a grid
3. Place on stage, focus on grid at 40X and then one square at 40X
4. Count number of cells in 5 squares - repeat if using multiple grids
5. Find the average number of cells in 5 squares and the dilution factor
6. Use the equation to find the stock culture concentration of cells/mL
56
What is the equation for cells/mL when using a hemocytometer
(av number of cells in 5 squares x dilution factor)/total volume of cells in 5 squares in mL
57
What is the purpose of spread plating?
To determine colony counts and back calculate the concentration of cells/mL in the original stock culture
58
How do you determine the TDF for a serial dilution?
TDF = stock culture concentration/required concentration of cells/mL
59
List the steps for a serial dilution, including the calculation steps
1. Determine your TDF
2. From the TDF, split into a) the 'weird numbers' and then multiples of 10 or 100. They should multiply together to give the TDF
- ie. if you have a TDF of 45000, split into 4.5 and 10000 = 100 x 100
3. Draw how many tubes you will need and list the TDF and dilution at each step
4. Determine how much isotonic saline will need to be added at each step, not below 10 uL and not above a total volume of 1.0mL
5. Determine how much of each culture will need to be added, not below 10 uL and not above a total volume of 1.0mL
6. Turn your bunsen burner on
7. Vortex culture and start diluting
8. Label plate with name, date, organism, section and incubation temp
9. Plate volume onto agar
10. Flame ethanol rod and wait to cool
11. Start in careful turning motions, 5 - 6x in middle, and then slowly make your way to the edges until all the agar and liquid is dry.
60
From the spread plate colony, how do you calculate the stock culture concentration?
Stock culture = (average number of colonies/volume plated) x (dilution factor/plating efficiency)
61
How do you calculate the plating efficiency?
Number of colonies/expected number of colonies
62
What are some possible human errors with spread plating?
- clusters/run on colonies
- spread plating technique was not performed properly, resulting in a density of colonies in the middle or too close to the edges
- if the ethanol rod was not cooled enough, some of the cells could have been killed
- if sterile technique was not used well enough, possible contaminants could have entered and grown on the agar, competing for nutrients
63
What are some possible biological errors with spread plating?
If the media is a complex general medium, the concentration of ingredients and the amount of each added are unknown. The agar may not be the optimized environment for the cells to grow.
64
What happens when a cell is placed into a hypertonic solution?
Water moves out of the cell, causing it to shrivel
65
What happens when a cell is placed into an isotonic solution?
There is no net loss or gain of water
66
What happens when a cell is placed into a hypotonic solution?
Water moves into the cell, causing it to lyse (burst).
67
What are the three components of blood that were separated?
First, the plasma, then the lysate (cytoplasm) and finally the cell membrane
68
How was blood separated into the three components?
By differential centrifugation and osmotic lysis.
69
How were blood cells lysed, and why was this a necessary step?
The isolated erythrocytes were treated with a hypotonic solution, causing water to move into the cell and lysing it. This allows for the cytoplasm (lysate) and cell membrane to be further isolated with more differential centrifugation.
70
Why did the protein samples have to be placed on ice?
Proteins easily degrade because of ubiquitous cellular proteases released following lysis. This can be prevented with glycerol and ice or expensive protease inhibitors. This slows down enzyme degradation.
71
How can protein concentration being measured using the bradford assay?
The acidic component of the bradford reagent (coomassie brilliant blue dye) reacts and binds quantitatively with certain amino acids like arginine and some aromatic ones; tryptophan, tyrosine and phenylalanine. They dye changes colour when it binds which can be measured with a spectrophotometer. They are compared to a set of standards prepared with BSA.
72
What amino acids does Coomassie Brilliant Blue Dye bind with?
Arginine, tyrosine, phenylalanine, and tryptophan
73
What are the features of Coomassie Brilliant Blue Dye unbound?
Brown, 470 nm and acidic,
74
What are the features of Coomassie Brilliant Blue Dye when bound?
Blue, 595 nm and decreases in pH. It interacts with arginine and aromatic AAs to shift to blue as the protein conc [BSA] increases.
75
How was the original concentration of the isolated plasma fraction adjusted to the ideal value of 6 mg/mL?
Using the C1V1 = C2V2 equation. Our C1 (plasma fraction) was 27.7 mg/mL, our C2 was 6mg/mL, and our V2 was 30 uL. We wanted to know how much of our C1 to add, and that was 6.5 uL. We subtrated that from the total ideal volume of 30 uL to find out how much hypotonic saline to add as well (23.5 uL).
76
What is electrophoresis?
Electrophoresis separates charged molecules that are subjected to an electric field.
77
How can proteins be identified with electrophoresis?
The proteins differing in charge and size create distinct bands while migrating in the gel that can be compared to a molecular ladder of known protein weights.
78
What pole to negatively charged molecules migrate towards during electrophoresis?
The positive pole (anode)
79
What pole do positively charged molecules migrate towards during electrophoresis?
The negative pole (cathode)
80
What does SDS PAGE stand for?
Sodium dodecyl sulfate polyacrylamide gel
81
What is the gel matrix that SDS PAGE forms?
It forms a gel matrix made of an acrylamide cross-link with N-N'methylene-bis-acrylamide.
82
Why is it important that SDS PAGE forms a charge-to-mass ratio with proteins?
When charge to mass is the same, the motility of proteins is related to molecular weight because of the molecular sieving properties of the gel.
83
What are the four components of the sample buffer?
The tracking dye, SDS, beta-mercaptoethanol, and sucrose or glycerol.
84
What is the role of the tracking dye in the sample buffer?
Proteins are invisible and can only be tracked with a dye that runs just ahead of the the fastest running proteins during electrophoresis.
85
What is the role of SDS in the sample buffer?
SDS is a detergent that denatures and coats proteins with a negative charge, leading to the same charge to mass ratio so the motility of proteins can be compared to the molecular weight. It allows the now negatively charged proteins to migrate towards the positive pole (anode).
86
What is the role of beta-mercaptoethanol?
Beta-mercaptoethanol dissociates multichain proteins (reduces disulfide linkage) so SDS can bind.
87
What is the role of sucrose or glycerol?
They add density to prevent the sample buffer from mixing with the running buffer.
88
What is the role of the chamber during electrophoresis?
Holds the cast gel in a buffered solution (running buffer) with electrodes and voltage.
89
What is the difference between the sample and the running buffer?
The running buffer is added to the chamber, while the protein samples were prepared with the sample buffer
90
What were the protein standards used in the SDS PAGE?
A molecular protein ladder contains known molecular weights of proteins that run alongside the samples.
91
How can unknown protein weights be determined?
A standard curve using the log of the weights can be used to determine the other unknown weights.
92
After casting, how were the gels visulized?
They were stained with Coomassie blue dye and microwaved under water since they are not visible without dye.
93
What is western blotting?
Western blotting uses antibodies to detect specific proteins that give off light detectable by X-ray film.
94
What does WHMIS stand for?
Workplace hazardous materials information system
95
What are the six types of fires we could see?
Trash, wood, paper, grease, liquid, electrical
96
What are Type A fire extinguishers used on?
Trash, wood and paper; liquid based
97
What are Type B fire extinguishers used on?
Grease and liquid; liquid detergent based to mix with the oil, grease, liquid
98
What are type C fire extinguishers used on?
Electrical fires; nonconductive, low evapouration point liquid based
99
What are Type ABC fire extinguishers used on
All fires
100
What are the four main components of WHMIS?
Product labels, labware labels, material safety data sheets and training programs
101
When would you use a compressed gas logo?
When the pressure in a gas tank is above 40 psi
102
When would you use a flammable/combustion logo?
When there is a material capable of catching fire in the presence of a spark or open flame.
103
When would you use a poisonous and infectious division one logo?
When there is a material that could cause immediate and serious death or injury when exposed in small amounts.
103
When would you use an oxidizing materials logo?
When there is an increased risk of fire if it comes into contact with a flammable and combustible material
104
When would you use a poisonous and infectious division two logo?
When the material could cause other toxic effects that could cause life threatening injury or serious long term harm in larger amounts
105
When would you use a poisonous and infectious division three logo?
When there are biohazardous, infectious and harmful microorganisms
106
When would you use a corrosive materials logo?
When the material could self-react dangerously when standing or exposed to outward elements like pressure, heat, or water/
BIOL 225
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