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CMB Practical Test Flashcards

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1
Q

What is the Laboratory Safety Conduct?

A

All students must comply with these requirements:
1. Laboratory overalls must be worn while in the laboratory.
2. Covered shoes must be worn at all times in the laboratory.
3. Safety goggles must be worn when you work with any chemicals or work within 3m of anyone working with chemicals. This rule applies to laboratory users who are not wearing glasses.
4. All volatile solvents, acids, ammonia and smelly chemicals must be handled in the fume hoods to avoid building up of such volatile chemicals in the laboratory.
5. Broken glassware must be swept up immediately and placed in designated boxes for broken glasses.
6. Food and drinks must not be consumed in the laboratories

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2
Q

Good Laboratory Housekeeping and Procedures

A
  1. Hazardous waste must not be poured into the sink drain. They must be deposited into labeled receptacles designated for separate classes of hazardous waste, i.e. water soluble solvents, water insoluble solvents, toxic chemicals, acids etc.
  2. Wash your hands thoroughly before you leave the laboratory.
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3
Q

What are some common equipment bench top items? (7 examples)

A
  1. Micropipette
  2. Latex gloves
  3. Face mask
  4. Safety goggles
  5. Vortex Mixer
  6. Ethanol / water spray bottle
  7. Bench top centrifuges (provides centrifugal force to separate a fluid from a fluid or from a solid substance)
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4
Q

What are some common waste disposal bench top items? (4 examples)

A
  1. Sharps bin
  2. Biohazard waste bag
  3. Normal waste bag
  4. Broken glass bins
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5
Q

What are examples of chemical safety and clean work? (2 examples)

A
  1. Fume hood
  2. Laminar flow hood
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6
Q

What is a fume hood?

A

Ventilated box used when working with hazardous substances

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7
Q

What is a laminar flow hood?

A

Workspace with HEPA filter used to protect samples from contamination
(HEPA - high efficiency particle)
*Only laminar flow hood has HEPA filter to help maintain a sterile environment

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8
Q

What are some common symbols found in the lab? (5 examples)

A
  1. Biohazard
  2. Radioactive
  3. Toxic
  4. Corrosive
  5. Flammable
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9
Q

What do you do after each lab session? (3 steps)

A
  1. Ensure that the lab is clean and tidy
  2. Wipe the bench tops
  3. Wash your hands before leaving the lab
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10
Q

A. Proper use of micropipettes
How to withdraw fluid? (4 steps)

A
  1. Fit the pipette with the correct tip.
  2. Depress plunger to first stop and hold. Dips tip into fluid and gently release thumb.
  3. Slide pipette tip out along wall of tube to dislodge remaining droplets adhering to the outside of the tip.
  4. Check that there is no air space at the very end of the tip.
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11
Q

B. Proper use of micropipettes
How to expel sample into reaction tube? (4 steps)

A
  1. Touch tip to wall of tube.
  2. Slowly depress plunger to first, and then to the second stop to expel fluid.
  3. While keeping the plunger at the second stop, slide the tip out the fluid, along the tube wall and out of the tube.
  4. Eject the tip into the trash.
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12
Q

C. Proper use of micropipettes
How to prevent cross contamination of reagents? (6 steps)

A
  1. Never rotate volume adjustor beyond the upper or lower range.
  2. Never use without a tip in place.
  3. Never lay pipette down with filled tip – fluid could run back into the piston.
  4. Never let the plunger snap back after withdrawing or ejecting fluid. This could damage the piston.
  5. Never immerse anything other than the disposable tip in liquid.
  6. Never flame the micropipette tip.
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13
Q

Practical 1 Lab Experiment
What is a micropipette?

A

The micropipette is a specialized tool that if properly used will allow you to accurately transfer small volumes of liquid.

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14
Q

Practical 1 Lab Experiment
What is a spectrophotometer?

A

A spectrophotometer is an instrument that measure the amount of light absorbed by molecules in a solution at a given wavelength.

It can be used to indirectly determine the amount of a compound present. The higher the concentration of the solutes in the solution, the greater will be the absorbance.

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15
Q

Practical 1 Lab Experiment
What is the range of a P200 micropipette and what coloured tip to use?

A

Range: 20 - 200 microliter
Tip: Yellow tips

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16
Q

Practical 1 Lab Experiment
What is the range of a P1000 micropipette and what coloured tip to use?

A

Range: 200 - 1000 microliter
Tip: Blue tips

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17
Q

Practical 1 Worksheet
Briefly describe how you would aspirate or draw up 1070 microliter of reagent using only two different micropipettes and least number of volume adjustments on the micropipette.

A
  1. Firstly, I will draw up 1000 microliter of reagent using a P1000 micropipette (using a blue pipette tip).
  2. Secondly, the remaining 70 microliter of reagent, I will draw it up using a P100 micropipette (using a yellow pipette tip).
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18
Q

Practical 1 Worksheet
Given a stock solution of 5% KMNO4, show how you would prepare 200 microliter of a solution with a concentration of 0.5% KMNO4. Distilled water is used as a diluent.

A

C1V1 = C2V2
(C1 = starting concentration), (V1 = starting volume)
(C2 = final concentration), (V2 = final volume)

C1 = 5%, V1 = ? microliter
C2 = 0.5%, V2 = 200 microliter
C1V1 = C2V2
V1 = C2V2/C1 = 0.5% * 200 microliter / 5%
V1 = 20 microliter

Distilled water = 200 - 20 = 180 microliter
Therefore, add 20 microliter of 5% of KMNO4 solution to 180 microliter of distilled water to get 200 microliter of 0.5% KMNO4 final solution.

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19
Q

Practical 1 Assignment
What is the volume reading on this micropipette dial?
a) P200 => 067
b) P1000 => 020
c) P100 => 070
d) P1000 => 100

A

Volumes:
a) 67 microliter
b) 200 microliter
c) 70 microliter
d) 1000 microliter

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20
Q

Practical 1 Assignment
Fill in the following volume readings in the micropipette dial
a) P10 => 7.8 microliter
b) P200 => 167 microliter
c) P20 => 12.4 microliter
d) P100 => 34 microliter

A

Readings on dial:
a) 078
b) 167
c) 124
d) 034

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21
Q

Practical 1 Assignment
Given a stock solution of 2.5% KMNO4, show how you would prepare 150 microliter of a solution with a concentration of 0.5% KMNO. Distilled water is used as a diluent.

A

C1V1 = C2V2
(C1 = starting concentration), (V1 = starting volume)
(C2 = final concentration), (V2 = final volume)

C1 = 2.5%, V1 = ? microliter
C2 = 0.5%, V2 = 150 microliter
C1V1 = C2V2
V1 = C2V2/C1 = 0.5% * 150 microliter / 2.5%
V1 = 30 microliter

Distilled water = V2 - V1 = 150 - 30 = 120 microliter
Therefore, to prepare 150 microliter of solution with a concentration of 0.5% KMNO4, add 30 microliter of 2.5% KMNO4 to 120 microliter of distilled water.

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22
Q

Practical 2 Lab Experiment
What is a dissecting microscope?

A

A dissecting microscope can be used to examine larger objects and a compound microscope to view smaller specimens.

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23
Q

Practical 2 Lab Experiment
What is a compound microscope?

A

The compound microscope uses the magnifying powers of two lenses to produce a greatly enlarged image of structures too small to be viewed by the naked eye. Using the compound microscope, we can view plant and animal cells. Cell walls and plasma membranes and nuclei can be easily visualized.

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24
Q

Practical 2 Lab Experiment
Disadvantage of compound microscope?

A

Most structures within the cell are too small to be resolved by the compound microscope. However, these can be resolved by the use of more powerful microscopes such as the electron microscope.

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25
Practical 2 Lab Experiment What are the rules to use compound microscope? (5 steps)
Notes to students: 1. The compound microscope is a precision instrument. Handle it with care! 2. When carrying the equipment always use two hands. Place one hand on the arm and the other to support the base. 3. Place it down on the workbench gently without jarring. Always lift to move it. 4. Keep the lenses and stage clean and dry. 5. Only use lens tissue to wipe the lenses.
26
Practical 2 Lab Experiment What are the parts of a compound microscope? (10 examples)
1. Eyepiece 2. Objective lenses 3. Revolving nosepiece 4. Microscope stand 5. Mechanical stage 6. Condenser (aperture iris diagram) 7. Coarse focus adjustment knob (big) 8. Fine focus adjustment knob (small) 9. Light source 10. Base
27
Practical 2 Lab Experiment What are the types of lenses of a microscope? (4 types)
- 4x objective - 10x objective - 40x objective - 100x oil immersion objective
28
Practical 2 Lab Experiment How can the overall magnification be determined?
The overall magnification is determined by multiplying the power of eyepiece lens (10x) and the objective lens.
29
Practical 2 Lab Experiment What is 100x objective lens?
100 x objective is an “oil immersion” lens and can be identified by the presence of a black or white band on the lower part of the lens.
30
Practical 2 Lab Experiment What is oil immersion?
Oil immersion refers to the fact that “immersion oil” must be used to improve resolution. When using this lens, oil is placed on the slide and the slide is then viewed through the oil and not through the air.
31
Practical 2 Lab Experiment Can we use oil with non-oil immersion lenses?
Never use oil with non-oil immersion lenses and always remove the oil with lens paper after use.
32
Practical 2 Lab Experiment How do we care and maintain the microscope? (5 steps)
1. Care should be taken not to scratch the lens surface. 2. Clean lens externally with a lens tissue. Never disassemble an objective lens. 3. Always remove immersion oil immediately after use, since it hardens upon drying. 4. Stains should be removed by gentle cleaning with lens tissue moistened in distilled water. 5. The microscope should be clean, dry and in good order before replacing it in its box
33
Practical 2 Lab Experiment At the end of the class, do we need to clean the lenses and how?
At the end of your class you must clean the lenses with lens paper sprayed with 70% alcohol and then put the microscope away.
34
Practical 2 Lab Experiment Describe what would you do to view a slide? (4 steps)
1. Examine these living cells using LOW POWER. 2. Select one cell that shows its contents clearly and move it to the center of the microscopic field. 3. Using HIGH POWER, examine all the parts of the cell. 4. Remember to only use FINE ADJUSTMENT when using the high power objective.
35
Practical 2 Worksheet Which part of the microscope is known as the supporting stand and rest on the bench?
Base
36
Practical 2 Worksheet Which part of the microscope contains a system of lenses that focuses light on your specimen?
Condenser
37
Practical 2 Worksheet Which part of the microscope has a horizontal surface on which the slide is placed?
Mechanical Stage
38
Practical 2 Worksheet Under the microscope, what is the shape and color of a blood cell? (epithelial cells)
Circle and Red
39
Practical 2 Worksheet Under the microscope, what is the shape and color of an elodea? (onion cells)
Rectangular and Green
40
Practical 3 Lab Experiment What is the protocol for chlorophyll extraction? (11 steps)
1. Turn on the spectrophotometer. Use 80% acetone as the reference blank. 2. Wash spinach leaves and place about 30 g of deveined leaves in a blender with 150 ml ice-cold buffer. 3. Blend the tissues at high speed until it forms a homogenous “soup” (about 30 s). 4. Filter the homogenate through 4 layers of cheese cloth lining a filter funnel and collect the filtrate in a beaker standing in ice. 5. Transfer equal quantities of filtrate into two centrifuge tubes and centrifuge at 3000 rpm for 2 min at 40C. 6. Discard supernatant and gently resuspend the green pellet (combine from both tubes) in 10 ml of ice cold buffer. Stand on ice. 7. Take 100 microliter of the chloroplast suspension and add 900 microliter of deionized water in a fresh test tube. 8. Add 4 ml of acetone. Mix well. 9. Transfer to a centrifuge tube and cap tightly. 10. Centrifuge at 3000 rpm for 3 min at 40C. (Ensure tubes are balanced). 11. Take 1 ml of supernatant into a quartz cuvette to measure the absorbance at 645 and 663 nm.
41
Practical 3 Lab Experiment How to use the spectrophotometer? (5 steps)
i. Go to the photometric mode. ii. Key in the desired wavelength and then press “Go to” iii. Open the cover and place cuvette containing reference blank into the cuvette holder. Make sure cuvette is in the right direction. iv. Close the cover and press the “Auto Zero” button. The reading should be zero. v. Open cover and remove cuvette. Replace with another cuvette containing unknown. Close the cover and once the reading stabilizes, record. vi. When completed, ensure that the compartment is clean before switching off.
42
Practical 3 Lab Experiment What is cold buffer used for?
To ensure that that chloroplast do not burst
43
Practical 3 Lab Experiment What is spin?
Separation based on density
44
Practical 3 Lab Experiment What is supernatant and pellet?
Supernatant is a liquid and pellet is a solid
45
Practical 3 Lab Experiment What does it mean to dissolve pellet in 10ml cold buffer?
Resuspend, and do not want to burst the chloroplast yet
46
Practical 3 Lab Experiment Why do we add 900microliter of deionized water?
So that H2O bursts the chloroplast to release chlorophyll
47
Practical 3 Lab Experiment Why do we add 4ml of acetone?
Acetone is an organic solvent that helps to bring chlorophyll into solution (blend solution)
48
Practical 3 Lab Experiment What does A645 and A663 mean?
A645 means absorbance value at wavelength 645 A663 means absorbance value at wavelength 663
49
Practical 3 Worksheet What is the formula to calculate amounts of chlorophyll A and B in the green and red lead samples? (use average values for calculation)
Chlorophyll A = (12.7 x A663) - (2.69 x A645) Chlorophyll B = (22.0 x A645) - (4.68 x A663) Total chlorophyll = (20.2 x A645) + (8.02 x A663)
50
Practical 3 Worksheet What is the formula to calculate Chlorophyll A?
Chlorophyll A = (12.7 x A663) - (2.69 x A645)
51
Practical 3 Worksheet What is the formula to calculate Chlorophyll B?
Chlorophyll B = (22.0 x A645) - (4.68 x A663)
52
Practical 3 Worksheet What is the formula to calculate Total chlorophyll?
Total chlorophyll = (20.2 x A645) + (8.02 x A663)
53
Practical 4 Lab Experiment What is Iodine Test for?
Test for starch
54
Practical 4 Lab Experiment What are the steps for Iodine Test? (3 steps)
1. Use iodine to test for the presence of starch 2. Add dropwise of iodine to the sample 3. Positive test observation = solution turns from orange to blue/black
55
Practical 4 Lab Experiment What is the positive test observation after an Iodine Test?
Positive test observation = solution turns from orange to blue/black
56
Practical 4 Lab Experiment What is Biuret Test for?
Test for proteins
57
Practical 4 Lab Experiment What are the steps for Biuret Test? (3 steps)
1. Use biuret solution to test for the presence of proteins 2. Add dropwise of biuret to the sample 3. Positive test observation = solution turns from blue to purple (light violet)
58
Practical 4 Lab Experiment What is the positive test observation after a Biuret Test?
Positive test observation = solution turns from blue to purple (light violet)
59
Practical 4 Lab Experiment What is Ethanol Emulsion Test for?
Test for lipids/fats
60
Practical 4 Lab Experiment What are the steps for Ethanol Emulsion Test? (4 steps)
1. Use ethanol emulsion to test for fats 2. Add 2cm^3 of ethanol to the sample and shake (mix properly) 3. Followed by adding 2cm^3 of distilled water 4. Positive test observation = white emulsion forms
61
Practical 4 Lab Experiment What is the positive test observation after an Ethanol Emulsion Test?
Positive test observation = white emulsion forms
62
Practical 4 Lab Experiment What is Benedict's Test for?
Test for reducing sugars and non-reducing sugars
63
Practical 4 Lab Experiment What are the steps for (reducing sugars) Benedict's Test? (3 steps)
1. Use benedict's reagent to test for reducing sugars 2. Add several drops of benedict's reagent to the sample and heat 3. Positive test observation = solution turns from blue > green > yellow > orange > brick red precipitate
64
Practical 4 Lab Experiment What are the steps for (non-reducing sugars) Benedict's Test? (6 steps)
1. Firstly, confirm that it isn't a reducing sugar 2. Following a negative benedict's test, where the reagent remains blue 3. Add acid and boil for 2 minutes (this is called acid hydrolysis) 4. Cool the solution and then add an alkali to neutralize 5. Afterward, add benedict's reagent and heat 6. Positive test observation = solution turns from blue > green > yellow > orange > brick red precipitate
65
Practical 4 Lab Experiment What is the positive test observation after a Benedict's Test?
Positive test observation = solution turns from blue > green > yellow > orange > brick red precipitate
66
Practical 4 Lab Experiment What are some examples of a reducing sugars? (5 examples)
1. Glucose 2. Fructose 3. Galactose 4. Lactose 5. Maltose
67
Practical 4 Lab Experiment What is an example of a non-reducing sugars? (1 example)
Sucrose
68
Practical 4 Lab Experiment What is DCPIP Test for?
Test for vitamin C
69
Practical 4 Lab Experiment What are the steps for DCPIP Test? (3 steps)
1. Use DCPIP to test for vitamin C 2. Add sample to DCPIP while shaking it 3. Positive test observation = DCPIP turns colourless
70
Practical 4 Lab Experiment What is the positive test observation after a DCPIP Test?
Positive test observation = DCPIP turns colourless
71
Practical 5 Lab Experiment What are the purines bases of DNA and RNA? (2 types)
Adenine (A) and Guanine (G)
72
Practical 5 Lab Experiment What are the pyrimidines in DNA and RNA? (3 types)
In DNA = Thymine (T) and Cytosine (C) In RNA = Uracil (U)
73
Practical 5 Lab Experiment What is the protocol for DNA extraction? (18 steps) [DNA Isolation + Quantitation of DNA]
1. Place 0.5g of minced chicken meat into a 2ml eppendorf tube containing 1ml cell lysis solution. 2. Add 15 microliter Proteinase K solution to the lysate and mix by inverting 25 times. 3. Incubate at 55oC for 1 h and periodically invert tube during incubation. 4. Add 2 microliter RNase A solution to the cell lysate. 5. Mix sample by inverting the tube 25 times and incubate at 37 degree celsius for 30 min. 6. Cool sample to room temperature. Add 300 L protein precipitation solution to the cell lysate. 7. Invert the tube gently to mix the contents for 20sec. 8. Centrifuge at 14000 g for 3 min. 9. Remove the supernatant containing DNA using a micropipette and transfer to a new 1.5ml eppendorf tube. (Proteins and RNA are removed in the pellet). 10. Add 300 microliter of 100% isopropanol to precipitate the DNA. 11. Mix sample by inverting gently for ~10 times. 12. Incubate the tubes at -20 degree celsius for 5 minutes 13. Centrifuge at 14000 g for 1 min. The DNA will be visible as a small white pellet. 14. Pour off the supernatant and drain the tube by inverting and standing it vertically on clean absorbent paper for 1-2 min. (Pellet may be loose, so pour slowly and watch the pellet). 15. Add 1 ml TE buffer and resuspend the pellet. 16. Centrifuge at 14000 g for 1 min to pellet insoluble impurities. 17. Transfer the solubilized DNA (in TE buffer, pH 8.0) to a 1-mL cuvette. 18. Measure A260 and A280.
74
Practical 5 Lab Experiment What is the formula to calculate the DNA content in your sample?
A260 reading of 1 = 50 micrograms of double-stranded DNA/ml DNA content = A260 x 50 micrograms Example: A260 is 0.846 Answer: 0.846 x 50 = 42.3 micrograms DNA
75
Practical 5 Lab Experiment What is the formula to calculate the yield of DNA per gram of sample used?
Yield per gram / Weight of meat = DNA content Yield per gram meat = ( DNA content / Weight of meat ) x 1 gram Example: DNA content is 42.3 micrograms DNA and Weight of meat is 0.5089 grams Answer: ( 42.3 micrograms DNA / 0.5089 grams ) x 1 gram = 83.12 micrograms of DNA
76
Practical 5 Lab Experiment What is the A260/280 ratio of Pure DNA?
Pure DNA has an A260/280 ratio of between 1.65 and 1.85
77
Practical 5 Lab Experiment What is the A260/280 ratio of Pure RNA?
Pure RNA has an A260/280 ratio of 2.0
78
Practical 5 Lab Experiment What does the lower ratios indicate?
Lower ratios indicate protein contamination
79
Practical 5 Lab Experiment What does the higher ratios indicate?
Higher ratios indicate RNA contamination
80
Practical 5 Lab Experiment How pure is your DNA sample?
Example: Our A260/280 sample is 1.373 Answer: This means that our DNA sample has protein contamination as the ratio is below 1.65
81
Practical 5 Lab Experiment What is the formula to calculate protein content?
Protein content (mg/ml) = (1.45 x A280) - (0.74 x A260) Example: A280 is 0.616 and A260 is 0.846 Answer: (1.45 x 0.616) - (0.74 x 0.846) = 0.8932 - 0.6260 = 0.267 mg/ml
82
Practical 5 Lab Experiment What does protein content indicate?
Protein content will give an indication of the level of protein contaminants present
83
Practical 5 Lab Experiment What is the purpose of adding Cell lysis solution?
To break open your cell (lysis cell)
84
Practical 5 Lab Experiment What is the purpose of adding Proteinase K?
A protein enzyme that breaks down (degrade) protein
85
Practical 5 Lab Experiment What is the purpose of adding RNase A?
RNA enzyme to break down RNA
86
Practical 5 Lab Experiment What is the purpose of adding Protein precipitation solution?
Gather all your protein (debris) together
87
Practical 5 Lab Experiment What is the purpose of adding Isopropanol?
To crystallize (precipitate) DNA
88
Practical 5 Lab Experiment What is the purpose of spinning (centrifuge)?
To separate the debris and DNA by density
89
Practical 6 & 7 Lab Experiment What is cell division responsible for?
Cell division is responsible for growth and reproduction in all organisms
90
Practical 6 & 7 Lab Experiment What is are the two main types of cell divisions?
Mitosis and Meiosis
91
Practical 6 & 7 Lab Experiment What is mitosis?
Mitosis results in the formation of two daughter cells with identical chromosome number to the parent cell
92
Practical 6 & 7 Lab Experiment What are the four stages in mitosis?
- Interphase 1. Prophase 2. Metaphase 3. Anaphase 4. Telophase - Cytokinesis
93
Practical 6 & 7 Lab Experiment What is meiosis?
Meiosis occurs in reproductive cells and results in the formation of gametes (egg cells and sperms)
94
Practical 6 & 7 Lab Experiment How many daughter cells are formed in meiosis and what do they contain?
The four daughter cells formed have nuclei containing the haploid number of chromosomes or half the complement number of chromosomes found in the 'parent' cell
95
Practical 6 & 7 Lab Experiment What is prophase? (based on video)
Chromosomes become visible in the chromatin network as elongated threads (chromosomes pair up)
96
Practical 6 & 7 Lab Experiment What is late prophase? (based on video)
- Chromosomes become thicker and start to coil - Nuclear membrane and nucleolus disintegrates - Centrioles separate and move to opposite poles
97
Practical 6 & 7 Lab Experiment What is metaphase? (based on video)
Pairs of chromatids arrange at the equator and become attached to the spindle threads (chromosomes line up at the equator)
98
Practical 6 & 7 Lab Experiment What is anaphase? (based on video)
Chromatids completely separates and pull to opposite poles of the cell (sister chromatids are pulled apart)
99
Practical 6 & 7 Lab Experiment What is telophase? (based on video)
- Telophase is the final stage of cell division - Chromatids are referred to again as chromosomes - When chromosomes reach the opposite end of the cell, a nuclear membrane reforms around them - During telophase, chromosomes condenses and revert to their thread-like form - After completion of nuclear division, a plate can be seen to grow across the equator of cell, which are separating the two halves (cell pinches in the middle)
100
Practical 6 & 7 Lab Experiment What is cytokinesis? (based on video)
- Telophase is ended by the process called cytokinesis - Cytokinesis is the division of the cytoplasm - The result is to complete an identical daughter cells which then goes to interphase
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