Page 1 of 12

162101 Biology of Cells 2014 Lab Theory Test Feedback.

General comments.

The statistics show that most people coped well with the test and have strengthened
their marks before the exam. Calculation questions were done very well on the whole. If
you see ECF or ecf (error carried forward) on your paper, this means that you made an
error early in a calculation, but you have been given credit for correct calculations that
follow, despite the wrong numbers. However, there is an additional penalty for a wrong
final answer.

Questions 1 and 2 had the same wording as for the 2013 paper; only the numbers were
changed. It was very clear that students who could not cope with these questions did
not prepare adequately for the test. Worked calculation answers and the expected
graph for the 2013 paper were available on Stream and there were many practice
examples of both types of question in the lab manual.

Question 1
A graph is constructed to show if there is a relationship between what is changed (e.g.
amount of protein) and what is measured (e.g. absorbance).
A straight line relationship shows that, within the linear range, more protein results in a
higher absorbance in a predictable way, shown by the line. However, the Biuret assay
has a very limited range over which this linear relationship applies, and at a certain point
(past the linear range of the graph), the absorbance values give you NO information
about how much protein is there. For this reason, we tell you not to extend the best fit
line beyond the end of the linear range. Not as a dotted line, and definitely not as a
solid line, no matter what you were taught at school.

Quite a few students calculated the protein concentration of the unknown solution from
the absorbances of both tubes 9 & 10 and got different answers. This shows a lack of
understanding of what the test was about you were trying to determine the protein
concentration of an unknown solution. All that was different between tubes 9 and 10
was the volume of unknown solution you added to the assay. You cannot have two
different answers to the concentration of protein in a single solution. One must be
wrong. The absorbance of tube 10 was beyond the linear range and should NOT have
been used to calculate concentration.

Best fit lines:
We do not expect you to calculate a statistical fit to your results in this paper. But we do
expect you to be able to draw a best fit line by eye to your results. This should be a
straight line that passes through or close to as many points in the linear range as
possible, including zero. No protein =absorbance of zero, so it is a valid point. To do
this, you use a ruler to find the line that looks right, with about as many points above the
line as below it, and usually some right on the line. You were given seven points on the
line (within the linear range) instead of the four you had in the lab, in order to make it
easier to do this. If you didnt know what we meant by best fit line, you should have
asked for help in the weeks prior to the test.
DO NOT join each point to the next with straight lines. Please note:
Lab manual, p24 for an explanation of a standard curve, p 27 3(ii) with a straight line of
best fit drawn through the experimental points and p39 5(ii) Using a ruler, draw a
straight line of best fit through the linear portion If there are any non-linear
absorbances at one end of the plot, do not incorporate these points in your line of best
fit otherwise your line will deviate from a straight line.
Lines drawn freehand were unacceptable.



Page 2 of 12
Horizontal (x) axis.
The lab book instructions are equally clear about this. You should have plotted amount
of protein (in mg), in preference to concentration, and certainly not volume in mL.
Plotting concentration is not wrong, but involves more calculations, so more chances for
errors. Many students plotted amount and called it concentration, which is NOT correct.
Part C of Experiment 1 explains how to plot the graph correctly, as do standard curve
example questions 1 & 2 (lab manual pages 49-50).

We are aware that some of the graphs in this course are not straight lines (the bacterial
growth curves and the DNA gel calibration curve. Sometimes the relationship is not a
straight line, but a logarithmic or some other mathematical relationship. This appears to
have confused some students. However, the instructions and examples for the
standard curve graphs were very detailed and explicit.

If you lost any other marks for question 1, please look at the file Plotting a good
graph which is in the file with previous lab test papers.

Comments about the other questions are shown with the questions and example
answers.

Important:
Markers were required to follow a strict marking schedule. Your marks will not be
altered if this would result in different marking for some students and not others.


Please Note.
Lab calculations and questions related to the experiments will not be tested in the final
exam, but parts of Experiments 7 and 8 are relevant to lectures and the paper content as
follows:
The principles of PCR: How it works, how it amplifies a specific piece of DNA,
the type of primers used.
How to interpret diagrams representing the results of DNA gel electrophoresis
(but not the calibration of gels).
Pedigrees and probabilities in genetics.
Why and how restriction endonucleases are used in DNA Technology.

Make sure you revise these before the exam.
Page 3 of 12
QUESTION 1
(10 marks)
Suppose you wish to determine the protein concentration of an unknown solution
using the Biuret assay. You have a stock protein solution with a concentration of
5 mg/mL. You prepare tubes 1 to 8 as described in the table below, and measure
their absorbance at 540 nm. The experimental results are shown in the second
table.

Tube # Volume of
stock solution
Volume of
H
2
O
Volume of
Biuret
reagent
Absorbance at 540 nm

1

0

4.0 mL

4.0 mL

0
2 0.2 mL 3.8 mL 4.0 mL 0.055
3 0.5 mL 3.5 mL 4.0 mL 0.170
4 1.0 mL 3.0 mL 4.0 mL 0.350
5 1.5 mL 2.5 mL 4.0 mL 0.470
6 2.0 mL 2.0 mL 4.0 mL 0.655
7 2.5 mL 1.5 mL 4.0 mL 0.840
8 3.0 mL 1.0 mL 4.0 mL 0.850

You assay two different volumes of your unknown protein sample at the same
time and obtain the results shown below.

Tube # Volume of
unknown
protein
solution
Volume of
H
2
O
Volume of
Biuret
reagent
Absorbance at 540 nm

9

0.7 mL

3.3 mL

4.0 mL

0.495
10 1.5 mL 2.5 mL 4.0 mL 0.870

Using a standard curve, calculate the concentration of protein in the unknown
solution to 3 significant figures. Show all your working. Graph paper is provided
on the next page.

Tube #9:
7.5 mg (7.5 0.5)
0.7 ml

Conc. unknown = 10.7 mg/ml


Calculate amount of protein in
each tube.
1: 0 mL x 5 mg/mL = 0 mg
2: 0.2 mL x 5 mg/mL = 1.0 mg
3: 0.5 mL x 5 mg/ml = 2.5 mg
4: 1.0 mL x 5 mg/mL = 5.0 mg
5: 1.5 mL x 5 mg/ml = 7.5 mg
6: 2.0 mL x 5 mg/ml = 10 mg
7: 2.5 mL x 5 mg/mL = 12.5 mg
8: 3.0 mL x 5 mg/mL = 15 mg

Page 4 of 12

Page 5 of 12
QUESTION 2

a) Suppose you start a new job working in a laboratory. You are asked to
prepare exactly 1150 mL of an 18 mg/mL solution from a stock solution of
475 mg/mL. How much stock solution will you need and how much distilled
water will you need to add to dilute it? (3 marks)

C
1
V
1
= C
2
V
2
V
1
= C
2
V
2

C
1
= (18 mg/mL) (1150 ml)
475 mg/mL
= 43.6 ml (43.57 acceptable)

Volume of stock = 43.6 mL
Volume of water (diluent) = 1150 mL 43.6 mL
= 1106.4 mL


b) Suppose 11 mL of a stock solution with concentration 27 mg/mL is added to
121 mL of diluent. Then 4 mL of the resulting solution is added to 20 mL of
diluent. What will be the concentration of the final solution? Show all your
working. (3 marks)

(V
2
= V
1
+ V
dil
so 35 + 7 = 42mL) (not essential to be shown)

Df
1
= 132 mL
11 mL
= 12

Df
2
= 24 mL
4 mL
= 6

TDF = 12 x 6 = 72

c) Convert the following to the units shown. (4 marks)

(i) 9.57 nm = _______________ m 9.57 x 10
-9


(ii) 78.5 L = _______________ mL 0.0785 or 7.85 x 10
-2


(iii) 0.019 mg/mL = _______________ g/mL 19

(iv) 567 m = _______________ nm 5.67 x 10
5


Manystudentsmadeerrorsofconversionandlostseveralmarks.Thisalsoshowedin
subsequentcalculations.Youneedtopracticeuntilyouareconfident.
Df = C
1

C
2

C
2
= C
1

TDF

= 27 mg/mL
72

= 0.375 mg/mL
Page 6 of 12
QUESTION 3

A student designed an experiment to test the hypothesis, washing hands with
soapy water removes more microorganisms than washing with water. An agar
plate was divided into 4 sectors and the following treatments were carried out:

1. No treatment
2. Hands were rubbed together then a finger was
touched to the plate
3. Hands were rubbed together using tap water
then a finger was touched to the plate
4. Hands were rubbed together using soapy
water then a finger was touched to the plate

The plate was inverted and incubated at 25
0
C for
24 hours.

(a) Was the hypothesis supported YES/NO? Give a reason for your answer.
(2 marks)
NO the hypothesis is not supported.
There is no (statistically significant) difference between the colony counts for tap
wash vs soapy wash treatments.
Or Both water and soapy water appear to be equally effective at lifting microorganisms
from the skin that are then readily transferred to the plate)
Moststudentsunderstoodthatthehypothesiswasn'tsupportedduetothesimilarityofthe
colonynumbersinsections3and4.

(b) What sort of control is sector 1 and what does it tell you? (2 marks)
Sector 1 is a negative control, indicating that the plate was not contaminated prior to
the experiment.
Studentsconfusedpositivecontrolsandnegativecontrols.Ifyougettheansweryouwant
fromacontrolthatdoesn'tmakeitpositive.
Therewasnotrue'positive'controlinthisexperiment.

(c) What is a possible explanation for the low colony count in sector 2. (2 marks)
A dry hand/finger does not transfer so many microorganisms to the plate as a
wet hand/finger.
Microorganisms may be lifted/suspended in water, resulting in greater transfer
to another surface/object than just leaving the hands dry.
Manystudentswrotemislabelled.Thiswasnotaccepted,asweexpectedyoutoassume
thiswasnotthecaseduetotheinformationlegendnexttotheplateandtofindother
reasonsforthisresult.
Rubbingyourhandstogetherwhendrydoesn'tproduceenoughfrictionheattokillthe
bacteriaonyourhands.
Manystudentsdidn'tunderstandhowwaterisabletomobilizebacteriaonthehandto
allowforbettertransferincomparisontodryhands.
Phenomenonisnotthecorrecttermwhendescribingtheprocessinanexperiment.

(d) List two good points about the experimental design (2 marks)
Rubbing hands together was a good idea to ensure uniformity of microorganisms on
hands. Butrubbingyourhandstogetherwillnottransferallthebacteriafromone
hand/fingertotheother.
Page 7 of 12
Wetting hands and testing with and without soap limited the number of variables.
A negative control was used (Sector 1)


(e) List two limitations of the experimental design (2 marks)
No time duration given for washing. Wash time for sectors 3 & 4 could have been
different, therefore potentially affecting the results
Unclear if a finger was from the same or different hands needed to specify this.
Lack of replicates
Colonies were counted, but different types of colonies were not recorded.Thenumbers
seenontheplatecouldstillbeduetonormalflora(seebelow).

Manystudentsdidn'tgetmarksforsections(d)and(e).ThinkaboutHOWyoucouldhave
improvedtheexperiment,andwhatfeaturesoftheexperimentyouthinkaregood
(controls,hypothesis,nottoomanyvariables).Thesearedifferentandspecifictoeach
experiment.Youcantrelyonmodelanswerstoapplytoallexperiments.

Basicmisunderstandingsinthisquestion/experimentalsession.
Wehavemanybacteriawhichnormallyliveonourskin,anddonoharm.Bacteriaareessential
forustolive.Whatwewanttoremovebyhandwashingaretheextrabacteriawepickupfrom
theenvironment,whichmaycausedisease,particularlyifwetransferthemtoourmouth,eyes,
earsetcorwhichcouldenterawound.However,whenwetouchanagarplateweare
transferringsomeofthenormalskinfloraandsomeoftheextradirtyhandsenvironmental
bacteria.
Youcannotsterilizeyourhandscertainlynotbywashingthem.
Antibacterialhandwashwillremovesome,butnotallbacteriafromtheskin(handsanitizeror
ethanolwouldkillmoreofthem,butnotALL).
Page 8 of 12
QUESTION 4

(a) Which two things need to be in focus for a microscope to be correctly set up
for critical illumination? (1 mark)
The object on the prepared slide and the light source.


(b) What procedure was used during slide preparation to make prokaryotic
(bacterial) cells visible under the microscope? (1 mark)
Gram staining

(c) Fill in the table below to distinguish between E. coli and M. luteus.
(4 marks)
Bacteria Gram
+or -
Colour Shape The cell wall has a
layer of
peptidoglycan that is
relatively thick/thin
(choose one)
E. coli - Red/pink Rod thin
M. luteus
+ Purple/black Spherical/coccus thick
Somemarksweregivenifthestudentmixedupthetwobacterialspeciesbuttheir
answerswereotherwiseconsistent.

(d) Write the correct term in the space to complete the sentence:
Use of immersion oil increases resolution because it has a higher
_________________________ than air. (seeexpt3) (1 mark)


(e) A student did serial dilutions of a bacterial culture of 10-fold, 6-fold and
another 10-fold. What was the total dilution factor? (1 mark)
TDF = 10 x 10 x 6 = 600 fold


(f) What volume of a bacterial suspension of 2 x 10
4
cells/mL would the student
have to use to plate out 300 cells? (1 mark)
Volume in mL = Cells/(cells/mL)
= 300/2 x 10
4
mL
= 150 x 10
-4
mL = 15 x 10
-3
mL = 15 L or 0.015 mL

(g) If a student spread 300 cells on a plate but only had 150 colonies grow, how
could they explain this result?
Not all viable (alive). Assumethecolonieswerespreadcorrectlyandlookforadeeperanswer.


Page 9 of 12
QUESTION 5

(a) A stage micrometer is etched with a 2 mm line that is divided into 100 equal
divisions. When viewed with an eyepiece micrometer at a total magnification
of 400x, it is found that 14 stage micrometer divisions correspond to 87
eyepiece micrometer divisions. Viewed at 400x magnification, a spherical
cell has a diameter of 21 eyepiece divisions. What is the diameter of the cell
in micrometers (m)? Show all of your working. (4 marks)
1 SMD = 2mm/100 x 1000 m/mm = 20 m
87 EPD = 14 SMD = 14 x 20 m = 280 m
1 EPD = 280 m/87 = 3.22 m
Cell diameter = 21 EPD = 21 x 3.22 m = 67.6 m

(b) A cytometer has the dimensions shown below. A student counted 486
yeast cells in the shaded portion of the cytometer grid. Calculate the cell
density of the yeast cell suspension in cells/mL. Show all of your working.
Note that 1 mL =10
3
mm
3
. (3 marks)






Depth=0.2mm
Volume of grid used :
2.5 mm x 0.5 mm x 0.2 mm = 0.25 mm
3
or 0.5 x 0.5 x 0.2 x 5 = 0.25 mm
3

or (2.5 mm x 2.5 mm x 0.2 mm)*5 = 0.25 mm
3

0.25 mm
3
/10
3
mm
3
x 1 mL = 2.5 x 10
-4
mL

Cell density = # cells/volume
=486/2.5x10
4
cells/mL
=1.94x10
6
cells/mL

(c) If the cell density of a yeast cell suspension was 9.97 x 10

5
cells/mL, and the
cells were counted in the same shaded area of the grid shown above in (b),
how many cells would be counted? ( 2 marks)
The volume would be the same: 2.5 x 10
-4
mm
3
.
# cells = cell density x volume
= 9.97 x 10
5
cells/ml x 2.5 x 10
-4
ml
= 249 cells (no part cells! )
Ananswerof10
6
cells(=1millionthofacell)orsimilarshouldhaverungalarmbells!
orStudentscouldhavedonethisasaratioofcelldensitiesusingthecellnumberfrompart
(b):
486 x 9.97 x 10
5
/(1.94 x 10
6
) = 249 cells

(d) Would it be possible to count E. coli cells using a cytometer? Explain.
(1 mark)
No, they are too small, cant use immersion oil with cover slip, lens too long, cant treat
cytometer with gram stain, cant usually see bacteria without the stain.
Somanyreasons,andyouonlyneededone.Thistypeofquestionwaswhywe
recommendedyoutrytovisualisebeingbackinthelablookingdownthemicroscopeand
nottrytolookforonemodelanswer.
2.5mm
Page 10 of 12
QUESTION 6
(a) Vicia faba root tips were microscopically examined to determine the number
of cells in each stage of the cell cycle. The treatment consisted of root tips
incubated in 0.1% colchicine for 5 hours at 20C. 400 cells were examined at
random for the untreated control and the colchicine treatment. Calculate the
length of the cell cycle for this organism. Show all of your working. (3 marks)
Number of cells in .
Interphase Prophase Metaphase Anaphase Telophase
Control
(Slide 1)
299 28 22 38 13
Treatment
(Slide 2)
319 35 46 0 0

length of cell cycle = 100% x time (hr)
% cells in met after - % cells in met before

% met after = 46/400 = 11.5% % met before = 22/400 = 5.5%
Treatment time = 5 h
Cell cycle length = 100/(11.5 5.5) x 5 = 83.3 h
Themostcommonmistakewastousethenumbersinthetableandnottocalculate
percentages

(b) Using your result from part (a) above, calculate how long untreated cells of
these beans will spend in: (3 marks)
(i) Prophase.
% untreated cells in interphase = 28/400 = 7%
Time in interphase = 7% (0.07) x 83.3 h = 5.83 h

(ii) Anaphase
38/400 x 83.3 h = 7.91 h
Fullmarksweregivenforcorrectcalculationsevenifthecellcyclelengthfrom(a)was
incorrect.

(c) Why is it necessary to select only the very end of a root tip when preparing a
slide to study mitosis? (1 mark)
Because this is the only part of the root containing rapidly dividing cells (meristem).
This is where the root is growing, so mitosis is occurring in many cells. Mitosisisinthe
question,soanexplanationofwhythiswasoccurringintheroottipwasrequired.
Avoidsweepingstatements:theroottipisnottheonlyplaceintheplantwherecell
division(ormitosis)isoccurring.

(d) The frequency of penicillin-resistant mutants in a particular bacterial culture is
7.45 x 10
-6
. How many penicillin-resistant cells will there be in 500 l of the
culture with a total cell density of 8.95 x 10
8

cells/ml? Show all of your
working. (3 marks)
500 l = 0.5 ml or total cells = cells/mL x cells
0.5 ml x 8.95 x 10
8
cells/ml = 4.475 x 10
8
cells in total
(Bestnottoroundto3sigfigsatthisstageitaffectstheanswerquitealot)
If mutant freq = 7.45 x 10
-6

then no. of cells in the culture is 4.475 x 10
8
cells x 7.45 x 10
-6

= 3.33 x 10
3
cells (3333 or 3334 cells)
Page 11 of 12
QUESTION 7
(a) Complete the table to show how you detected the following cellular
components after differential centrifugation of pea homogenate. Include the
colours you observed. Include microscopic evidence, biochemical assays or
stains as appropriate and mention any diagnostic colours observed in each
case. (3 marks)
Components
detected
Methods used to detect component
Starch granules
Blue/black (notjustdark) with iodine stain,
observed using microscope. Notgramstain
procedure.
Chloroplasts
Green colour observed in pellet and in microscope
Notaspecifictestforgreen
Mitochondria
Red colour in tetrazolium assay.
Mitochondriaarenotredcolouredandaretoosmallto
seebyeye.
Hadtostatemethods,assaysandcolours.

(b) Explain what differential centrifugation is and why it was used. (2 marks)
Samples are centrifuged, and the supernatant and precipitate separated.
Further steps of centrifugation for longer and at faster speeds are used to
separate fractions of smaller and smaller particles.
Used as a method of separation by density (weight, size) (and often as a start for
purification of some cell components).
Manypeopledescribedasinglecentrifugationstep,butnotmultistepdifferential
centrifugationorwhyitwasused.

(c) Explain whether pure preparations of the components listed in the table
above were obtained. (2 marks)
Pure components are not obtained
- some components (eg starch granules) vary in size/mass density so are not totally
separated
- some different components may have the same size/mass density as each other so not
be separated.
Manystudentsaskedaboutthisquestionbeforethetest.Puremeanspure.Considerwhat
isbeingseparatedandthebasisofseparationofthemethodandthinkaboutwhatis
happeningduringtheproceduretoansweraquestionofthistype.

(d) Name and briefly describe what happens in each of the three steps in each
PCR cycle. (3 marks)
(i) Denaturing/denaturation - to separate double-stranded DNA molecules into
single strands
(ii) Annealing/primer attachment - of primers to the single-stranded
template DNA
(iii) Extension/DNA synthesis - DNA synthesis extending from the end of each
primer, making a double-stranded DNA molecule (the PCR product).
PrimersdonotmovealongtheDNAstrand.TheDNAprimershydrogenbondtothe
complementaryantiparallelsequenceoftheDNAandthentheTaqDNApolymerase
catalysesformationoftherestoftheDNAstranduntiltherearenomorebasestocopy.
RNAIsnotinvolved.
Page 12 of 12
QUESTION 8

A 15.3 kb fragment of linear DNA was cut with the restriction enzymes EcoRI and
BamHI to yield the DNA fragment sizes shown in the table.

Restriction
enzyme(s)
EcoRI BamHI EcoRI +BamHI
Sizes of fragments
(kb)
6.0, 5.3, 4.0 7.5, 4.8, 3.0 6.0, 4.0, 3.0, 1.5, 0.8

a) Draw the pattern of DNA fragments you would expect to see after gel
electrophoresis of vector cut with EcoRI or BamHI or with both enzymes
together. Clearly indicate their sizes by reference to the size marker ladder
shown. (5 marks)
Plasmid vector digested with
Size marker EcoRI BamHI EcoRI+BamHI
Ladder

EasymarksmoststudentsdidthisOK

b) Draw lines on the diagram below to indicate where these enzymes cut the
vector. Label the distances (kb) between the cutting positions. (5 marks)





Unitswererequiredandeachcuthadtobelabelledwiththecorrectenzyme.


EcoR1 EcoR1
BamH1 BamH1
5kb 3kb
4kb
1.5kb 0.8kb