Beruflich Dokumente
Kultur Dokumente
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
(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