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Unit 15 – Assignment 3 – P3 & P4
Serial Dilution of Milk
Equipment
Glass petri dish
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Method
1
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Using same syringe throughout without putting it down, do serial dilution as follows:
3
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Remember to flame the
necks of the bottles briefly each time you take the top off
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Take 3 milk agar plates
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Label each with initials, then one each of 10-4 , 10-5 , 10-6
6
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2cm3 from each of these 3 tubes (10-4 , 10-5 and 10-6 )
and put in centre of correctly labelled petri dish
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Pour small amount of 100% alcohol into glass petri dish AWAY FROM THE BUNSEN
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Use to spread the milk round the agar
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Dip in alcohol and flame again
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9
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10
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Results

862 colonies on a 106 plate
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2ml
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Use of liquid media and colorimetry to investigate the effect of
glucose concentration on the growth of yeast
Method - Measuring absorbance of the yeast cultures
1
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You will need to carry out a serial dilution of this
culture
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To do this, follow the instructions on the PowerPoint presentation, making 5 dilutions in
total
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Measure 3cm3 of the 10-5 dilution into a cuvette
4
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Set the filter to 440 (blue)_
6
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7
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8
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Record the absorbance in the table

Method: How to count the cells in each culture
1
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3
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5
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Place one drop of each culture on a haemocytometer
Count the number of yeast cells that you can see in a representative 3-lined (type B) square
The volume of this square is 0
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0mm3 (Cell count x 250)
Repeat this for each of the yeast cultures
If you have to dilute the yeast even more in order to be able to count them then you must
record how much you have diluted it by
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Record cell counts in the table

Yeast results
Type of
tube

0

1

2

3

A

B

C

Dilution

Pure

1:50

1:25

1:625

Pure

Pure

Pure

Absorbance 1
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34

0
...
00

0
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70

0
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8

2

1

0
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200

38

25

3

Number/ mm3

2,000

380

250

30

Number/ cm3

2,000,000

38,000

250,000

30,000

Cell count/
concentration

8,000,000

2,000,000

38,000

250,000

Absorbance

1
...
25

0
...
04

Yeast results

To count the number of cells or concentration in each culture, I placed one drop of each culture onto
a haemocytometer
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I then calculated
the number of cell in 1
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Using the result we obtained we plotted a graph, the graph e plotted
can be seen below:-

Conclusion
Based on the result I obtained from the experiment, I’d conclude that the higher the absorbance
rate of the culture being tested, the higher the amount of cells found in the culture
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Method:
1
...

2
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D
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3
...

4
...

5
...

6
...

7
...

8
...

9
...

10
...

11
...

12
...

13
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By looking at these photographs we can see that at different temperature controlled conditions
show different results, as you can see there is a significant different the photograph on the left hand
side shows that the enzymes become inactive at 40°, unlike the photograph on the left which slows
the enzyme activity down and the middle picture shows perfect conditions for mycelia growth
...
For the 4° the conditions are too cold meaning the enzymes will not work as well and slow
down there process
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Pouring agar plates to prepare ‘seeded’ plates for antibiotic discs
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They are stored in water baths at 50o C to keep them liquid
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1
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2
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coli’ and the other ‘B
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Remove a bottle of agar from water-bath
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coli to the agar, using the technique of flaming the wire loop
and bottle necks
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Turn the
Petri dish labelled ‘E
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Remember to flame the neck of the bottle
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Repeat with another bottle of agar, but using B
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5
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6
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7
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8
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Results:

As soon as the samples were incubated, the areas surrounding he antibiotic discs were examined
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There circles were measures to identify which antibiotic was the most effective
on which plate of culture
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Nutrient agar plates
2
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coli & B
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Glass spreaders, cork borers & forceps ( for making wells in agar) plus 100% alcohol for
flame-sterilising these
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Sterile 1 ml syringes
5
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Anti-bacterial surface cleaner (A
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C
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Label agar plate with bact
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Use sterile 1ml syringe – put 0
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culture in centre of agar
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Put spreader and syringe in ‘WASTE + contaminated pot’
3
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4
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S
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using sterile pipette
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Tape lid carefully without spilling liquid from wells
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Bacteria was grown on the rest of the agar and there were circles around the discs which did not
have grown bacteria
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Unit 15 – Assignment 3 - M2
Aseptic techniques
Aseptic techniques (such as positive and negative biocontainment) are carried out to prevent harm
to people or the environment around them, whilst eradicating the risk of contamination
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On the other hand, negative bio-containment protects the people and the

environment from being contaminated by the sample
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In addition, secondary
bio-containment is carried out to protect the environment outside of the lab from being
contaminated by the sample
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Operational practices are also carried out to prevent contamination
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The air pressure
inside is higher than the air pressure outside
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The positive air pressure will
move to the negative air pressure
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Negative biocontainment – Using a flow cabinet with the higher air pressure inside and a lower air
pressure outside
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The possibility of the air inside the cabinet escaping is eradicated
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Unit 15 – Assignment 3 – M3
Temperature and pH
35oC
350C
25oC
25oC

pH7
pH5
pH7
pH5

Log10number of cells at time
zero
0
...
2
0
...
1

Log10number of cells after two
hours
1
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5
1
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0

1
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Explain the calculations and set
your calculations out clearly
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806 - 0
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301 * 2) = (1
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602)) = 2
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83 times every hour
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5 - 0
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301 * 2) = (1
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602)) = 2
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159 times every hour
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3 - 0
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301 * 2) = (1
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602)) = 1
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827 times every hour
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1) / (0
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9 / (0
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495
This set of bacteria divided 1
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2
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Explain the calculations and set the calculations out clearly
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83 = 0
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35 hours to double in size
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159 = 0
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49 hours to double in size
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827 = 0
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547 hours to double in size
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495 = 0
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67 hours to double in size
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Which were the most favourable conditions for the growth of the bacteria? Explain your
answer
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This reason for this
was that under these conditions over the two hours, the bacteria was multiplied the fastest
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4
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The least favourable conditions for the growth of the bacteria was 25oC & pH5
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The
least favourable conditions were: a temperature lower than body temperature and a pH which was
acidic; this would make the bacteria use more energy to overcome the harsh conditions rather than
multiplying
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I held the loop in the outside part of the
roaring flame (as the outside is hotter than the middle), ensuring that the loop glows red
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Flaming glass neck of the bottle – Also, I ensured after every use of the glass bottle, that I passed the
neck of the bottle through the roaring flame
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The heat of the Bunsen
burner will also cause the air the area to increase
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However, firstly I did not perform this step efficiently
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If done for
too long, it would increase the temperature of the whole glass bottle and kill the bacteria inside,
instead of simply sterilising the neck of the bottle
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Then I simply passed the neck of the bottle through the flame
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As
I held the glass spreader in the flame for too long, the whole glass got heated and as soon as it hit a
cold surface, it shattered
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I learnt from this
error and instead of leaving the glass spreader in the flame, I simply passed it through the flame (like
the neck of the glass bottle); this improved the yield of which each colony was grown
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So I ensured that the lid was sealed to stop microorganisms from the air getting in and
contaminating the culture
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So, after I realised, I ensured that I washed my hands and sterilised the desks before and
after the procedures
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How Penicillin is manufactured
Small production method:
1
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It will start out grey but then overtime it will turn blue/green
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After you can see this, cut the bread up into pieces and place it into a sterilised flask
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Incubate this flask for around 7 days at 21 degrees Celsius
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Sterilise another flask and put the following things in:
o
o
o
o

500ml of cold tap water
44
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0 grams cornstarch
3
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25 grams magnesium sulfate
0
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75 grams glucose monohydrate
0
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044 grams manganese sulfate
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After this, add enough cold tap water to make one litre
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0 and 5
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The pH can be measured with litmus paper
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Then add the spores from the mouldy bread, and incubate for another seven days
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After incubation, the penicillin will be left floating in the liquid portion of what is there
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Industrial method:

Reference: http://scienceaid
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uk/biology/micro/biotechnology
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Also, if I were to compare the time it would take for each method to be
completed, I would find that the small scale method would take much longer to carry out, due to the
incubation periods and the time taken for the mould to grow
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However, in an industrial site, these errors would be kept to a
minimum, due to the advanced machinery used
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Unit 15 – Assignment 3 – D3
Liquid media and colorimetry
By observing my results, I can say that the growth of the culture of yeast (fungus) has a grows a lot
more, when a higher level of glucose is given, as opposed to when a smaller level of glucose is given
...
Oxygen is also used to release the energy from the glucose during respiration
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Preparation of media and inoculation of liquid media
By observing my results, I can say that the streptomycin disc had a greater effect on preventing the
growth of E-coli
...


Spreading on solid media
By observing my results, I can say that B-sub had a greater effect with the alcohol, whilst the E-coli
had a greater effect with the surface cleaner
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Growing mycelia discs
By looking at these photographs we can see that at different temperature controlled conditions
show different results, as you can see there is a significant different the photograph on the left hand
side shows that the enzymes become inactive at 40°, unlike the photograph on the left which slows
the enzyme activity down and the middle picture shows perfect conditions for mycelia growth
...

For the 4° the conditions are too cold meaning the enzymes will not work as well and slow down
there process
...


The effects of the antibiotics on the bacteria
Streptomycin: E-coli was effected the most by streptomycin
...
Streptomycin binds to the ribosome of the bacteria, which as a
result, prevents protein synthesis from occurring, causing the bacteria to be unable to multiply or
grow
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An outer membrane surrounds the thin layer of peptidoglycan
in the cell wall of the gram-negative bacterium
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An oxygen-dependant active pump
transports the streptomycin through the inner cytoplasmic membrane
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Penicillin: B-sub was effected the most by penicillin
...
This is because penicillin prevents the peptidoglycan molecules to cross-link in the
bacterium cell wall
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This means that because of the high external osmotic pressure, the cell wall of the bacterium will
burst
...
B-sub (and other
gram-positive bacteria) does not have any external layers of the cell wall, but on the other hand, Ecoli (and other gram-negative bacteria) does; it has a lipopolysaccharide layer surrounding the cells
wall, which protects the bacteria and prevents penicillin from having an effect
...
The results of this experiment would be used to
determine the optimum level of glucose, which is required for the fermenter in the initial growth
medium
...


Preparation of media and inoculation of liquid media
This experiment would be very useful for the industry, as they can be carried in order to find out
which antibiotic has the best effect of different forms of bacteria, and which would be able to fight
against which illness/bacterial infection
...
The experiment
could also be used to examine the effectiveness of the product, compared to other manufacturers
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Each
disinfectant would be tested to compare the areas around each disinfectant-soaked disk
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This would be used if a specific amount of an antibiotic is required of them
...


Growing mycelia discs
An experiment similar to this could be carried out in industry, as the manufacturers would need to
carry out these tests to find out what temperature is ideal for the incubation of penicillium
chrysogenum in the fermenters
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