Battling with Bacteria
Exploring the pH that kills bacteria
Contents
Background ........................................................................................................ 3
Planning a Test ................................................................................................... 4
Rules for Using Bacteria ................................................................................. 5
Culturing Bacteria .......................................................................................... 8
Testing the Bacteria at Different pH Levels .................................................... 9
Controlling the Variables.............................................................................. 11
Aim .................................................................................................................. 12
Hypothesis ....................................................................................................... 12
Materials.......................................................................................................... 12
Method & Setup .............................................................................................. 13
Safety Requirements........................................................................................ 24
Results ............................................................................................................. 26
Observations after 3 days in experiment vials.............................................. 26
Observations of agar plates after 3 days of growth...................................... 27
Observations of agar plates after 6 days of growth...................................... 32
Table of agar plates selected for counting colonies ..................................... 37
Graphs ............................................................................................................. 38
Discussion ........................................................................................................ 43
What have I found?...................................................................................... 43
Thinking about my results ............................................................................ 44
How could I make my test better? ............................................................... 45
Future Ideas ..................................................................................................... 45
Conclusion ....................................................................................................... 45
Project Acknowledgements ............................................................................. 46
Bibliography ..................................................................................................... 47
2
Background
My sisters always tell me that I am gross when I eat food that has fallen on the
ground. But I tell them not to worry because of the 5-second rule. This game
me and idea: Can I prove the 5-second rule.
I research on the internet to see if anyone had done this before. I found that
someone had won a Nobel Prize. It was Julian Clarke in 2003. I learnt food
would have to touch the surface for less than 1 Femtosecond. This is how long
it takes for the bacteria to stick to the food.
My question had already been answered so I had to think of a new testable
question. Even though there is bacteria on the food we don’t get sick. I could
try to find out what our immune system must does to protect us. Maybe I
could find out what our immune system does to protect us.
I know that our stomach has acid in it. Acid can kill bacteria. So I thought:
“Does the acid in our stomach kill all the bacteria that gets onto food in five
seconds?” Now I had a new testable question:
At what pH will bacteria be killed?
3
Planning a Test
I needed to make sure that I know what I am testing. If the stomach has a pH
of 1.5- 3.5 then I want to know if bacteria will be killed at this pH.
I had to think about how I would test this question. I spoke to two science
teachers to see what they thought.
They said I needed to:
1.
2.
3.
4.
Find out the rules for working with bacteria
Culture bacteria
Test the bacteria at different pH levels
Control the experiment
4
Rules for Using Bacteria
I talked with the school lab technician and she looked up the safety
requirements for microbiology activities in schools.
She said we could not subculture bacteria from wild cultures.
She explained to me that we were allowed to subculture bacteria that is
bought as a pure strain of non-pathogenic bacteria. (That means a bacteria
that will not cause disease.)
She also said that year 6 students should not do the subculturing. I would have
to find someone who knew how to subculture bacteria for me.
This meant I needed to purchase bacteria for this investigation.
Together with two of my science teachers and the lab technician we looked up
safe bacteria from Southern Biological.
I chose two types of bacteria:
1. B3B - Micrococcus luteus (previously Sarcina lutea), live broth
2. B5 - Bacillus subtilis, live slope
I chose M.luteus because it is commonly found in soil and vegetation. I chose
B.subtilis because it is found in soil, dust, water and air, and on skin. (See
Description of Bacteria on the following page.)
These two bacteria are probably some of the types of bacteria found on the
ground. These bacteria represent the bacteria on the ground that goes into
your mouth when you eat food that has been dropped on the ground.
5
They are also two different types of bacteria:
-
One is bacilli (these are rod shaped)
The other is cocci (these are spheres)
I looked up the information for the two bacteria on the Southern Biological
Website:
Bacillus subtilis is a Gram-positive, rod shaped bacterium commonly found in
soil and vegetation. It has the ability to form a tough protective endospore,
allowing the organism to tolerate extreme environmental conditions. B. subtilis
is not a human pathogen; it may contaminate food but rarely causes food
poisoning. The optimal temperature for growth is 25-35°C.
Item sold as a slope.
Micrococcus luteus is a Gram-positive, spherical bacterium, found in soil, dust,
water and air, and as part of the normal flora of mammalian skin. It also
colonizes the human mouth, mucous membranes, oropharynx and upper
respiratory tract. M. luteus can grow well in environments with little water or
high salt concentrations. They grow optimally at 37°C.
Item sold as a 10mL broth.
6
When you work with bacteria you must use the Safe Handling Techniques. I
found these in the safety document that comes with the specimens from
Southern Biological. This series of steps is called aseptic technique:
1. You must clean the loop in
the flame before and after
using it to transfer bacteria.
You must also flame the top
of the glass bottles before
and after transfer of bacteria.
2. You must also dip the spread
in ethanol and then burn it in the
Bunsen burner before and after
spreading bacteria on the plate.
3. The Bunsen burner keeps the air
clean so you have to work
within the area of the
flame.
4. You must have an
autoclave available to
sterilise the broth and
agar before and after the experiment.
5. When you apply the sample to the agar plate you have to hold the lid
over the plate to stop bacteria falling in. To apply the sample to the agar
plate we are using the spreading pattern shown below:
7
Culturing Bacteria
I needed to understand what culturing means. My science teacher told me that
I needed to be able to get bacteria from a culture and subculture it into a
medium that can be kept at different pH’s.
Here are some words I need to look up:
1.
Culture
The growing of microorganisms in a culture medium such as ager.
2.
Subculture
The process of transferring bacteria from one growth container to another.
This subculturing allows the analyst to change the conditions that the bacteria
grows in.
3.
Culture Medium
A liquid substance containing nutrients in which microorganisms, cells, or
tissues are cultivated for scientific purposes.
Here are some mediums that I found:
Medium
Cooked meat broth
Nutrient agar slopes
Semisolid nutrient agar stabs
Heated blood agar slopes
Feeley Gorman agar
Use
In general, used for preservation and
storage of bacterial cultures
Specialized media for isolation of
Legionlla
8
Testing the Bacteria at Different pH Levels
I now had a list of different types of culture mediums, so I needed to decide
which mediums I would use for my experiment.
I needed a culture medium that I would be able to control the pH of.
One of the science teachers that I spoke to found a way of growing and testing
bacteria in a liquid. It is called a broth culture.
My other science teacher said we could use buffers in a liquid to make it a
certain pH.
She explained to me that buffers are a solution that will stop a pH from
changing. She said that we could see if it worked in beef broth.
I needed to go to the laboratory with my science teachers to see if this new
idea would work.
At the laboratory, I made beef broth using beef stock and water. I had to filter
out the leaves. I put the broth culture into three small beakers. I used Universal
indicator paper to see what pH the broth was at. Then I crushed up buffer
tablets using a mortar and pestle. I had three different tablets. One made a
solution at pH 4, another at pH 7 and the third one at pH 10. I dissolved the
crushed buffer tablet in the beef broth. Then I used the Universal indicator
paper to see what pH the solutions were. It worked!
9
Observations:
The buffer solutions were pinky brown for pH 4, yellowy brown for pH 7 and
greeny brown for pH 10. When I checked the pH with the indicator paper it
game the following results:
pH Buffer
Indicator pH
value
4
7
10
5
7
9
I also decided to dilute the beef broth by adding more water because my
science teacher said it looked too concentrated and it might not work at that
concentration.
10
Controlling the Variables
What are the variables?
Variables:
pH
Bacteria in beef broth
Effect of buffer on bacteria
growth
Wild bacteria contaminating
agar plate
Temperature of lab
Light exposure to vials and
agar plates
Different concentration of
beef broth
Time for bacteria to grow in
vials
Time for bacteria to grow on
agar
Control:
Buffer tablet
One vial no bacteria
One vial has only beef broth and no buffer
Agar plate without subcultured bacteria
added
Cannot control this
Same cupboard same shelf
Made up one big amount then dilute it
Subculture on the same day for same time for
growth
Plated up on the same day for the time for
growth
To get an accurate number for bacteria colonies on an agar plate you have to
be able to count the number of colonies on the plate. If there are too many to
count you have to dilute the sample. We will dilute the sample so that I can
count the bacteria on the plate.
11
Aim
To find at what pH will bacteria be killed.
Hypothesis
Both types of bacteria will not survive in a medium of pH 4.
Materials
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Mass balance
Beef stock powder
Water
500mL beaker
Heat mat
Conical flasks
Filter papers
Filter funnels
Universal indicator paper
pH Indicator strips
Small glass vials
Marking pen
Autoclave
Buffer tablets: for pH 4, 7 and 10
A number of sterile syringes
Spatulas
Hot plate
Bunsen Burner
Inoculating loop
Nutrient agar powder
Glass stiring rod
Agar plates
Glass spreader
Cavicide
Ethanol
Micrococcus luteus sample from Southern Biological
Bacillus subtilis sample from Southern Biological
Sticky tape
 Computer
12
Method & Setup
Part A: Making the Beef Broth
This was done by me.
1. Use a mass balance to measure 20g of Beef Stock Powder. Put that
in 500ml of boiling water.
2. Stir till all powder has dissolve completely and wait till boiled.
3. Leave on a heat mat to cool down and then filter.
4. Use a conical flask, funnel and filter paper to filter the leaves and
herbs out.
5. Dilute the beef stock with 1 part Beef Stock and 7 parts water. (My
science teacher assisted me with this step.)
6. Use Universal indicator paper to measure the broth.
7. Get three vials and use a 10mL syringe to put 10mL of beef broth
into each vial.
8. Label one of the vials “CULTURE”, the second one “CONTROL –
BACTERIA – NO BUFFER”, and the third one “CONTROL – NO
BUFFER + NO BACTERIA”.
9. Put these to the side so they can be sterilised using the autoclave.
13
Part B: Making the Buffer Beef Broths
This was done by me with my science teachers.
1. Use a pH 4 buffer tablet.
2. Use a 10mL syringe three times to put 30mL’s of beef broth into a
vile.
3. Then put it straight into 30mL of the Beef Broth in a conical flask.
4. Put lid on and swirl till buffer is mostly dissolved.
5. Use Universal indicator paper to test what pH it is and to see if it
stays at the same pH. Test it once then wait two minutes then test
it again to make sure.
6. Repeat steps 1-4 for pH buffer tablet 7 and 10.
7. Get six vials and use a 10mL syringe to put 10mL of the buffer beef
broth into a vial. Do this twice for each buffer.
8. Before drawing the liquid, the solids at the bottom must have
settled down. Draw the liquid without getting any solids from the
bottom. (Make sure you use a different syringe for each of the
three buffer solutions so the solutions don’t mix.)
9. Label the vials “pH 4#1”, “pH 4#2”, “pH 7#1”, “pH 7#2”, “pH 10#1”
and “pH 10#2”.
10. Put these to the side so they can be sterilised using the
autoclave.
14
Part C: Preparing the Blanks.
This was done by my science teacher.
1. Get 16 vials and put 9.9mL of water in each vial using a syringe
(white lid so that it doesn’t get mixed up.)
2. Each of these vials will be labelled as we do the experiment.
3. Put these to the side so they can be sterilised using the autoclave.
4. Get 16 vials and put 9mL of water in each vial using a syringe (black
lid so that it doesn’t get mixed up with the white lidded vials.)
5. Each of these vials will be labelled as we do the experiment.
6. Put these to the side so they can be sterilised using the autoclave.
15
Part D: Sterilising all the Beef Broth Vials and the Blanks
This was done by my science teacher.
1. Put 2L of water into the autoclave.
2. Put the autoclave on a hot plate. Make sure the hot plate is
turned on high.
3. Place each vial into the autoclave.
4. Wipe Vaseline around the rim to seal the lid (stops gas getting
out.)
5. Close the lid of the autoclave and lock it down tight.
6. Heat until the autoclave reaches sterilising pressure (121°C). Let it
stay at this temperature for 20min.
7. Turn it off and let it cool off.
8. Once it has cooled down take the vials out ready to be used for
the experiment.
16
Part E: Subculturing the Bacteria into the Four Experiment Vials
This was done by my science teacher.
1.
2.
3.
4.
5.
6.
7.
8.
Make sure you are working within the flame area at all times.
Sterilise the bench using ethanol/ Cavicide.
Make sure you put the ethanol bottle right away from flame.
Flame the loop using a Bunsen burner on blue flame.
Flame the vial also using the Bunsen burner put the lid back on.
Flame the experiment vial Put loop into bottle to transfer bacteria.
Flame the vial again and put lid straight back on the vial.
Repeat this for each of the four vials.
17
Part F: Diluting the Bacteria
This was done by my science teacher.
1. Make sure you are still working within the flame area.
2. Open the lid of the experiment vial pH 4#1 and flame the vial.
3. Put the sterile syringe into the vial and get 0.1mL of experiment vial
pH 4#1.
4. Flame the vial before putting the lid back on.
5. Get the blank vial and flame it then put the 0.1mL of experiment
vial pH 4#1 into the blank vial with white lid (9.9mL).
6. Flame the blank vial before putting the lid back on.
7. Label the blank vial pH 4#1 – dilution 1.1
8. Repeat steps 1-7 to make a second dilution. Label it pH 4#1 –
dilution 1.2
9. Make sure you are still working within the flame area.
10. Open the lid of the experiment vial pH 4#1 – dilution 1.1 and
flame the vial.
11. Put a new sterile syringe into the vial and get 0.1mL of
experiment vial pH 4#1 – dilution 2.1.
12. Flame the vial before putting the lid back on.
13. Get a new blank vial with white lid (9.9mL) and flame it then
put the 0.1mL of experiment vial pH 4#1 – dilution 1.2 into the new
blank vial.
14. Flame the new blank vial before putting the lid back on.
15. Label the new blank vial pH 4#1 – dilution 2.2.
16. Get a new blank vial with black lid (9mL) and flame it then put
the 1mL of experiment vial pH 4#1 – dilution 2.1 into the new blank
vial.
17. Flame the new blank vial before putting the lid back on.
18. Label the new blank vial pH 4#1 – dilution 3.1.
19.
Get a new blank vial with black lid (9mL) and flame it then put
the 1mL of experiment vial pH 4#1 – dilution 2.2 into the new blank
vial.
18
20. Flame the new blank vial before putting the lid back on.
21. Label the new blank vial pH 4#1 – dilution 3.2.
22. Get a new blank vial with black lid (9mL) and flame it then put
the 1mL of experiment vial pH 4#1 – dilution 3.1 into the new blank
vial.
23. Flame the new blank vial before putting the lid back on.
24. Label the new blank vial pH 4#1 – dilution 4.1.
25. Get a new blank vial with black lid (9mL) and flame it then put
the 1mL of experiment vial pH 4#1 – dilution 3.2 into the new blank
vial.
26. Flame the new blank vial before putting the lid back on.
27. Label the new blank vial pH 4#1 – dilution 4.2.
28. Repeat the steps 1-27 and do it to the experiment vials pH 7#1,
pH 10#1, CONTROL- BACTERIA- NO BUFFER and CONTROL- NO
BUFFER + NO BACTERIA.
19
Diagram showing how the serial dilutions work:
Repeat this to make more dilutions if you need to.
20
Part G: Preparing Agar Plates and Putting Diluted Samples onto the
Agar Plates
This was done by me and my science teacher.
1. Measure 28g of nutrient agar powder.
2. Put 11.2g per 400mL of hot water (bring to boil-make up 1L of agar
solution)
3. Stir with glass stirring rod.
4. Let it cook.
5. When it begins to bubble take it off the hot plate.
6. Pour into glass bottle near a Bunsen burner flame.
7. Undo the lid of the glass bottle a little.
8. Put into the autoclave.
9. After it has been sterilised, set up a sterile area. (make sure that
during this process you work within the sterile area)
10. Put Bunsen burner on heat mat
11. Put the bottle of agar over the flame.
12. Pour the agar onto the agar plate – fill half way (hold the lid
over the plate to stop bacteria falling in)
13. Repeat steps 1- 12 to make 26 agar plates.
14. Use a syringe to place 1 mL on the agar plate.
15. Sterilise the glass spreader by dipping then end in alcohol and
then holding it over a Bunsen burner.
16. Use the glass spreader to spread the solution around the plate.
Close the lid of the plate and seal with tape.
21
Part H: Counting the Bacteria Colonies on the Agar Plates
This was done by me.
1. Look at each agar plate and see if you can count the number of
bacteria colonies. If the number of colonies is bigger than 50
than you go to the next dilution level.
2. A good range for counting bacteria colonies is 10 – 50 colonies.
3. Record your observations of the types of bacteria and other
microorganisms.
22
Part I: Calculating the Amount of Bacteria in the Experiment Vials.
This was done by me using Excel on a computer.
1. Choose the agar plates for each vial that have the most bacteria
but less than 50.
2. Enter the number of bacteria colonies into Excel. Multiply each
count by the dilution level. (Level 2 is x1000, Level 3 is x
100 000, Level 4 is x1 000 000)
3. Find the average of the two copies for each vial.
4. Graph the average bacteria count against the pH of the
solution.
You can make different types of graphs depending on what you want
to see.
23
Safety Requirements
Material
Agar plate
Alcohol
thermometer
Autoclave
Beakers
Beef Broth
Powder
Bunsen Buner
Box of Matches
Risk
Agar is harmless,
bacteria may be
dangerous
Breakage of
thermometer.
Control
Don’t incubate at above 37°C.
Only use group 1 organisms.
Autoclave may release
steam violently from
the emergency vent. If
it becomes blocked of if
it fails, it might explode.
Make sure you are standing
away from the emergency
vent. Check it is fitted with an
emergency over pressure.
Don’t put small items in the
autoclave that could block it.
Check that it has no cracks (no
matter how small the damage.)
sweep up broken glass.
Do not eat in lab, due to
chemical contamination.
Do not put hand near the
flame.
Keep dry. Used matches should
never be returned to the box.
Count boxes out and in.
Do not heat up flammable
liquids on hotplate.
Breakage of beaker, cut
from sharp rim.
Harmless
It may burn your hand
at roaring flame.
Box burns violently if
ignited.
Electronic
hotplate
Ignition source, may
cause burns
Filter paper
Harmless
Conical flask
Breakage of beaker, cut
from sharp rim.
Sweep up broken glass with a
brush and dustpan.
Dispose of residue and filter
paper in bin.
Check that it has no cracks (no
matter how small the damage.)
sweep up broken glass.
24
Glass bottle
Breakage of beaker, cut
from sharp rim.
Glass funnel
Breakage of beaker, cut
from sharp rim.
Glass stirring rod
Breakage of beaker, cut
from sharp rim.
pH Indicator
paper strips +
Universal
indicator
Syringe
Bacillus subtilis
Harmless
Micrococcus
luteus
Not pathogenic.
Harmless
Not pathogenic.
Check that it has no cracks (no
matter how small the damage.)
sweep up broken glass.
Check that it has no cracks (no
matter how small the damage.)
sweep up broken glass.
Check that it has no cracks (no
matter how small the damage.)
sweep up broken glass.
Store in a dry place
Dispose of in bin after use
Follow safe handling
procedure for subculturing
bacteria. Wear labcoat, gloves
and safety glasses when
looking at plates with bacteria
cultures.
Follow safe handling
procedure for subculturing
bacteria. Wear labcoat, gloves
and safety glasses when
looking at plates with bacteria
cultures.
25
Results
Observations after 3 days in experiment vials
Vial pH
pH 10 pH 8.5
Description
It has a very thin line of cloud at
the bottom of the vial. This
means there is bacteria in this
vial.
pH 7 pH 6.5
It almost as same as pH 10 but
the cloud is a bit thicker.
pH 4 pH 4.5
This vial has a thicker line of
cloud than pH 10 and pH 7 even
though it is at a pH of 4.5 and is
very acidic.
No Bacteria pH 7
No Buffer pH 6.5
It has more cloud at the base
than pH 4, 7, 10.
There is no sign of bacteria at all.
26
Observations of agar plates after 3 days of growth
2.1
Close to 17 very
small specks of
bacteria.
Agar Plates for pH 4
2.2
About 6 black
specks of
bacteria.
3.1
1 large cream dot
and 1 large
feathery type of
bacteria.
3.2
10 small dots of
bacteria and lots
of black specks of
bacteria.
4.1
1 cream dot of
bacteria and lots
of small black
specks of
bacteria.
4.2
NO BACTERIA
27
2.1
2 small specks of
bacteria
Agar Plates for pH 7
2.2
NO BACTERIA
3.1
1 feathery white
type of bacteria.
3.2
2 cream dots of
bacteria and 2
black specks of
bacteria.
4.1
3 small specks of
bacteria.
4.2
1 cream speck of
bacteria.
28
2.1
Cloudy but no
but no obvious
growth.
Agar Plates for pH 10
2.2
NO BACTERIA
3.1
About 20 small
black specks of
bacteria.
3.2
About 9 small
black specks of
bacteria.
4.1
About 14 small
black specks of
bacteria.
4.2
2 cream dots of
bacteria.
29
1.1
Lots of small
black specks of
bacteria (too
many to count.)
Agar Plates for No Buffer
1.2
Almost the same
as 1.1 but the
colony is a bit
smaller
2.1
About 15- 20
small black
specks of
bacteria and 3
small cream
dots.
2.2
NO BACTERIA
3.1
NO BACTERIA
3.2
About 10 small
black specks of
bacteria and 1
cloudy spot.
4.1
NO BACTERIA
4.2
1 big colony with
too many to
count in it.
30
Agar Plates for No Bacteria
No bacteria (just beef broth)
1 large cream dot of bacteria
No Subculture #1 (just agar)
Cloudy patches with lots of very, very tinny specks of
bacteria.
No Subculture #2 (just agar)
About 20- 30 specks of bacteria growing.
31
Observations of agar plates after 6 days of growth
2.1
0 bacteria
3.1
1 brown on the
outside & white
(centre) fungus
4.1
1 med bacillus
white o.5mm
Agar Plates for pH 4
2.2
6 pink wild
1 micrococcus
yellow
1 bacillus 0.5 mm
white
3.2
6 bacillus
8 micrococcus
4.2
0 bacteria
32
2.1
2 fungi
1 is white [fungi]
1 is grey [fungi]
Agar Plates for pH 7
2.2
0 bacteria
3.1
1 fungi really lge
white fungi.
1 bacillus 0.5
3.2
7 bacillus white 1
is med about
0.5mm
4.1
0 bacteria
4.2
1 cream wild
strain bacteria.
33
2.1
3 micrococcus
yellow
O bacillus
Agar Plates for pH 10
2.2
1 micrococcus
yellow
0 bacillus
3.1
0 bacteria
3.2
0 bacteria
4.1
1 bacillus med
0.5mm wild
4.2
4 bacillus med
0.5mm wide
34
1.1
Too many
2.1
18 micrococcus
yellow
1 bacillus white
3.1
No bacteria
4.1
1 bacillus
Agar Plates for No Buffer
1.2
Too many
2.2
5 micrococcus
yellow
3 bacillus white
3.2
2 micrococcus
On the rim 1 lge
colony of pink wild
strain another lge
wild strain of
yellow (may be micrococcus)
4.2
1 wild strain smll
pink
1 lge white fungi
35
Agar Plates for No Bacteria
No bacteria (just beef broth):
1 lge white fungi (hairy)
1 lge wild strain bacteria
No Subculture #1 (just agar):
no bacteria
No Subculture #2 (just agar):
no bacteria
36
Agar Plates for Bacteria That We Bought
Micrococcus: lge colonies white circular bacteria
Bacillus: lge colonies yellow circular bacteria
Table of agar plates selected for counting colonies
No Buffer
Experiment
Vials
pH 4
pH 7
pH 10
(only beef broth +
bacteria)
Agar plates
used for
the count
3.1
(x100 000)
3.2
(x100 000)
3.1
(x100 000)
3.2
(x100 000)
2.1
(x1000)
4.2
(x1 000 000)
2.1
(x1000)
2.2
(x1000)
The count for the selected agar plates were calculated then I took the average
of the two plates for each vial. I also recorded the true pH reading that I got
from the pH indicator strips.
37
Graphs
Here are the graphs I made with my results:
Graph 1:
pH v Number of Bacteria Colonies
Total Number Of Bacteria Colonies
2500000
2000000
1500000
4.5
6.5
1000000
8.5
6.5
500000
0
1
2
3
4
5
6
7
8
9
pH
Table for Graph 1:
Information
Average Total
Bacteria
True pH
pH 4
pH 7
pH 10
No Buffer
750000
450000
2001500
13500
4.5
6.5
8.5
6.5
38
Graph 2:
pH v Total Number of Bacteria Colonies for Buffer Vials Only
Total Number Of Bacteria Colonies
2500000
2000000
1500000
4.5
6.5
1000000
8.5
500000
0
1
2
3
4
5
6
7
8
9
pH
Table for Graph 2:
Information
Average Total
Bacteria
True pH
pH 4
pH 7
pH 10
750000
450000
2001500
4.5
6.5
8.5
39
Graph 3:
pH v Number of Micrococcus Luteus Colonies
Total Number Of Bacteria Colonies
450000
400000
350000
300000
250000
200000
150000
100000
50000
0
-50000
1
2
3
4
5
6
7
8
9
pH
Table for Graph 3:
Information
Average Total
Bacteria
True pH
pH 4
pH 7
pH 10
400000
50000
1500
4.5
6.5
8.5
40
Graph 4:
pH v Number of Bacillus Subtillus Colonies
Total Number Of Bacteria Colonies
2500000
2000000
1500000
1000000
500000
0
1
2
3
4
5
6
7
8
9
pH
Table for Graph 4:
Information
Average Total
Bacteria
True pH
pH 4
pH 7
pH 10
350000
400000
2000000
4.5
6.5
8.5
41
Graph 5:
Bacteria Colonies Count on Experiment Vials
2500000
Number of Bacteria Colonies
2001500
2000000
1500000
Wild Strain
Bacillus
1000000
Micrococcus
750000
450000
500000
13500
1
0
no buffer
no bacteria
only agar
0
pH4
pH7
pH10
Table for Graph 5:
Average
Count
Average Total
Micrococcus
Average Total
Bacillus
Average Total
Wild Strains
Average Total
Bacteria
pH 4
pH 7
pH 10
No
Buffer
(only beef
broth +
bacteria)
No
Bacteria
Only
Agar
(only beef
broth)
(no beef
broth + no
bacteria)
400000
50000
1500
11500
0
0
350000
400000
2000000
2000
0
0
0
0
0
0
1
0
750000
450000
2001500
13500
1
0
42
Discussion
What have I found?
By just looking at the graphs I get the following results:
 The trend line for Graph 1 shows that as the pH reaches around 6, the
growth of the bacteria decreases. This was different to my hypothesis.
Bacteria growth is better at a lower pH than 6 and a higher pH than 6.
Growth is best at around pH 8.5.
 The vial with bacteria and no buffer grew bacteria colonies really well.
But my graphs seem to show that the Buffer vial pH 7 grew better. If you
look at the photos this is not true.
 This is really strange. So my graphs are not telling the truth.
 From my graphs I could not calculate how much bacteria would grow in
a solution with a pH similar to the human stomach because my graphs
are not telling the truth.
 The buffer vials did not have a dilution pattern. But the No Buffer vials
worked really well. You can see this from the photos.
 There was a very small amount of wild strain bacteria in the beef broth
but this must have contaminated it after it was sterilised in the
autoclave. It could have fallen out of the air into the plate.
 The agar plate was clean so the agar was sterile.
43
Thinking about my results
 My results are very random. I know this because the results for the No
Buffer Vial show a good dilution of bacteria. However, the buffer vials
for pH 4, 7 and 10 do not have a good dilution pattern.
 The real thing that told me there was something wrong, was the No
Buffer vial and the Buffer pH 7 vial had the same pH but the results were
so different.
 If my test was accurate they would have had similar results. This means
that the graph would have had their points close together.
 This means that my data for the buffer vials are not accurate at all.
 Graph 3 shows the trend line for Micrococcus. Micrococcus looks like it
grows better in more acidic pH conditions.
 Graph 4 shows the trend line for Bacillus. Bacillus looks like it grows
better in more basic pH conditions.
 It is hard to tell whether this is true because the two bacteria may be
competing with each other. This means that the trend I am getting may
be because of the competition of the bacteria not the pH conditions.
 I know that the buffer vials are not accurate so something is wrong with
my method.
44
How could I make my test better?
 I think I might need a better buffering technique. The buffer solutions
that I used are for calibrating pH probes so they may be toxic for
bacteria.
 There were not really enough bacteria colonies to count in any of the
buffer vial agar plates for my results to be accurate.
 The results for the No Buffer Vial show that the technique that was used
for subculturing and diluting the bacteria was correct.
 To see if the bacteria are competing with each other I would grow them
separately in different pH conditions.
Future Ideas
 Find out what buffers are used by people who grow bacterial cultures.
 I cannot test pathogenic bacteria in school so maybe when I get to
university I could do further research using wild types of bacteria.
 Test the two bacteria separately because they may be competing against
each other.
 Give myself more time so that I can repeat the experiment or adjust how
I do it to try and get better results.
Conclusion
I was not able to answer my testable question. This was because the buffer I
used was not the right one and I think it killed the bacteria. I did find out that
the technique used for subculturing the bacteria and diluting it for agar plates
was correct. By changing my method I may be able to find out if the two
bacteria are competing and see how they behave when grown separately in
different pH conditions. If I could find a better way of controlling the pH
without killing the bacteria, then I may be able to find out the pH that bacteria
will die in. I think the real title of my project should be battling with the
buffers.
45
Project Acknowledgements
I would like to thank:
 Ian my cousin for helping me think of an idea for my science
project.
 The lab technician who helped me plan my experiment and make
sure it was safe.
 My science teacher, Joel Poyitt for helping me carry out the
subculturing and serial dilutions and helping me with my
calculations.
 My other science teacher, Mursell Cannon for helping me write up
my report and helping me count the bacteria. She also showed me
how to make the graphs in Excel.
 Southern Biological for sending the bacteria so quickly.
46
Bibliography
Websites that I used:
 https://en.wikipedia.org/wiki/Five-second_rule
 http://news.aces.illinois.edu/news/if-you-drop-it-should-you-eat-itscientists-weigh-5-second-rule
 http://laboratoryinfo.com/wpcontent/uploads/2015/03/bacteria-types.jpg
 http://www.southernbiological.com/specimens/microbiological
-specimens/bacteria-risk-group-1-suitable-for-schools/b5bacillus-subtilis-live-slope/
 http://www.southernbiological.com/specimens/microbiologicalspecimens/bacteria-risk-group-1-suitable-for-schools/b3b-micrococcusluteus-previously-sarcina-lutea-live-broth/
 medical-dictionary.thefreedictionary.com/bacterial+culture
 https://www.reference.com/science/purpose-subculturingmicrobiology-f4811e04e877cbdf
 www.thefreedictonary.com/culture+medium
 www.microbiologyinfo.com/list-of-culture-media-used-in-microbiologywith-their-uses/
 Microbiology Safety Considerations.This guide for Australian schools is
made available through the courtesy of the author, S K
Hoffmann. http://file.southernbiological.com/Assets/Products/Specime
ns/Microbiological_Specimens/MicrobiologySafetyConsiderations.pdf
Online videos that I used:
 https://www.youtube.com/watch?v=rYXdsOEWBj0
Pictures that I have used:
 http://www.duskyswondersite.com/wpcontent/uploads/2012/02/bacteria-12.jpg - Front Cover Picture
47
Books that I have used:
 Chemical Safety in Schools Manual
 Laboratory Exercises in Organismal and Molecular Microbiology
Alexander, S.K. Strete, D and Niles, M.J. (2004) Laboratory Exercises in
Organismal and Molecular Microbiology Boston: McGraw Hill higher
Education, p123.
 MICROBIOLOGY - Safety Considerations by Sheryl K. Hoffmann,
Concordia College
http://file.southernbiological.com/Assets/Products/Specimens/Microbio
logical_Specimens/MicrobiologySafetyConsiderations.pdf
 Microbiology 1 - Laboratory Manual University of Western Sydney Autumn Semester 2011, Methods for Sampling from a broth Culture
p.49 - 79.
48