APPARATUS:
RISK ASSESSMENTS:
During an experiment, precautions have to be taken to prevent yourself, and people around you getting hurt.
For this experiment, I will put all bags under desk to prevent anyone tripping up; I will wear a lab coat and gloves. I will wipe down my work surface and clean all apparatus. I will also discard the agar piece I removed properly to prevent infection and contamination. The bacteria have to be killed. When I am not using the flame on the Bunsen burner I will change the flame from blue to yellow so people are aware that it is on.
PRELIMINARY WORK:
Preliminary work is carried out to test my techniques and make sure concentrations are all suitable. During my preliminary work I just tested the two brands of toothpaste on one bacterium. Staphylococcus albus.
My preliminary work proves that my experiment works and I can now go on to test the two toothpastes on four more bacteria.
The results to my preliminary work were:
AREA OF INHIBITION (mm2)
The preliminary work helped me to plan my experiment and gain experience with the experiment’s processes. The technique works.
Through looking at my results I can see that the more expensive toothpaste has killed the most bacteria because there are more squares covered by the clear space. There is a larger area of inhibition. This means that the preliminary has proved my hypothesis which I hope to go on to do in the real experiment.
Improvements:
The preliminary experiment has allowed me to make note of improvements I need to make for my real experiment. To make sure I don’t contaminate the bacteria with unwanted bacteria I will make sure to use a different cork borer each time, therefore ensuring I do not encounter cross-contamination. I only did the preliminary once but for the real thing I will definitely repeat the experiment to get an average and my results should be more accurate. From the preliminary experiment I realised that pH, and oxygen conditions could not be controlled, these factors will have to be taken into consideration when I analyse my results. The results should still prove my hypothesis but they won’t be as accurate as I had hoped.
STATISTICAL TEST:
I have decided to use the Mann Whitney U Test for my statistical analysis.
Null hypothesis:
There is no significant difference between the effects on the toothpaste with a high alcohol content on bacterial growth compared to the toothpaste with a low alcohol content.
The Mann Whitney U Test is used to test the difference between two sets of data. If the U value is lower than or equal to the critical value then it is positive to reject the null hypothesis and accept that there is a significant difference between the two sets of data.
INTRODUCTION:
I will test two different brands of toothpaste on five different gram-positive bacteria and analyse the results of growth.
I have chosen to use gram-positive bacteria rather than gram-negative because of the effect that the toothpaste will have on the gram-positive. The experiment will work much better, this is because of the structure of a gram-positive bacteria.
From a previous gram staining experiment I know that the two bacterial cells look very different following staining with the Gram stain. Gram-positive cells are purple and gram-negative are red.
The basis for this differential reaction relates to the cell wall. The knowledge gained from gram staining helps me with the scientific background to apply to my experiment. Looking at the two different electron micrograph pictures below I can see that the gram-negative cell has an additional layer and from the external view of the cell the outside is convoluted. (Not really obvious in the below photo).
The gram-positive wall is much thicker then the gram-negative and from the external view has a smoother appearance. The gram staining experiment helps us realise that there is a difference in structure of the two bacteria which can then lead to certain effects. In both gram-positive and negative cells they both have peptidoglycan in common. Most cell walls contain peptidoglycan; it is a thick rigid layer. It is composed of an overlapping lattice of two sugars that are cross linked by amino acid bridges. The two sugars are N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM). A distinguishing factor among gram-positive bacteria is that roughly 90% of their cell wall is comprised of peptidoglycan and a gram-positive bacteria can have more than twenty layers of peptidoglycan stacked together to form the cell wall which causes it to be very thick. In the gram-negative bacteria the peptidoglycan is much thinner with only 15-20% of the cell wall being made up of peptidoglycan and this is only intermittently cross-linked.
Here are two diagrams of the gram bacteria showing there structure and contents:
As seen in the above diagrams, the gram-negative bacteria are a much more complicated organism and therefore the toothpaste would have a better effect on the gram-positive bacteria. My results would be clearer. The complexity of the gram-negative bacteria would lessen the effect of the toothpaste.
We brush our teeth in order to remove bacteria from the mouth and to remove left over food particles. Bacteria use left over food particles as an energy source. Bacteria break down these food particles producing acids which attach tooth enamel. This bacterial break down of food particles also produces sulphur compounds which give breath its foul smell. Looking at the ingredients of both brands of toothpastes may help me come to a conclusion for my experiment.
SAINSBURYS OWN BRAND:
Active ingredients: Sorbitol
Aqua
Hydrated silica
Glycerin
Aroma
Sodium lauryl sulphate
Sodium monofluorophosphate
Cellulose gum
Sodium saccharin
Hydroxyethylcellulose
Sodium hydroxide
Cl 18965, Cl 4205
COLGATE:
Active ingredients: Sodium fluoride
Triclosan
Ingredients: Copolymer
Hydrated silica
Glycerine
Sorbitol
Sodium lauryl sulphate
Cellulose gum
Titanium dioxide
Sodium saccharin
Carrageenan
Sodium hydroxide
Water
When comparing the toothpastes there is not much difference between the ingredients. The main ingredient that has the biggest effect on the experiment would be Triclosan. Colgate total is currently the only toothpaste containing the ingredient. It is an antibacterial agent. It is also clinically proven to fight gingivitis in adults by inhibiting the growth of bacteria.
In toothpastes the different types of alcohol has a major effect on the growth of bacteria. The alcohols, especially Triclosan, denature the proteins and dissolve the lipids in the cell membrane in the gram-positive bacteria. This is why I think that the experiment will prove my hypothesis.
“The toothpaste containing higher levels of alcohol ingredients will have a greater effect of inhibition on the growth of bacteria than the toothpaste containing low levels of alcohol ingredients. This means that Colgate toothpaste will work better than the Sainsbury’s own brand.
METHOD:
This method is exactly what I did and why I did what I did.
The first thing to do was make sure that experiment was under aseptic conditions, this meant wiping down all the work surfaces with ‘Milton’, and washing hands at the start. Next thing I did was to heat the inoculating loop in the Bunsen burner, on the blue flame, until the loop was red hot. When I was heating the loop I held the loop upright so that the bacteria did not fall on my hands. This killed any unwanted bacteria. Then I left the loop for about ten seconds. This allowed it to cool down and prevented killing the wanted bacteria.
I then collected the five different bacteria already growing on the agar plate. I removed the lid slightly from the plate containing the bacteria. This reduced the contamination from outside air. When the loop had cooled, I inserted it into the plate avoiding contact with the stem of the loop and the top of the plate. Once again, avoiding cross-contamination. I wiped the loop over the slime until the loop was full will bacteria. Then using the prepared set agar plate, I opened the lid and swiped the loop over the set jelly without breaking it.
I swiped the loop down the middle only third of the way. Then I swiped it across so the jelly was covered. Whilst doing this I moved the Petri dish around and for the last third of the plate I moved the loop in a wider zigzag motion until the loop ends up where the streaking had began. I added cello tape at three different points around the dish and then flamed the loop again to kill the bacteria.
Next I labelled the Petri dish with its contents and put it in the incubator. This was repeated for all five bacteria.
Once all five of the bacteria were incubated for twenty four hours and allowed to grow, I transferred the bacteria into sterile distilled water. I then checked the growth of the bacteria on the cloudiness of the water. The bacteria were transferred from the perti dish to the distilled water bottles by using the inoculating loop. I heated it over the Bunsen burner until red hot which killed any microbes. The loop was then cooled and swiped across the incubated bacteria. Then I opened the lid of the first distilled water bottle with thumb and fourth finger trying not to touch the neck of the bottle. I flamed the neck of the bottle to keep it sterile and then inserted the loop into the bottle and gently shaked the loop so that the bacteria was released into the water. To finish I replaced the lid and labelled the bottle and incubated for a further 24 hours. This was once again repeated for each bacterium.
When the water had been incubated it was added to one of the twenty fresh agar plates. To ensure a fair test, the whole surface of the agar plate was covered with the bacteria before the toothpaste was inserted.
The twenty agar plates had been labelled with the bacteria being tested on and the brand of toothpaste so that the experiment had been organised and it avoided and cross-contamination.
I flamed the neck of the bottle I was about to use and then I used a 1ml syringe I transferred 1 ml of the liquid containing the bacteria from the bottle, into the centre of the first new agar plate. I then swirled the bacteria around so eventually a thin layer covered the whole dish.
After the bacterium was covering the whole plate I sterilised a cork borer by flaming it and then I used it to make the single hole in the centre of the dish. The hole is where the toothpaste was added later.
Next I placed the piece of agar jelly that had been removed by the cork borer into an empty Petri dish so that it was disposed properly without contaminating others. I then inserted 1cm3 of one of the toothpastes into the hole made. I tried to keep the toothpaste as neat as possible and avoided spilling it over the sides. I then incubated all twenty plates for another twenty four hours. Bacillus stereothermophilus was incubated at 50°C because this was its optimum temperature.
After the incubation process I had to measure the results. I measured the area of inhibition around the hole to the nearest millimetre by holding the dish on a light box with 1mm2 paper on top. I then drew around the area of inhibition and counted how many squares that the clear area had covered.
RESULTS:
- Staphylococcus Albus
- Micrococcus luteus
- Bacillus subtilis
- Bacillus megaterium
- Bacillus stereothermophilus
1. Colgate total:
Bacteria
2. Sainsbury’s own brand:
Bacteria
I used a histogram to record my results. A histogram could be thought of as a graph of frequency distribution.
Using my results I have put them into a table of frequency. I did a histogram for each of my toothpastes.
Colgate:
Histogram on graph paper.
Sainsbury’s
Histogram on graph paper
ANALYSIS:
Statistical test:
Table of the average area of inhibition for each toothpaste
Table of data in increasing order for COLGATE TOTAL:
Table of data in increasing order for SAINSBURYS OWN BRAND:
Data Rank Toothpaste
1. 58 1 Sainsbury’s
2. 253.5 2 Sainsbury’s
3. 341 3 Sainsbury’s
4. 370.5 4 Sainsbury’s
5. 433 5 Sainsbury’s
6. 439.5 6 Colgate
7. 569.5 7 Colgate
8. 624 8 Colgate
9. 673 9 Colgate
10. 699.5 10 Colgate
Sum of rank 1= 15
Sum of rank 2= 40
Formulae to calculate U1 and U2
R1 (Rank 1) R2 (Rank 2)
n1 = number of average Sainsbury’s values
n2 = number of average Colgate values
Sainsbury’s toothpaste:
U1 = n1 x n2 + n2 (n2+1) - R1
= 5 x 5 + 5 (5+1) - 15
= 25 + 5 (6) - 15
2
= 25 +15 -15
U1 = 25
Colgate toothpaste:
U1 = n1 x n2 + n2 (n2+1) - R2
= 5 x 5 + 5 (5+1) - 40
= 25 + 5 (6) – 40
2
= 25 +15 – 40
U1 = 0
The smallest U value is taken U = 0
When the value of n1 is 5 and the value of n2 is 5, the critical value is 2. The smallest U value, 0 is less than the critical value of 2. The null hypothesis is rejected.
“There is no significant difference between the effects on the toothpaste with a high alcohol content on bacterial growth compared to the toothpaste with a low alcohol content.”
In order to eliminate terms like certainly or probably, and state whether or not there is a significant difference, the critical value is used as a cut-off point along the scale of overlap.
As the smallest U value is under the critical value it means there is no overlap and it is possible to be certain there is a significant difference.
The null hypothesis is rejected at the p=0.005 or 5% level.
P< 0.005
This means there is a 5% confidence level that there is only a 5% chance that the null hypothesis is incorrect.
