The independent variable was the Pasteur pipettes which I had to keep changing every time I used one so that my solutions will not get contaminated. My variables are continuous. This means that each time I done the experiment I had to do the same thing over again, therefore they are continuous.
APPARATUS:
- Six universal bottles, or capped containers – each containing 9.0cm3 of sterile, distilled water
- Twelve sterile Pasteur pipettes – plugged with cotton wool
- 1cm3 plastic syringe, fitted with a silicon rubber connector, to attach to Pasteur pipettes
- Six sterile Petri dishes
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Suitable culture for counting, e.g. E.coli or sample of pasteurised milk
- Supply of suitable agar medium, molten, kept in water bath at 45°C
- Bunsen burner
- China graph pencil or spirit marker pen
- Discard jar containing disinfectant
- Incubator at 30°C
- Adhesive tape
- Alcohol
- Ruler
The different items must be the same amount as each other and these measurements must be accurate due to incorrect results. To make sure my results are reliable I will make sure I count the cells of bacteria twice so I know if I have made any errors.
HEALTH AND SAFETY:
- Wear protective clothing (gloves)
- Wear eye protection (safety glasses)
- Tie hair up
- Make sure you don’t throw the plastic syringe and sharpened pencil around due to people being stabbed by a pencil
- Make sure hands are washed before and after the experiment, thoroughly with soap and water.
- Working area must be clean during work
- Must be aware of contamination
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Everything must be labeled correctly due to confusion and a mix up in solutions
- Industrial Methylated Spirit is highly flammable to be careful
- If the alcohol in the beaker catches on fire, cover the beaker with a damp cloth
PROBLEMS WITH PLATE COUNTS:
- They need long incubation for colonies to even show
- When cell clump, they can guide to an error in counting the viable cells
- It is extremely simple to have too less or too many colonies on a plate to precisely measure viable count
- Avoidance of squashing usually involves serial dilution
TO AVOID CONTAMINATION OR OTHER PROBLEMS:
- Wash hands with soap thoroughly before and after experiment
- Disinfect table before and after experiment
- Ensure lid of the plate is not took off completely
- Do not even put the lid on the table so other bacteria does not get onto plate
- Do not cough or sneeze on the plates
- Work near bunsen burner
METHOD:
Set up equipment.
Label containers of sterile distilled water 10-1, 10-2, 10-3, 10-4, 10-5 and 10-6 and the Petri dishes similarly.
Label the Petri dishes on their bases.
Shake the sample thoroughly to ensure that it is evenly mixed. Then using aseptic technique, transfer 1.0cm3 of the container labeled 10-1, using the sterile pipette.
After use, place the pipette into the discard jar of disinfectant.
Mix this first dilution carefully then.
Using a fresh sterile pipette each time, transfer a 1.0cm3 sample of each dilution separately to each appropriate, labelled Petri dish.
Again, using aseptic technique, carefully pour cooled, but molten, sterile agar medium into each Petri dish.
Swirl each Petri dish very carefully to ensure that the samples and the agar are evenly mixed.
Gently move each dish in a figure of eight pattern, but do not allow the agar to spill over the edge of the dishes.
Allow the agar to set, and then fasten each lid with 2 pieces of adhesive tape.
Invert the dishes, and incubate at 30°C.
After incubation, count the number of colonies present in a dish containing a suitable dilution.
Calculate the number of viable cells present in 1.0cm3 of the original culture.
As an alternative to pipetting a 1.0cm3 sample into each Petri dish and then adding molten medium, a 0.1cm3 sample may be transferred to a ready poured agar plate.
The sample is then spread uniformly over the surface of the agar medium using an alcohol flammed glass spreader.
Following a couple of days, various sorts of microbes grow as divided colonies. Cells from separate colonies could be picked up for a subculture.
IMPLEMENTING
This was a very quick process in which everything had to be complete straight after another. Therefore, measurements also should have been done quickly during the experiment straight away to put into whichever solution it may have been.
My results have been recorded according to how much attempts I made. In each attempt, I have shown the dilution factor and how many cells I saw in each square using the see through round scale. The see through round scale had 64 squares in it. Some squares were completely filled therefore I have written that down too.
I done three replicate to ensure my results were accurate.
RESULTS:
Experiment attempt one:
10-1 → 144 = 1 square
10-2 → 96 = 1 square
10-3 → 65 = 1 square
10-4 → 33 = 1 square = 14 boxes complete
10-5 → 30 = 1 square = 11 boxes complete
10-6 → 22 = 1 square = 14 boxes complete
Experiment attempt two:
10-1 → 170 = 1 square
10-2 → 145 = 1 square
10-3 → 111 = 1 square
10-4 → 44 = 1 square
10-5 → 8 = 1 square
10-6 → 5 = 1 square
Experiment attempt three:
10-1 → 187 = 1 square
10-2 → 100 = 1 square = 2 boxes complete
10-3 → 55 = 1 square = 15 boxes complete
10-4 → 23 = 1 square = 31 boxes complete
10-5 → All squares complete
10-6 → 17 = 1 square
In one see through circle were 64 squares so…
Experiment attempt one:
10-1 → 144 x 64 = 9216
10-2 → 96 x 64 = 6144
10-3 → 65 x 64 = 4160
10-4 → 33 x 64 = 2112 + 14 = 2126
10-5 → 30 x 64 = 1920 + 11 = 1931
10-6 → 22 x 64 = 1408 + 14 = 1422
Experiment attempt two:
10-1 → 170 x 64 = 10880
10-2 → 145 x 64 = 9280
10-3 → 111 x 64 = 7104
10-4 → 44 x 64 = 2816
10-5 → 8 x 64 = 512
10-6 → 5 x 64 = 320
Experiment attempt three:
10-1 → 187 x 64 = 11968
10-2 → 100 x 64 = 6400 + 2 = 6402
10-3 → 55 x 64 = 3520 + 15 = 3535
10-4 → 23 x 64 = 1472 + 31 = 1503
10-5 → All squares complete
10-6 → 17 x 64 = 1088
FINAL RESULTS
My results show that as the dilution factor increases the amount of colonies decrease, as stated in my hypothesis. The decrease is shown as exponential, also there no peaks.
According to my results, the values are quite variable, but as predicted. The maximum value in average is at the 10-1 dilution factor, 10688 and the minimum value in average is at the 10-6 dilution factor, 943.3.
Here are my results shown on a line graph:
ANALYSING
CONCLUSIONS:
My hypothesis stated that the more the dilution factor would be, the lesser the number of colonies. Well, according to my results, I was correct.
As my dilutions increase, my colonies decrease. This is because, during the experiment when I had to take out 1cm3 of solution from 10-2 and put it into the next, which was 10-3, the E.coli was being shared, and decreased as it was let out through the syringe. When I poured it into 10-3, I had to shake it so it was mixed properly. Subsequently, I did the same again but to the next aseptic technique, which was 10-4. Again, the E.coli was being shared. Obviously, it was lesser than it was in 10-2 because it was also being shared in 10-3 and 10-2. This is why as the dilution factor raises, the colonies fall.
ANOMALOUS RESULTS:
As shown by my results, I only had one error. This was in my third attempt of the experiment at dilution 10-5. It may have been due to contamination while carrying out that particular part of the experiment. For example, I may have left the lid of the plate on the table, which could have not been disinfected, therefore it picked up other bacteria. Alternatively, it could have just been due to my infective flu, I probably sneezed unintentionally on the plate, which caused the whole plate to be filled with colonies. Other reasons include my hands being dirty. Next time I will make sure I wear gloves, or I sneeze to the side if I do and I ensure that I keep the desk disinfected encase I by chance leave the plate’s lid n the table.
However since there was only one error, I do not think it made a huge difference to the experiment since my prediction was still correct. But next time I will be aware of these little mistakes.
EVALUATION
I think my results were reliable since I just made one error and did not have any other anomalies. However, i think if I was to do the experiment, again, I would improve on avoiding contamination and I would do more replicates to show my results as more reliable.
My results do not have a specific trend or pattern in which they decrease in, but the fact that they do not keep increasing and decreasing shows its reliability. My replicate values are not very close together; therefore tell I should have done more replicates for accuracy.
I think I may have made parallax errors when counting the cells. This means I may have miscounted the results or over counted them. This may have been because of my bad eyesight or due to distraction while counting. This could have been improved to accuracy if I counted each plate 3 times at least. So the correct amount of colonies in each plate would be certain and not doubted on. On the other hand, I could have used a different method to count the cells to make it easier for me, like using a counting meter.
To achieve much accurate results I think, other than avoiding contamination, I could have changed around my method a little so it could have been done quicker or much accurately. For example, I could have just left the petri dishes in the incubator for a little longer or lesser period; I could have also used a different culture for counting. If I were to do the experiment again, I would repeat it more than just 3 times so my results can show more accuracy and I can identify where/when I went wrong. Furthermore, the next time I would limit the temperature to see if that would make a difference in allowing my results to be precise and I would also avoid causing any errors.
BIBLIOGRAPHY
- Class notes
- Class hand outs
- Micro Organisms and Biotechnology, John adds. Erica Larkcom. Ruth Miller (Nelson) ISBN 0-17-448269-8
