Counting Cells Using The Pour Plate Method

In the start of this assignment, I was told to choose one of seven other experiments to do. I chose the Counting cells using the pour plate method because I find it much easier than the other ones. In addition, I have had past experience therefore; it should be straightforward. I also have more knowledge of it than the other experiments.

I will be testing the effects of various items on the growth of bacteria. I will investigate using the pour plate method in which I will be counting the cells of bacteria produced, of which are viable.

The pour plate method can be used to establish the amount of microbes/mL or microbes/gram in a sample. It has the benefit of not have need of earlier arranged plate, and is usually used to examine bacterial contamination of foodstuffs.

While using the pour plate method, a diluted specimen is pipetted in a sterile Petri plate, and next melted agar is tipped in and combined with the specimen. Using this technique permits for a bigger volume of the diluted specimen. This is normally in the choice of 0.1 – 1.0ml. This technique yields colonies, which produce colonies all over the agar, not only on the surface. Caution has to be taken with this technique to guarantee that the organism to be counted is able to resist the temperatures linked with the melted agar.

Dilution Factor

The dilution factor is a number used for getting the whole number of infected cells from the observed data.

Microorganisms are usually counted in the laboratory using methods like the viable plate count, where a dilution of a sample is plated onto an agar medium. Following the incubation, plates with 30-300 colonies per standard-sized plate are counted. This number of colonies was selected because the number counted is high enough to have statistical accuracy, so far low enough to avoid nutrient competition among the developing colonies.

Each of the colonies is supposed to have arisen from only one cell, but this may not be true if chains, pairs, or groups of cells are not entirely broken apart before plating. The sample has to be controlled so that it consists of a number of cells in the right range for plating. If the cell number is high, the sample is diluted; but if too low, the sample is concentrated.

Dilutions are carried out by careful, aseptic pipetting of a known volume of sample into a known volume of sterile water, buffer, or saline. This is mixed well and can be used for plating or further dilutions. If the number of cells is unknown, then a range of dilutions is usually ready and plated.

HYPOTHESIS:

I predict that the more the dilution is, the lesser the number of colonies.

VARIABLES:

I have considered the accuracy of my measurements and come to the conclusion hat the dependent variable is the aseptic technique, which in this case was E.coli. This is because I had to measure how much I had to put into each of the sterile distilled water bottles. I did not have to make many measurements but other than measuring, the E.coli and a sample of dilution into the next solution then transfer 1.0cm3 into the petri dish. Obviously, other events took place among these measurements.

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

* Suitable culture for counting, e.g. E.coli or sample of pasteurised milk

* Supply of suitable agar medium, molten, kept in water bath at 45C

* Bunsen burner

* China graph pencil or spirit marker pen

* Discard jar containing disinfectant

* Incubator at 30C

* 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

* 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 30C.

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

Attempt one

Attempt two

Attempt three

AVERAGE

10-1

9216

10880

11968

10688

10-2

6144

9280

6402

7275.3

10-3

4160

7104

3535

4933

10-4

2126

2816

1503

2148.3

10-5

1931

512

N/A

1221.5

10-6

1422

320

1088

943.3

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

* http://www.bio.fsu.edu/courses/mcb4403L/dilution.pdf

* http://filebox.vt.edu/users/chagedor/biol_4684/Methods/platecounts.html

* http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Meat_Milk/Pour_Plate.htm

* http://www.microbiologyprocedure.com/microbiological-methods/pour-plate-method.htm

* Class notes

* Class hand outs

* http://www.mansfield.ohio-state.edu/~sabedon/biol4038.htm

* Micro Organisms and Biotechnology, John adds. Erica Larkcom. Ruth Miller (Nelson) ISBN 0-17-448269-8

* http://books.google.co.uk/books?id=AtjDUn5KfG0C&pg=PA185&lpg=PA185&dq=Counting+cells+using+pour+plate+method&source=web&ots=H1ulPxFpd3&sig=S9pvM8ulJXfrta7nuKb74VX4H5w&hl=en&sa=X&oi=book_result&resnum=10&ct=result#PPA186,M1