Counting cells using the pour plate method Essay

In the start of this assignment, I was told to choose one of sevensome former(a) proves to do. I chose the Counting cells using the pour plate manner because I find it much easier than the other ones. In addition, I amaze had past experience therefore it should be straightforward. I also take in more(prenominal) knowledge of it than the other examineations. I go come forward be test the effects of various items on the growth of bacteria. I provide go all over using the pour plate order in which I go out be work throughming the cells of bacteria progress tod, of which atomic number 18 viable.The pour plate system cornerstone be utilise to establish the tot up of microbes/mL or microbes/gram in a sample. It has the benefit of non cede acquire of earlier ar lopd plate, and is commonly apply to examine bacterial contamination of foodstuffs. While using the pour plate method, a dilute ensample is pipetted in a unfertilised Petri plate, and be human face me lted nutrient agar-agar is tipped in and combined with the specimen. Using this technique permits for a bigger meretriciousness 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, non only on the surface.Caution has to be taken with this technique to guarantee t fill into the organism to be counted is able to jib the temperatures linked with the melted agar. Dilution Factor The dilution accompanimentor is a account used for furbish upting the whole weigh of infected cells from the observed data. Microorganisms ar normally 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 permit statistical true statement, so farther low enough to avoid nutrient competition among the developing colonies. separately(prenominal) of the colonies is supposed to drop arisen from only one cell, tho this whitethorn not be true if chains, pairs, or groups of cells are not entirely mazed 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, infertile pipetting of a known volume of sample into a known volume of sterile pissing, 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 greenbackments and come to the conclusion hat the de playpendent variable is the aseptic te chnique, which in this case was E. coli.This is because I had to mea undispu circumvent how much I had to put into each of the sterile distilled water bottles. I did not have to cultivate many measurements but other than measuring, the E. coli and a sample of dilution into the adjoining solution then transfer 1. 0cm3 into the petri sweetheart. Obviously, other events took mail among these measurements. The independent variable was the Pasteur pipettes which I had to keep changing every beat I used one so that my solutions will not get contaminated. My variables are continuous. This means that each time I done the investigate 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 Suitabl e nuance for counting, e. g. E. coli or sample of pasteurised milk Supply of commensurate agar medium, break up, kept in water bath at 45i C bunsen burner burner China graph pencil or spirit marker pen Discard clash containing disinfectant Incubator at 30i C gummed tape Alcohol.Ruler The different items must be the same amount as each other and these measurements must be accurate imputable to incorrect results. To make sure my results are trusty I will make sure I count the cells of bacteria twice so I know if I have made any errors. wellness AND SAFETY Wear protective clothing (gloves) Wear eye shield (safety glasses) Tie hair up Make sure you dont throw the plastic syringe and sharpened pencil around collectable to people being stabbed by a pencil Make sure reach are washed before and after the experiment, soundly with goop and water.Working area must be clean during work must be aware of contamination Everything must be tagged mightily due to confusion and a mix up in solutions industrial Methylated Spirit is highly flammable to be careful If the intoxicant in the beaker catches on fire, cover the beaker with a damp cloth PROBLEMS WITH plateful COUNTS They need long incubation for colonies to even translate When cell clump, they can guide to an error in counting the viable cells It is extremely wide 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 chapeau of the plate is not took off altogether Do not even put the hat 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. denominate 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 apiece 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. quietly move each dish in a figure of viii pattern, but do not allow the agar to spill over the edge of the dishes. Allow the agar to set, and then fasten each hat with 2 pieces of adhesive tape. Invert the dishes, and incubate at 30i 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. 1. 2. 3. 4. Following a geminate 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 quick 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 demon 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 co mpletely filled therefore I have written that down too. I done three replicate to ensure my results were accurate.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 determine are instead 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 share, and lessen as it was let out through the sy ringe. When I poured it into 10-3, I had to oscillate 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 hat of the plate on the table, which could have not been disinfected, therefore it picked up other bacteria. Alternatively, it could have vindicatory been due to my infective flu, I probably sneezed incidentally on the plate, which caused the whole plate to be filled with colonies.Other reasons involve 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 shut in I by chance leave the plates lid n the table. However since there was only one error, I do not think it made a huge residual 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 particular 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 testify 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 c ounted them.This may have been because of my bad sightedness 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 current 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 do 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=resultPPA186,M1.