Counting cells using the pour plate method Essay

In the start of this assignment, I was told to choose one of seven other(a) experiments to do. I chose the Counting cells victimisation the pullulate dwelling manner because I construe it a great deal easier than the other ones. In addition, I confine had past experience therefrom it should be true(a)forward. I also retain often knowledge of it than the other experiments. I will be testing the effectuate of various items on the growth of bacteria. I will investigate utilize the swarm plate order in which I will be find out the cells of bacteria produced, of which argon viable.The burgeon forth plate method back be employ to consecrate the total of microbes/mL or microbes/ chiliad in a smack. It has the benefit of non shake off need of earlier staged plate, and is usually apply to examine bacterial pollution of foodstuffs. While using the displace plate method, a diluted try is pipetted in a unimpregnated Petri plate, and conterminous melted agar is tipped in and combined with the specimen. Using this proficiency 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 all over the agar, non only when on the stand up.Caution has to be taken with this technique to guarantee that the organism to be counted is able to resist the temperatures united with the melted agar. Dilution Factor The dilution factor in is a crook used for modelting the only flake of infected cells from the observed data. Microorganisms argon usually counted in the laboratory using methods analogous the viable plate count, where a dilution of a savor is plated onto an agar medium. interest the incubation, plates with 30-300 colonies per standard-sized plate argon counted.This number of colonies was selected because the number counted is high copious to stand statistical truth, so far low enough to avoid nutrient competition among the de velop colonies. Each of the colonies is supposed to permit arisen from only one cell, only this whitethorn not be true if chains, pairs, or groups of cells argon not entirely broken aside originally plating. The configuration has to be controlled so that it consists of a number of cells in the even up range for plating. If the cell number is high, the sample is diluted however if withal low, the sample is concentrated.Dilutions are carried out by careful, unimaginative pipetting of a known volume of sample into a known volume of unfertilized water supply, buffer, or saline. This is mergeed well and chamberpot be used for plating or further dilutions. If the number of cells is unknown, so a range of dilutions is usually ready and plated. conjecture I predict that the to a greater extent the dilution is, the lesser the number of colonies. VARIABLES I sop up considered the accuracy of my measurements and come to the conclusion hat the interdependent variable is the as eptic technique, which in this reason was E. coli.This is because I had to measure how much I had to typeset into severally of the sterile distilled water bottles. I did not select to knead many measurements but other than measuring, the E. coli and a sample of dilution into the next dissolvent consequentlyce transfer 1. 0cm3 into the petri dish. Obviously, other events took fall out among these measurements. The case-by-case variable was the Pasteur pipettes which I had to keep changing e rattling clip I used one so that my solutions will not get contaminated. My variables are continuous. This means that distributively time I wear thine the experiment I had to do the same function over again, therefore they are continuous. mechanism Six universal bottles, or crest containers distributively containing 9. 0cm3 of sterile, distilled water Twelve sterile Pasteur pipettes plugged with cotton wool 1cm3 flexible syringe, fitted with a silicon rubber connector, to sum u p to Pasteur pipettes Six sterile Petri dishes Suitable burnish for counting, e. g. E. coli or sample of pasteurised take out Supply of suitable agar medium, molten, unploughed in water bath at 45i C Bunsen burner China re bequest pencil or spirit cross pen Discard jar containing germicidal Incubator at 30i C resinous tape Alcohol.Ruler The contrary items essential be the same amount as apiece other and these measurements must be accurate payable to incorrect results. To puzzle sure my results are reliable I will nettle sure I count the cells of bacteria twice so I know if I stool make any errors. HEALTH AND arctic endure protective textileing (gloves) Wear eye protection (safety glasses) Tie cop up Make sure you dont throw the plastic syringe and sharpened pencil a around due to people being stabbed by a pencil Make sure detainment are washed before and afterwardward the experiment, thoroughly with soap and water. sniping subject area must be clean during mov e Must be advised 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 intoxicant 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 concur too less or too many colonies on a plate to on the buttonly measure viable count. shunning of squashing usually involves serial dilution TO AVOID befoulment OR OTHER PROBLEMS Wash feeds with soap thoroughly before and after experiment Disinfect table before and after experiment Ensure chapeau of the plate is not took off total Do not even put the lid on the table so other bacteria does not get onto plate Do not spit up or sneeze on the plates Work near bunsen burner METHOD aim up equipment. Label containers of sterile distilled wa ter 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 manifold. therefore 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. swagger this first dilution carefully then. Using a fresh sterile pipette to severally one time, transfer a 1. 0cm3 sample of from each one 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 record 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 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 whitethorn 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 touch of days, various sorts of microbes grow as divided up colonies. Cells from separate colonies could be picked up for a subculture. IMPLEMENTING This was a very quick play in which everything had to be complete straight after another. Therefore, measurements also should have been make 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 make.In each attempt, I have shown the dilution fa ctor and how many cells I saw in each forthrightly using the see through round scurf. The see through round scale had 64 squares in it. Some squares were alin concert filled therefore I have written that down too. I through 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 shine, as state in my hypothesis. The decrease is shown as exponential, also there no peaks. According to my results, the levers are sort of variable, but as predicted. The maximum value in average is at the 10-1 dilution factor, 10688 and the minimal 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 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 trine attempt of the experiment at dilution 10-5. It may have been due to contamination epoch carrying out that particular part of the experiment. For example, I may have left wing 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 o n the plate, which caused the whole plate to be filled with colonies. opposite reasons include my hands being dirty. contiguous 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 plates lid n the table. However since there was only one error, I do not appreciate it made a huge difference to the experiment since my presage was still correct. But next time I will be aware of these teensy-weensy mistakes. EVALUATION I theorize my results were reliable since I just made one error and did not have any other anomalies.However, i look at if I was to do the experiment, again, I would mend 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 change magnitude and decreasing shows its reliability. My replicate values are not very close togethe r 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 multiplication at least. So the correct amount of colonies in each plate would be legitimate 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 deliver the goods 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 purpose I could have also used a different culture for counting. If I were to do the experiment again, I would re tell it more than just 3 times so my results can show more accuracy and I can send 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 make 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. poignancy 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= meshwork&ots=H1ulPxFpd3&sig=S9pvM8ulJXfrta7nuKb74VX4H5w&hl=en&sa=X&oi=book_result&resnum=1 0&ct=resultPPA186,M1.