Experiment

Aim: The aim of the experiment is to determine the relationship between Cell Number and Optical Density. Abstract: This experiment examines the number of cells per unit volume of a sample of yeast culture. A spectrophotometer, a device for measuring light intensity, is being used to determine the Optical Density, OD, of the cell. The optical density is associated with the absorbance of the cell culture. A microscope and a counting chamber, hemocytometer, are being used to obtain the number of cells per unit volume of the yeast culture. Spectrophotometer works on the principles of measuring light absorption of the sample tested. Due to the large quantity of cells present in a concentrated sample of the yeast culture, dilution is done to minimize the time spent and also counting errors. After the construction and calibration of a standard curve, the results show the direct relationship between the Optical Density and Cell Number. Principles: In this experiment, yeast culture in YPUAD and fresh medium YPUAD are used to determine the quantity of yeast cells in a given sample. Under the light microscope, the yeast cells can be seen as ‘grey dots’. The yeast samples are diluted to an extent that the cells are widely and evenly distributed. This is to obtain a more accurate result and also reduce the time spent as counting a large number of cells can be quite time consuming. A spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity as a function of the color (or more specifically the wavelength) of light. Important features of spectrophotometers are spectral bandwidth and linear range of absorption measurement. This is a widely used method to obtain the cell density of any cell sample. The given yeast sample is placed in a quartz cuvette and into a spectrophotometer. When light shines through, the Optical Density (OD) of the given sample will be determined by the absorbance of light that passed through the sample. The greater the amount of light absorbed by the sample, the higher the OD value. In order to obtain accurate readings, the spectrophotometer has to be ‘zeroed’ by measuring the absorbance of light of a reference substance, which is the fresh medium YPUAD in this experiment. After the initial calibration, this absorbency of YPUAD is set as a baseline value, so the absorbencies of all other substances are recorded relative to this value. A Hemocytometer is an etched glass chamber with raised sides that can hold a quartz cover slip exactly 0.1 mm above the chamber floor. Calculation of the concentration of a sample of cells is based on the volume underneath the cover slip. One large square has a volume of 10⁻⁴ cm³ (Length (0.1cm) x Width (0.1cm) x Height (0.1cm)). A sample of cells, in this case, the diluted yeast culture in YPUAD was loaded into one of the two counting chambers and covered over by the cover glass. The Hemocytometer was then placed onto the microscope stage of a light microscope and focused until a region of 16 big squares can be observed. We count the number of cells from top to bottom, left to right, to avoid multiple counting of cells. The total number of cells counted in the 4 regions will be averaged out, divided by the volume of 1 region, which is 10⁻⁴ cm³, and multiplied by the dilution factor (10X) in order to obtain the cell densities of the different samples of yeast culture of YPUAD. Discussion: As shown in the results and the graph plotted, there is a linear relationship between the Optical Density and the Cell Density of a cell sample. An increase in OD is directly proportional to an increase in cell density. This shows that for dilution samples with higher OD, the cell density is higher because more cells were present to absorb more light. With this relationship, the corresponding cell density of a particular cell sample can be found once the OD values are known. Generally, the experimental curve still defer from that of an ideal curve. This is probably due to any errors that might have occurred during the whole course of the experiment. Possible sources of errors include: Non-homogenous yeast suspension The yeast suspension may not have been shaken substantially to obtain a homogenous suspension. This will thus affect the OD readings taken when the samples were put into the spectrophotometer. Contaminated cuvette The cuvette may not have been cleaned (with ultra pure water) and wiped properly to remove all stains (fingerprints) and substances. OD readings obtained will thus be affected Contamination of the Hemocytometer The yeast samples may not have been completely cleaned off from the surface of the chambers. Thus, the value of cell density may be affected when there is contamination. Errors in counting of the cells Cells may be counted wrongly due to various human error and different perceptions of how a cell can be taken into account. Thus, the eventual cell count may differ a lot from each other, especially when a different person takes over in any instance. Suggestions for any improvements: * Ensure that the procedures are being closely followed, and with necessary precautions taken. For example, the ultra pure water must be used to ensure a thorough cleaning of the equipment and also Kim Wipes to clear away any stains. * There should only be a specific person assigned to count the cells as that will reduce errors owing to different individuals having different perceptions of a cell that is to be counted. * Repeat the counting of every set of cell density readings obtained. This is to minimize random errors that may occur during counting of the cells. Accuracy of the results will be enhanced when there is an average taken from different sets of readings. Conclusion It can be concluded that there is a linear relationship between the Optical Density and the Cell Density of the yeast sample, which is applicable to other types of cell samples. This is a very useful deduction. The hemocytometer has proven to be useful in determining the cell density but it is not very efficient in the aspects of time-consumption and tedious preparation work for the cell samples to be diluted.