Bacterial Growth Measurements II: Total Cell Count


Aim:
In this experiment, cell counting with direct (direct cell count) and indirect (spectrophotometer) methods were realized as the purpose.
Introduction:
Cell counting,
Cell counting is essential basic at microbiology. Cell counting has different methods.
As a classical method, direct microscopic count is known. A counting chamber or hemocytometer is used for this method. It is a kind of specialized slide including squares with certain lines in it. A drop of cells is poured onto chamber. After placing under microscope, cells are counted. If possible, all cells on the camber are counted to obtain the best results. It can be done by counting all of them instead of a certain part. These squares have a certain area and depth. The number of cells per milliliter can be calculated by this volume by using these certain area and depth at the end of the cell count calculated. According to non-written rules, this counting is done by including the cells in the upper left corner. (1)
As indirect method, turbidity measurement is used often. Cell suspensions have turbid structure. Cell suspensions have an absorbance value according to cell concentrations. In spectrophotometer, cells can absorb or scatter light. These amounts of absorbance can give information about biomass in sample. The light should have a certain wavelength for better results. 600nm-wavelength is used for this measurement.(2)
Cell growth,
When cells were placed onto fresh culture, they don’t grow immediately. For a while, cells can adapt to environment and then prepare for dividing. This phase is called lag phase. At lag phase, a growing cell culture is not observed. (3,4)
Main growing on culture starts with log phase or exponential phase. Cell population increases rapidly by using fresh media. This increasing depends on other conditions such as temperature, pH. This temperature is changeable according to kind of microorganism. E.coli prefers 370C mostly. (3,4)
After a while, population is found in stabile line. Equal amounts of dividing and dying cells form this balance. Limited conditions creates stationary phase. (3,4)
At a point, lacks of growth factors cause a reducing. Cells can’t survive and death rate increases by time. This phase is named death phase because of that. (3,4)

Material and Methods:
Direct microscopic counting,
The yeast culture prepared for this experiment after an overnight was placed onto chamber and then the cover slide was put onto the camber. The chamber was observed under microscope and cells on 3 squares were counted.
OD measurement,
Firstly, 1 ml LB was measured in spectrophotometer for blank value at 600 nm. After getting “0” value at blank, same process was applied with 1 ml of bacterial sample for 3 times for each sample at 600nm.
400 l of inoculum of E. coli was diluted with 19,6 ml LB broth to get 2% inoculum of E. col for examinations of Group 1, 2 and 3 by using micropipette and aseptic techniques. Shaking aeration; shaking and no aeration; no shaking with aeration was examined the samples at 2% respectively. For Group 4, 5 and 6, 4 ml of E. coli was transferred into 16 ml LB broth to be diluted to 20%. These tubes were prepared in same conditions. In order, shaking aeration; shaking and no aeration; no shaking with aeration were applied. A tube including only LB broth was used as a blank in spectrophotometer and then each sample was measured for 3 times. After first measurement, the samples were measured by time intervals. Each 3-measurement was used for getting average value. Finally, calculations were applied on a graph.

Result:
Counting 1 Counting 2 Counting 3
Number of cell 76 60 81
Table 1-Cell counting of 10µl of samples at 10-2 dilution under 100x microscope.

Calculation for direct cell counting,
(76+60+81)/3=72
72x104x100=72×106 = 7,2x 107 cells/ml
Calculations for values of OD measurements,
Average measurement= (measurement 1+ measurement 2+ measurement 3)
Measurement 1 Measurement 2 Measurement 3 Measurement 4
Group 1 0.032 0.164 1.149 0.742
Group 2 0.034 0.151 0.914 1.043
Group 3 0.021 0.084 0.825 1.182
Group 4 0.350 0.563 1.173 0.794
Group 5 0.254 0.480 0.917 0.960
Group 6 0.304 0.472 0.809 1.102
Table 2-The values of absorbance by time intervals; 0, 1.30, 18 and 25 hours by spectrophotometer, each value on table is average of 3 measurements.

Discussion:
The purpose of this experiment is to count cells in culture by using direct and indirect methods. At first step, direct cell counting, cell culture prepared before was used. For direct cell counting, yeast(S. cerevisiae) cells were used because of their size. Yeast cells are bigger than bacteria cells. They can be seen easily under microscope. Cell culture should be diluted. Diluted cell culture can prevent to incorrect counting. Naturally, some cells will be cluster in high concentrations and this will affect our results. Low concentration is a problem too. Distance between cells will give too low counting and this won’t be clean results. In our examination, at first attempt with 10-2 diluted solution didn’t give expected. Cells were so far from each other. Because of forgotten vortex process, observation was repeated with vortex. After that, cells could be distinguished. They weren’t so close or so far from each other. 76, 60 and 81cells could be counted at this part. After calculation, average of these counting was found 72. 100 mm and 10000 mm; of lengths and depth of chamber were used for calculated volume between chamber and cover slide. The dilution factor was calculated with 106 ml of volume of chamber calculated by multiplying 100 mm and 10000mm. The cell/ml was achieved as 72×106. As significant number, it is 7,2x 107 cells/ml.(Table 1)
Secondly, OD measurement was used for cell counting. The light passing through a turbid solution and having a certain wavelength can’t pass totally. Particles in solution can prevent to a total passage. If absorbance of particle is used, mass of particles can be known. Same principles can be used for cells. Cells can absorb 600 nm optimally. Spectrophotometer measures this absorbance. For our experiment, different dilutions and conditions were used. For 2% E.coli, shaking aeration; shaking without aeration; no shaking with aeration was applied. Same processes were prepared for 20% E.coli too. When only Tube 1 and 4 was investigated, population of bacteria increased and then decreased at last point was seen. E.coli is a facultative anaerobic bacterium. E.coli can live with oxygen or not, however it prefer anaerobic environment much more. In Tube 1 and 4, shaking causes contact with oxygen and that reduce number of bacteria in tubes. Shaking and aeration can decrease number of bacteria. In the other tubes (2, 3, 5 and 6), bacteria can reproduce, because less contact of bacteria with oxygen give a good environment to grow. Even in the tubes including aeration (3,6), bacteria can increase. If there is no shaking, bacteria can grow in middle sites of solution to not touching oxygen.

References:
[1] Priego, R.; Medina, L.M.; Jordano, R. (2011). “Bactometer system versus traditional methods for monitoring bacteria populations in salchichon during its ripening process”. Journal of Food Protection. 74 (1): 145–148.
[2] Grossi, M.; Lanzoni, M.; Pompei, A.; Lazzarini, R.; Matteuzzi, D.; Riccò, B. (2010). “An embedded portable biosensor system for bacterial concentration detection”. Biosensors & Bioelectronics. 26: 983–990.
[3] Fankhauser, David B. (17 July 2004). “Bacterial Growth Curve”. University of Cincinnati Clermont College. Archived from the original on 13 February 2016
[4] Zwietering MH, Jongenburger I, Rombouts FM, van ‘T Riet K (1990). “Modeling of the Bacterial Growth Curve”. Applied and Environmental Microbiology. 56 (6): 1875–1881

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