This lab investigates the use of numerous methods to determine cell density of Pichia pastoris present in the fermenter. The 5 methods explored in this study to estimate the biomass include: optical density, manual cell counting, viable plate counting and measuring dry and wet cell weight.
OD600 and OD700
In this experiment, both the diluted and undiluted samples were analyzed under OD600 and
OD700 to determine yield of biomass as shown below:
Sample OD600 OD670
Water 0 0
Media (YEPD) 0.078 0.056
Neat Sample Off Scale Off Scale
10X Sample 1.989 0.380
20X Sample 1.089 0.247
50X Sample 0.625 0.122
100X Sample 0.485 0.072
Table 1: OD600 and OD670 sample readings of sample flask 4 under various dilutions, with water as negative control.
Table 1 shows an indirect relationship between the dilution rate and cell density. As dilution rate increases to 100 times, cell density decreases to 0.485.
Cell density is determined by collecting raw sample readings between 0.05 to 0.5 absorbance unit. To calculate the diluted results of each reading, the following formulas was used:
Diluted Result600nm = Dilution factor x (Dilution sample reading) – blank readingSample OD600 OD670
10X Sample – 3.74
20X Sample – 4.88
50X Sample – 6.04
100X Sample 40.7 7.14
Table 2: Diluted results of sample readings obtained from flask 4 with different dilutions
Table 2 shows the calculated OD values under their respective dilution factors. These readings were obtained from raw absorbance values that fall between 0.05 and 0.5.
Cell counting using a hemocytometer Grid Cell Counted Cell Concentration (cells/l)
1 529 5.29 x1011
2 650 6.50 x1011
3 963 9.63 x1011
4 995 9.95 x1011
Table 3: Results gathered from cell counting using haemocytometer on 4 grids
The mean cell concentration can be calculated by the average of all the cell concentration in all 4 grids, resulting in a mean cell concentration of 7.84 x1011 cells per litre. The standard error of the mean is obtained using the formula:
Where s is standard deviation while n represents sample size. The standard deviation of the values obtained in Table 3 is 2.31 x1011 approximately. Using the formula s√n , we are able to estimate the standard error of the experiment to be 1.16 x1011. Hence, viable cell density can be estimated from the standard error to be 7.84 ± 1.16 x 1011 cells/l.
Measuring wet cell weight
Tube Empty weight (g) Weight with cell pellet (g) Cell pellet weight (g) Wet cell weight per L
1 0.98 1.04 0.06 60
2 0.98 1.05 0.07 70
Table 4: Data showing wet cell weight of cell cultures in different tubes
The measurement of wet cell weight requires proper pipetting technique to ensure smooth removal of the supernatant without disturbing the cell pellet. The mean wet cell concentration is 65g/L. The standard deviation gathered from the data is 7.07. Using the formula s√n , the standard error of the mean is approximately 5.00g/L of wet cell weight.
Hence, the viability cell density is determined to be 65±5 g/L.
Measuring dry cell weight
Mass filter (g) Mass filter + Biomass (g) Biomass (g)
0.0818 0.157 0.0752
0.0842 0.183 0.0988
Table 5: Data of weight of mass filter and biomass for dry cell weight estimation
The mass of the dry cell weight was determined using the formula:
X (g.l-1) = [ (Mass filter + biomass) – (Mass filter) ] (g) / 0.010 (l)
The 2 measurements obtained were 7.52 gX/l and 9.92 gX/l.
The mean cell weight is the average of the 2 measurements, which is 8.72 gx/l.
The standard deviation is calculated to be 1.70 g/l.