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Introduction

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.

Results

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:

s√n

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.