Inoculum density

Adherent insect cell release from solid surfaces generally does not require trypsinization, unlike anchorage-dependent mammalian cells. Insect cells, like mammalian cells, need rigorous aseptic manipulation during cell transfer, inoculation, and propagation in bioreactors. A minimal inoculum density is required for both cell types. Typically, insect and mammalian cell cultures are initiated with inocula of 1-2 X 105 cells per milliliter of liquid medium (Agathos, 1991). Table 2.3 summarizes the major differences and similarities between these cells. 

Dean, R. and Kuc, J. 1986. Induced systemic protection in cucumber effects of inoculum density on symptom development caused by... 

Fig. 11.2 Survival of Enterococcus faecalis exposed to a fluoroquinolone for 4 h at 37°C. Three initial bacterial concentrations were studied, 107cfu/ml ( ) 10 ctli/ml (A) and 105 cfu/ml (O). This clearly demonstrates a paradoxical effect (increasing antimicrobial concentrations past a critical level reveal decreased killing), and the effects of increased inoculum densities on subsequent killing (courtesy of Dr Z. Hashmi).

Influence of Inoculum Density on Culture Growth Rate. The slopes of the growth curve data were used to obtain a value for the maximum growth rate (Mimx) of the cells in LB media as summarized in Table I. Only sample cell densities of between 5 x 10 and 5 x 10 CFU/mL were used in the regression in order to avoid lag phase and stationary phase data. 

Figure 2. Apparent lag time as a function of inoculum density for CY15070/pWHA43, ( ) and CY15070 (o) ceUs.

Influence of Inoculum Density on the Fraction of Cells Containing Inclusion... 

As the cell density increased to 2 x 10 CFU/mL there was a rapid increase in the fraction of cells containing inclusion bodies in all cultures. Regardless of inoculum density, all cultures reached about 90-95% of the cells containing at least one inclusion in stationary phase. 

Figure 4. Growth (o) and inclusion body expression ( ) for a typical culture. The inoculum density was 6 CFU/mL.

Figure 5. Influence of inoculum density on growth (open symbols) and inclusion body expression (closed symbols) in CY15070/pWHA43 (a) 140 CFU/mL (b) 14 CFU/mL (c) 1.4 CFU/mL (d) 0.6 CFU/mL.

Figure 6. Influence of inoculum density on the fraction of cells containing inclusion bodies during exponential growth (10 - 10 CFU/mL).

Figure 7. Relationship between the fraction of cells containing inclusions during exponential growth and the relative prochymosin content of the cells. Inoculum density 0.14 CFU/mL (a) inoculum density 140 CFU/mL ( ).

Lag time effects. The relationship between the apparent lag time and the inoculum density observed in these experiments seems to be the result of a biphasic growth pattern in the batch cultures. This is seen more clearly in a composite growth curve for several different inoculum sizes as shown in Figure... 

We found that the apparent lag time had no relationship to the size of the reactor inoculated but only depended on the inoculum density. (Data not shown.)... 

Inoculum Density Versus Fraction of Cells with Inclusion Bodies. Cultures from higher inocula showed a significantly smaller fraction of cells containing visible inclusion bodies than inocula from low inocula. The reason for this is not clear at this time but it not due to the presence of plasmidless cells in the culture. Due to the low transformation efficiency, the inoculum contained 10 times as many plasmidless cells as plasmid containing cells. However, the former were rapidly killed in the growth medium. By 6 hours into the culture there were less than 1% plasmid free (antibiotic sensitive) cells in the culture. No plasmidless cells were detected by comparative plating on selective and non-selective plates at the cell densities where the inclusion body fraction was determined. 

The pole age model does not explain why low inoculum densities resulted in a higher fraction of the cells with visible inclusion bodies and higher synthesis rates for prochymosin without decreasing the exponential specific growth rate in the cells. 

Ozeki, Y. and Komamine, A. 1985. Effect of inoculum density, zeatin and sucrose on anthocyanin accumulation in carrot suspension cultures. Plant Cell Tissue Organ Culture 5, 45-53. 

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