Growth of Neisseria meningitidis in the FLUOstar OPTIMA with Microprocessor Controlled Gas Vent

Kerry L. Cutter
University of the West of England, Bristol, UK.

Automated cell growth and culture studies using FLUOstar OPTIMA equipped with Microprocessor Controlled Gas Vent

Neisseria meningitidis used to test Microprocessor Controlled Gas Vent as it requires optimally 5% CO₂ to grow and thrive

Improved growth rates observed in dilute cultures using the Gas Vent as compared to using a CO₂ controlled incubator

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Note: The Microprocessor Controlled Gas vent used in this application note has since been replaced with the Atmospheric Control Unit (ACU) for independent control of CO₂ and O₂. Click here for more information on the ACU.

Introduction
It is well known that all organisms need a certain, but not necessarily a similar, level of carbon dioxide for growth and reproduction.(1) The period during which this level is being increased corresponds to the lag phase as the organism is unable to divide until the critical concentration of CO₂ is reached.(2)

The meningococcus has been considered one of the most fastidious micro-organisms with respect to growth requirements and it has long been recognized that most strains of Neisseria meningitidis require or benefit by, a concentration of CO₂ greater than atmospheric.(3,4) Therefore, this organism is especially suitable for the study of CO₂ effect.

In this study, a strain of Neisseria meningitidis was used to assess the efficiency of a FLUOstar OPTIMA multimode plate reader coupled with a Microprocessor Controlled Gas Vent to deliver 5% CO₂. This was done by comparing growth of Neisseria meningitidis in the FLUOstar OPTIMA (figure 1) with a carbon dioxide incubator (set to deliver 5% CO₂) and in an incubator at atmospheric CO₂.

Fig. 1: BMG LABTECH's multimode microplate reader FLUOstar OPTIMA
equipped with the Microprocessor Controlled Gas Vent

Materials and Methods

FLUOstar OPTIMA, BMG LABTECH, Aylesbury, U.K.
Microprocessor Controlled Gas Vent, BMG LABTECH, Aylesbury, U.K.
Lucy1, Anthos, Salzburg, Austria

Neisseria meningitidis C751 was grown on Brain-Heart Infusion (BHI) agar supplemented with 10% foetal bovine serum (FBS) at 37°C and in 5% CO₂ overnight.

The following day, several colonies were resuspended in 10ml BHI broth supplemented with 10% FBS. This was serially diluted to 10^-6 and 200µl samples of each dilution were dispensed in triplicate into a sterile 96-well microplate. Absorbance readings were taken of each well every hour (ABS filter 405nm) in the FLUOstar OPTIMA with Microprocessor Controlled Gas Vent (set at 37°C, 5% CO₂) over a 24h time period.

Identical microplates were placed in a 37°C, 5% CO₂ incubator and a 37°C incubator with no supplemental CO₂, and absorbance readings (at 405nm) were taken at 0, 1, 2, 3, 4, 6, 8 and 24h using an Anthos Lucy 1 microplate reader (Salzburg, Austria).

Once absorbance readings had been collected for all treatments, the blank media control (values not shown) was used to adjust values to represent increase in OD. Percentage bacterial growth was then calculated using the maximum OD achieved for each treatment as 100%.

Data was taken from triplicate wells on two separate occasions to give six data sets for analysis. Microsoft Excel was used to plot graphs and Minitab 13 was used to carry out tests of statistical significance.

Results and Discussion

From the data shown in Figures 2-4 it is evident that there is a clear correlation between starting inocula and growth rate for all three treatments.

At the higher starting inocula there appears to be little difference in bacterial growth between treatments. This is most likely due to there being a sufficiently high number of organisms present to produce a critical level of CO₂, therefore enabling initiation of growth without the need for an external CO₂ source.

However, differences in growth rate of N.meningitidis C751 as a result of CO₂ effect are more apparent in the most dilute cultures. For example, when grown at 37°C in atmospheric CO₂ (figure 2), the most dilute culture (1 in 1,000,000 dilution) exhibits a prolonged lag period due to the inability of the small inocula to reach the critical level of CO₂ to initiate growth. This lag phase is appreciably shorter when the same culture is grown in either a carbon dioxide incubator (figure 3) or the FLUOstar OPTIMA with Microprocessor Controlled Gas Vent (figure 4) achieving 29% and 72% bacterial growth respectively after 24 hours incubation.

By comparing the data collected using a carbon dioxide incubator and the new FLUOstar OPTIMA Microprocesor Controlled Gas Vent (Figures 3 and 4) it can be seen that there is less difference between growth rates of the serial dilutions using the FLUOstar OPTIMA. This is most likely due to a constant temperature and level of CO₂ being maintained throughout the duration of the experiment. Whereas, when using a CO₂ incubator the samples have to be transferred to a microplate reader to measure absorbance resulting in both CO₂ level and temperature fluctuations which ultimately result in decreased growth rate of N.meningitidis.

Incubator with atmospheric CO₂

Fig. 2: Growth of serially diluted cultures of Neisseria meningitidis in BHI broth supplemented with 10% FBS at 37°C without supplemental CO₂. The data presented was calculated from triplicate optical density readings (at 405nm) taken in an Anthos Lucy 1 microplate reader from duplicate experiments.

Carbon dioxide incubator

Fig. 3: Growth of serially diluted cultures of Neisseria meningitidis in BHI broth supplemented with 10% FBS in an incubator at 37°C, delivering 5% CO₂. The data presented was calculated from triplicate optical density readings (at 405nm) taken in an Anthos Lucy 1 microplate reader from duplicate experiments.

FLUOstar OPTIMA with Microprocessor Controlled Gas Vent

Fig. 4: Growth of serially diluted cultures of Neisseria meningitidis in BHI broth supplemented with 10% FBS using a FLUOstar OPTIMA multimode plate reader coupled with a Microprocessor Controlled Gas Vent set to deliver 5% CO₂ at 37°C. The data presented was calculated from triplicate optical density readings (at 405nm) taken hourly over a 24h period from duplicate experiments.

The results shown in Table 1 illustrate that this increased growth rate in the FLUOstar OPTIMA compared with a carbon dioxide incubator is statistically significant for all dilutions.

The results in Table 1 also reveal a significant difference in bacterial growth between the FLUOstar OPTIMA at atmospheric CO₂ for all except one dilution (1 in 10: p=0.052). However, the results also reveal that there is no significant difference in bacterial growth between a CO₂ incubator and atmospheric CO₂ which is most likely attributable to the temperature and CO₂ level fluctuations described earlier.

Table 1: Comparing growth of a serially diluted culture of Neisseria meningitidis C751 over a 24 hour period when grown at 37°C in a carbon dioxide incubator delivering 5%, in a FLUOstar OPTIMA Microprocessor Controlled Gas Vent delivering 5% CO₂ and with no supplemental CO₂ (atmospheric incubator). Minitab 13 was used to carry out t-tests and results were considered significant (+) or not significant (-) at the 95% confidence interval.

Conclusion

In conclusion, by utilizing the CO₂ dependency of a strain of Neisseria meningitidis it has been demonstrated that the FLUOstar OPTIMA coupled with Microprocessor Controlled Gas Vent is able to both achieve and sustain a level of CO₂ required for growth of such a fastidious organism. This study has also highlighted the advantage of this automated system over a carbon dioxide incubator. For example, conditions are more stable and the fact that it is less labour intensive means that a greater amount of data can be generated. This system is not only ideal for experiments using CO₂ dependent organisms such as N.meningitidis but may also prove very useful in cell culture studies.

References

Gladstone, G.P., Fildes, P. & Richardson, G.M. (1935). Carbon dioxide as an essential factor in the growth of bacteria. British Journal of Experimental Pathology. 16, 335-348.
Tuttle, D.M. & Scherp, H.W. (1952). Studies on the carbon dioxide requirement of Neisseria meningitidis. Journal of Bacteriology. 64, 171-182.
Gates, F.L. (1919). The effect of carbon dioxide in the cultivation of the meningococcus. Journal of Experimental Medicine. 29, 321-328.
Glaister, D. & Athersuch, R. (1990). Apparent failure of brain heart infusion broth to cultivate meningococci from clinically infected patients. Medical laboratory sciences 47, 158-162.