Hi Prerna!

Growing Geobacter sulfurreducens to a higher density can be challenging due to its strict anaerobic nature and the specific conditions it requires. Here are some tips that might help you increase the cell density of your cultures:

1. **Optimize Media Composition**: Ensure that the NBAF medium is prepared exactly as described in the protocol, with all necessary nutrients and supplements. Sometimes, the quality or source of the medium components can affect growth. You might also consider trying different media formulations to see if G. sulfurreducens grows better in another type of medium.

2. **Inoculum Density**: Start with a higher density inoculum if possible. If you are transferring from a glycerol stock or a low-density culture, try to increase the initial cell density when inoculating the new culture.

3. **Anaerobic Conditions**: Double-check that the anaerobic conditions are being maintained properly. Even small amounts of oxygen can inhibit the growth of strict anaerobes. Make sure your anaerobic chamber is working correctly and that the gas mixture (N2:CO2::80:20) is being replaced regularly to maintain anaerobic conditions.

4. **Temperature Control**: Ensure that the incubator is set at a constant 30°C and that there are no fluctuations that could affect bacterial growth.

5. **pH Control**: Monitor the pH of the medium during growth. Some bacteria may alter the pH of the medium as they grow, which can affect their growth rate. Adjust the pH if necessary to maintain optimal conditions for G. sulfurreducens.

6. **Nutrient Limitation**: Check if there is any nutrient limitation in the medium. If the bacteria are running out of a specific nutrient before reaching a higher density, you may need to supplement the medium with that nutrient.

7. **Scale-Up**: If you are trying to grow G. sulfurreducens in large volumes, consider scaling up in stages rather than directly inoculating a large volume. This can help maintain the growth rate and potentially reach higher cell densities.

8. **Avoid Contamination**: Ensure that your cultures are not being contaminated by other microorganisms, which could compete with G. sulfurreducens for resources and space.

For growing Geobacter in 96-well plates, you will need to maintain the same anaerobic conditions and temperature control as you would for larger cultures. Here are some additional considerations:

- **Sealing**: Use an appropriate sealing film to maintain anaerobic conditions in the 96-well plate.

- **Mixing**: Gentle shaking or mixing might be necessary to ensure adequate oxygen transfer for the bacteria, as static conditions in the wells can lead to poor mixing.

- **Evaporation**: Be aware of potential evaporation from the wells, which can concentrate the medium and affect growth conditions.

- **Monitoring Growth**: Use a plate reader to monitor OD at 600 nm over time to track growth kinetics.

There are specialized 96-well plate sealers and anaerobic systems designed for working with anaerobic bacteria in multi-well plates that can be very helpful.

Lastly, if you haven't already, review the literature for specific growth conditions and techniques used by other researchers working with Geobacter sulfurreducens. Look for recent articles or methods papers that might provide additional insights or protocols that have been optimized for high-density growth.