I am running submerged fermentation of Bacillus with barley husk as carbon source in a bioreactor. When samples are taken, the barley husk particles are also included.However, the amount of barley husk particle that is included in the sampling varies ( not possible to standardise). Problem: The absorbance at OD 610 nm fluctuates too much ( suspected reason: unequal amounts of barley husk particle). I am thinking of filtering the sample with a muslin cloth or a filter paper to remove the barley husk particle before checking of the cell density. Is this a good move?
Do not filter in this way! You loose your cells. Better to use plate count method or nuclear counter instrument.
You cannot use the colorimeter method in this case. You can go with plate count method or protein estimation method for bacteria growth.
If you cant avoid costly instrument than there are few crude methods you can use, some of them mentioned above
1) plate count method would be the most sutable method. Be sure to vortex your sample in order to mixed you content properly and losen your cell from barley.
2) If the size of the barley is available, you can filter it from sieve and than use absorbance or plate count method. If bacteria is attached to barley than you cant use this method unless high vortexing can seperate it
3) Dry cell method: first you need to grow your bacillus in your respective media except replace your carbon source (Barley) with simple carbon source. Then take O.D at different time interval along with dry cell. Draw a graph.
Dry your centrifuged content of media and control (uninoculated) (do in 15 ml media). Subtract your actual fermentation result from control. Correlate it with above graph and get your cell count.
I strongly recommend that you use CFU. We have this same problem in the lab and we use CFU. Mr. Vohra made a good recommendation in item 3 but see that the substrate is solubilized during the fermentation and thus there is no way to use a control because it changes throughout the fermentation.
I think that you can use CFU but the best way is by oxygen consumption.
oxygen consumption is expensive rather than CFU, spending more money for oxygen measurement probe.
Since yoh have particles in your sample, it will always afffect the optical density of the cells. I think the best way to enumerate your cells is by CFU or if possible count the cell under the microscope with a counting chamber.
i think you will face difficult observation under microscopic measurement because you can face some particles and you can't clearly observe Bacillus.
I such case, viable count and/or total cellular DNA are the alternatives to score for growth.
You can also try to centrifuge the suspension before measuring the absorbance, but i think that filtering the sample with a filter paper is the best solution, easy and cheap.
Using centrifugal force at low RCF on broth sample can help in elimination of macro particles in the suspension with appropriate time intervals, as suggested by Prof Jai Ghosh. However this method has to be tested based on the sample and standardize the the RCF and time period for eliminating suspended macro particles. Following this step, biomass can be pelleted using appropriate centrifugal force ( say, 10000g for 10 min at 4oC) and then the pellet can be re-suspended in about 4ml 0.1% saline water for taking absorbance against the reference as 0.1% saline water.
All the best.
Yes, but use a standard material for the filtration process. A filter paper is better than muslin cloth. This will ensure uniform treatment of your samples. You may also consider diluting the samples after centrifugation with buffer before taking readings
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