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Sunday, December 23, 2012

Estimation of Biomass in Plant Cell Cultures

Biomass is an important parameter which needs constant monitoring to understand the growth process in plant cell and tissue cultures. Cell biomass itself is often the final product, or even if the desired product is a secondary metabolite and the compound is intracellular one needs to have a measure of biomass for several estimations related to the secondary metabolite production.

There are larger no. of methods for estimation of biomass which differ in their degree of accuracy, rapidity, convenience, instrumental requirements etc. some methods may not be suitable for a given culture system.

Measurement of cell number

In very fine suspension cultures cell number may be counted directly in a haemocytometer. Most cultures usually contains aggregates and clump. In such cases cell aggregates need to broken into individual cells prior to counting the number of cells.
  1. Add two volumes of 8% w/v aqueous chromic trioxide solution to one volume of culture suspension.
  2. Heat at 70oC for 2-15 mins.
  3. After cooling, shake the culture mixture vigorously to assist the loosening of cell clusters to individual cells. 
  4. dilute appropriately and count the number of cells under microscope using haemocytometer.
  5. Express the biomass as number of cells per ml of culture after correcting for the dilution factor.
Measurement of Packed Cell Volume (PCV)
  1. Transfer 10ml of culture suspension to a 15ml graduated centrifuge tube. tubes which taper at the bottom are preferable.
  2. Centrifuge for 5 mins at 200gm.
  3. Read pellet volume and express percentage of total volume of the culture suspension.
Measurement of Fresh Weight
  1. Filter the cell suspension through a pre-weighed whatman filter paper on a buchnel funnel under slight vacuum.
  2. wash the cells with distilled water.
  3. Drain fully under vacuum.
  4. Reweigh the cells per filter.
  5. Express the weight of cell biomass as a fresh weight.
Measurement of Dry weight
  1. Filter the suspension through a pre-weighed whatman filter paper on a buchnel funnel under slight vacuum.
  2. wash the cells with distilled water.
  3. Drain fully under vacuum.
  4. Dry cells plus filter paper in a hot air oven at 60oC to a constant weight.
  5. Reweigh the cells per filter.
  6. Express the weight of cell biomass as a fresh weight.