The growth results into synthesis of new cellular material. Therefore, it results into the increase in cell mass. Hence, methods used to determine cell mass can be useful to measure the growth.
Further, every cell possesses a specific amount of cell mass. Hence, this can be also useful to find out probable number of organisms present in the growth culture.
Criteria used to determine cell mass
Every cell possesses a specifie chemical composition, dry weight as well as the volume. Hence various methods based on determination of chemical constituents of cell, volume and dry weight are applied to measure cell mass.1]. Determination of cellular nitrogen and protein content
- Every cell possesses a fixed proportion of protein and nitrogen. To determine them, cells are first harvested, washed and then used for quantitative determination.
- Commonly micro Kjeldahl method is used to measure cellular nitrogen. This method is much laborious.
- Nitrogenous matter of the medium is also estimated together. Therefore this method is not used for routine cell mass determination.
2] Determination of cellular nucleic acid content
- Like nitrogen and protein, each bacterial cell possesses a fixed proportion of RNA and DNA. Hence their estimfuion can also be a useful criterion to measure cell mass.
3] Determination of cellular phosphorus and sulphur contents
- Phosphorus and sulfur are other major elemental constituents of cell, apart from carbon and nitrogen. They also occur in a specific proportion in cell.
- Hence, chemical estimation of cellular phosphorus and sulfur can be useful to measure cell mass.
4] Determination of dry weight of cell
- This is one of the highly useful methods to measure growth. However, it can be used only when cell population is much dense in the culture.
- The cells are separated from culture by centrifugation or filtration, washed thoroughly and then dried in oven at 50-55°C. Then their dry weight is determined.
- However in some instances, dry weight of cells may not be indicative of living matter.
- E.g. Bacteria can accumulate large amount of poly B hydroxy butyric acid as reserve material during late log phase without causing any increase in other cellular constituents. It may account for 70% of total dry weight in some instances.
5] Determination of packed cell volume (PCV)
- Each bacterial cell belonging to a particular species has a specific volume. Increase in cell mass and cell number results in to overall increase in cell volume.
- Hence, methods determining cell volume of bacteria can be used to measure growth.
- This is achieved by centrifugation of a fixed volume of growth culture and then the settled cell mass is measured as packed cell volume.
6] Determination by turbidimetric method
- Another method of measurement of cell mass is an optical method, by determining turbidity resulting due to growth in broth cultures.
- When, a beam of light is allowed to pass through a suspension of bacteria, the bacterial particles coming in the path of light cause scattering and absorption of light depending upon the cell density as per Beer-Lambert's law.
- Therefore amount of light transmitted through the culture suspension is reduced.
- The reduction in the amount of transmitted light, therefore, depends on the cell density or cell mass in the culture suspension.
- It is measured as % transmittance or optical density (O.D.) by the use of photoelectric colorimeter or spectrophotometer or densitometer.
- Usually the O.D. is measured at 540 nm wavelength of light. This is because at this wavelength light absorption by medium will be maximum and hence it will give more correct value of O.D. of the bacterial suspension.
- Another method of measuring cell density by applying same principle is through use of Nephelometer.
- Nephelometer does not measure transmitted light, passing through the culture suspension, but it measures intensity of light scattered by bacterial particles.
- The diagrammatic presentation of measurement of cell density by both photoelectric colorimeter and nephelometer is shown in figure below.
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| Diagrammatic presentation of measurement of cell density by photoelectric colorimeter. |
- a] Adjustment of instrument to read zero optical density by use of uninocculated culture medium as "blank" tube.
- b] Measurement of optical density of the growth tube, more the turbidity, greater the optical density.
Advantages and disadvantages of methods for determination of cell mass
- Of all the methods, cell mass determination by turbidimetric method is most convenient and reliable.
- Other methods especially involving determination of cell constituents such as cellular nitrogen, protein nucleic acids, phosphorous or sulfur has only academic value. In routine, these methods are complex, and impractical. Media constituents may interfere with estimation.
- The methods provide information about total cell mass; both living and non-living.
