Bacterial growth does not only comprise cell division but also increase in cell mass which is related to the synthesis of cell material. The growth is balanced if increase in biomass is proportional to the cell number. This is not the case in lag phase and late growth phases. In lag-phase, the cell biomass increases without increasing number of cells, whereas in the end of log phase, cell size is reducing and cell number increases more compared to biomass. For this reason, these two parameters describing microbial populations have to be distinguished.
1. 1. DIRECT METHODS
1.1. Determining biomass through weighing
This is the most straightforward and simple method, although also the most time-consuming.
Samples are drawn from the culture after certain periods of time, centrifuged at maximum speed to separate cells from the centrifugate or filtered through membrane or paper filter.
Biomass is determined either as wet or dry weight. Collected cells are washed and weighed in a pre-weighed container (pre-weighed together with filtering equipment). For dry matter determination, the tare weight of absolutely dry container and filtering equipment is taken and then the weight together with dry biomass is taken. Microwave oven or thermostat (100-105
°C) can be used for drying the matter to its dry weight. Dry matter (DM) content can be found from the following equation:
DM (mg/ml) = (A-B) / V,
where A is the laden weight with cells (mg), B the tare weight (mg) and V – the volume of the suspension (ml).
Weighing can give different results depending on the growth conditions of the microbe. Thus, microbes producing exogenous polysaccharides give higher dry weight if sugar concentrations in the media are higher. The limitation of this method is need for rather high volume of the centrifuged or filtered sample. At the same time it is the only method for evaluating the growth of species that have filamentous or flocculent growth.
2. INDIRECT METHODS
2.1. Turbidity of cell suspension
The most commonly used biomass determination method in laboratory practice is measuring cell suspension turbidity with spectrophotometry, which allows relatively quick and precise estimation of microbial biomass. The method is based on measuring the absorption of light by the cells, whereas the intensity of absorbed light is proportional to the cell number. According to the Lambert-Beer law, the attenuation of light transmitted through a medium is related
exponentially to the absorption coefficient (extinction coefficient), solution concentration and the optical path length (solution thickness). In standard conditions, the extinction (absorption) is called optical density (OD), whereas the numerical value of the subscript corresponds to the wavelength used for the measurement. The wavelength of the light must be chosen to ensure maximal absorption. The turbidity of microbial suspensions is measured at 420-600 nm. This higher wavelength light is used because the cells absorb it the best and the medium color (often yellow) is interfering the least. This law is only applicable if the particles (cells) do not disturb each other absorbing the light that is at low concentrations. To minimize the error, the high density suspension needs to be diluted. Most spectrophotometers have two scales: logarithmic OD scale and arithmetic transmission (T) scale. Optical transmission is the ratio of the intensity of transmitted light (I) to the intensity of incident light (I0) and OD=-logT.
The OD values do not provide us knowledge about cell number and thus it is rational to construct a calibration graph describing the relationship between the cell biomass and cell number, which is strictly species and even strain specific. Different dilutions are made for constructing a calibration curve for determining the cell number and if necessary, OD.
Depending on the method used for determining the cell number, the calibration curve illustrates the relationship between the optical densities of a specific microbial population and either it’s live or total cell number. The samples need to be kept on ice to prevent cell division and in case of motile bacteria, also fixed with 1% formaldehyde. The method is not applicable to microbial suspensions with very low cell number. The cell number has to be at least 107 cells/ml, so that the turbidity can be measured.
2.2. Amount of cell components
Biochemical analyses are used more often for biomass determination if the sample has low cell number. The amount of various microbial cell components is measured (e.g. total protein, DNA, specific genes, enzymes, RNA etc.). In general, determination of the amount of a single cell component is laborious and can vary depending on the growth phase. For example, cells in active growth phase contain more DNA per cell compared to cells in stationary phase.
QUESTIONS
1. Why is the cell number not expressed in cells/ml in plate count method?
2. Which inoculation methods are used if the cell number is determined with plate count method?
3. Which microbial groups number is determined with nutrient agar spread plate?
4. Find the CFU/ml in the initial sample, if 290 colonies grew on 20 ml of nutrient agar when 100 µl of a dilution (1 ml of the culture is transferred to 9 ml of water, 0.1 ml of the dilution obtained is transferred to 9.9 ml of water) was plated?
5. Why are the bacterial cell numbers different when determined with direct counting by microscopy and using a plating method? Which method is more precise?
6. Why is it necessary to fix microbiological samples and which fixing agents are used?
7. Is membrane filtration technique used with direct or indirect determination of bacteria?
8. Name advantages and disadvantages of a membrane filtration method?
9. How is MPN index determined with the most probable number method?
10. How many dilutions should be made for the MPN method?
11. What is the criterion for distinguishing live and dead cells with LIVE/DEAD kit?
12. What problem could arise with distinguishing live and dead cells with LIVE/DEAD kit?
13. Is it possible to determine the total number of microbes in drinking water using counting chamber? Explain.
14. What are the most common methods for determining the biomass?
15. What method would you use for determining the biomass of a microbial suspension with a flaky consistency?
16. What is spectrophotometric determination of biomass based on?
17. Are live or dead cells measured with spectrophotometry?
18. What is optical density (OD)?
19. What method would you choose for determining biomass if the cell number is very low?
20. What should you do if you want to determine live and total cell number based on OD values?