PART 1 Introduction
6.2. BATCH GROWTH
6.2.1. Quantifying Cell Concentration
The quantification of cell concentration in a culture medium is essential for the determina- tion of the kinetics and stoichiometry of microbial growth. The methods used in the quan- tification of cell concentration can be classified in two categories: direct and indirect. In many cases, the direct methods are not feasible due to the presence of suspended solids or other interfering compounds in the medium. Either cell number or cell mass can be quan- tified depending on the type of information needed and the properties of the system. Cell mass concentration is often preferred to the measurement of cell number density when only one is measured, but the combination of the two measurements is often desirable.
6.2.1.1. Determining cell number density. A Petroff–Hausser slide or a hemocytometeris often used for direct cell counting. In this method, a calibrated grid is placed over the culture chamber, and the number of cells per grid square is counted using a microscope. To be statistically reliable, at least 20 grid squares must be counted and av- eraged. The culture medium should be clear and free of particles that could hide cells or be confused with cells. Stains can be used to distinguish between dead and live cells. This method is suitable for nonaggregated cultures. It is difficult to count molds under the mi- croscope because of their mycelial nature.
Plates containing appropriate growth medium gelled with agar (Petri dishes) are used for counting viable cells. (The word viableused in this context means capable of reproduc- tion.) Culture samples are diluted and spread on the agar surface and the plates are incu- bated. Colonies are counted on the agar surface following the incubation period. The results are expressed in terms of colony-forming units (CFU). If cells form aggregates, then a single
mR N
dN
∫ 1 dt mnet =mg-kd
colony may not be formed from a single cell. This method (plate counts) is more suitable for bacteria and yeasts and much less suitable for molds. A large number of colonies must be counted to yield a statistically reliable number. Growth media have to be selected carefully, since some media support growth better than others. The viable countmay vary, depending on the composition of the growth medium. From a single cell, it may require 25 generations to form an easily observable colony. Unless the correct medium and culture conditions are chosen, some cells that are metabolically active may not form colonies.
In an alternative method, an agar–gel medium is placed in a small ring mounted on a microscope slide, and cells are spread on this miniature culture dish. After an incubation period of a few doubling times, the slide is examined with a microscope to count cells.
This method has many of the same limitations as plate counts, but it is more rapid, and cells capable of only limited reproduction will be counted.
Another method is based on the relatively high electrical resistance of cells (Fig. 6.1). Commercial particle countersemploy two electrodes and an electrolyte solu- tion. One electrode is placed in a tube containing an orifice. A vacuum is applied to the inner tube, which causes an electrolyte solution containing the cells to be sucked through the orifice. An electrical potential is applied across the electrodes. As cells pass through the orifice, the electrical resistance increases and causes pulses in electrical voltage. The number of pulses is a measure of the number of particles; particle concentration is known, since the counter is activated for a predetermined sample volume. The height of the pulse is a measure of cell size. Probes with various orifice sizes are used for different cell sizes.
This method is suitable for discrete cells in a particulate-free medium and cannot be used for mycelial organisms.
The number of particles in solution can be determined from the measurement of scattered light intensity with the aid of a phototube (nephelometry). Light passes through
Figure 6.1. Diagram of a particle counter using the electrical resistance method for measuring cell number and cell size distribu- tion. The ratio of volumes of a nonconduct- ing particle to the orifice volume (altered by changing orifice diameter) determines the size of the voltage pulse. (Adapted with per- mission, from D. I. C. Wang and others, Fer- mentation and Enzyme Technology, John Wiley & Sons, New York, 1979, p. 64.)
the culture sample, and a phototube measures the light scattered by cells in the sample.
The intensity of the scattered light is proportional to cell concentration. This method gives best results for dilute cell and particle suspensions.
6.2.1.2. Determining cell mass concentration. Direct methods. Determi- nation of cellular dry weight is the most commonly used direct method for determining cell mass concentration and is applicable only for cells grown in solids-free medium. If noncellular solids, such as molasses solids, cellulose, or corn steep liquor, are present, the dry weight measurement will be inaccurate. Typically, samples of culture broth are cen- trifuged or filtered and washed with a buffer solution or water. The washed wet cell mass is then dried at 80∞C for 24 hours; then dry cell weight is measured.
Packed cell volumeis used to rapidly but roughly estimate the cell concentration in a fermentation broth (e.g., industrial antibiotic fermentations). Fermentation broth is cen- trifuged in a tapered graduated tube under standard conditions (rpm and time), and the volume of cells is measured.
Another rapid method is based on the absorption of light by suspended cells in sam- ple culture media. The intensity of the transmitted light is measured using a spectrometer.
Turbidity or optical densitymeasurement of the culture medium provides a fast, inexpen- sive, and simple method of estimating cell density in the absence of other solids or light- absorbing compounds. The extent of light transmission in a sample chamber is a function of cell density and the thickness of the chamber. Light transmission is modulated by both absorption and scattering. Pigmented cells give different results than unpigmented ones.
Background absorption by components in the medium must be considered, particularly if absorbing dissolved species are taken into cells. The medium should be essentially parti- cle free. Proper procedure entails using a wavelength that minimizes absorption by medium components (600- to 700-nm wavelengths are often used), “blanking” against medium, and the use of a calibration curve. The calibration curve relates optical density (OD) to dry-weight measurements. Such calibration curves can become nonlinear at high OD values (> 0.3) and depend to some extent on the physiological state of the cells.
Indirect methods. In many fermentation processes, such as mold fermentations, di- rect methods cannot be used. In such cases indirect methods are used, which are based mainly on the measurement of substrate consumption and/or product formation during the course of growth.
Intracellular components of cells such as RNA, DNA, and protein can be measured as indirect measures of cell growth. During a batch growth cycle, the concentrations of these intracellular components change with time. Figure 6.2 depicts the variation of cer- tain intracellular components with time during a batch growth cycle. Concentration of RNA (RNA/cell weight) varies significantly during a batch growth cycle; however, DNA and protein concentrations remain fairly constant. Therefore, in a complex medium, DNA concentration can be used as a measure of microbial growth. Cellular protein measure- ments can be achieved using different methods. Total amino acids, Biuret, Lowry (folin reagent), and Kjeldahl nitrogen measurements can be used for this purpose. Total amino acids and the Lowry method are the most reliable. Recently, protein determination kits from several vendors have been developed for simple and rapid protein measurements.
158 How Cells Grow Chap. 6
Figure 6.2. The time-dependent changes in cell composition and cell size for Azotobac- ter vinelandiiin batch culture are shown. (With permission, from M. L. Shuler and H. M.
Tsuchiya, “Cell Size as an Indicator of Changes in Intracellular Composition of Azotobac- ter vinelandii,” Can. J. Microbiol. 31:927, 1975 and National Research Council of Canada, Ottawa.)
However, many media contain proteins as substrates, which limits the usefulness of this approach.
The intracellular ATP concentration (mg ATP/mg cells) is approximately constant for a given organism. Thus, the ATP concentration in a fermentation broth can be used as a mea- sure of biomass concentration. The method is based on luciferase activity, which catalyzes oxidation of luciferin at the expense of oxygen and ATP with the emission of light.
(6.4) When oxygen and luciferin are in excess, total light emission is proportional to total ATP present in the sample. Photometers can be used to detect emitted light. Small concentra- tions of biomass can be measured by this method, since very low concentrations of ATP (10-12g ATP/l) can be measured by photometers or scintillation counters. The ATP con- tent of a typical bacterial cell is 1 mg ATP/g dry-weight cell, approximately.
Sometimes, nutrients used for cellular mass production can be measured to follow microbial growth. Nutrients used for product formation are not suitable for this purpose.
Nitrate, phosphate, or sulfate measurements can be used. The utilization of a carbon source or oxygen uptake rate can be measured to monitor cellular growth when cell mass is the major product.
The products of cell metabolism can be used to monitor and quantify cellular growth. Certain products produced under anaerobic conditions, such as ethanol and lactic acid, can be related nearly stoichiometrically to microbial growth. Products must be either growth associated (ethanol) or mixed growth associated (lactic acid) to be correlated with microbial growth. For aerobic fermentations, CO2is a common product and can be related to microbial growth. In some cases, changes in the pH or acid–base addition to control pH can be used to monitor nutrient uptake and microbial growth. For example, the utilization of ammonium results in the release of hydrogen ions (H+) and therefore a drop in pH. The amount of base added to neutralize the H+released is proportional to ammonium uptake and growth. Similarly, when nitrate is used as the nitrogen source, hydrogen ions are re- moved from the medium, resulting in an increase in pH. In this case, the amount of acid added is proportional to nitrate uptake and therefore to microbial growth.
In some fermentation processes, as a result of mycelial growth or extracellular poly- saccharide formation, the viscosity of the fermentation broth increases during the course of fermentation. If the substrate is a biodegradable polymer, such as starch or cellulose, then the viscosity of the broth decreases with time as biohydrolysis continues. Changes in the viscosity of the fermentation broth can be correlated with the extent of microbial growth. Although polymeric broths are usually non-Newtonian, the apparent viscosity measured at a fixed rate can be used to estimate cell or product concentration.