Growth is defined as an orderly increase in cellular components. Microorganisms grow in a variety of physical and chemical environments. To distinguish orderly growth with that of in orderly growth, in recent years the term “balanced growth” has been used. “Balanced growth” as defined by Campbell, is doubling of every biochemical unit of the cell within the time duration of a single division without a change in the rate of growth.
A number of methods are available for measuring microbial growth. The choice depends upon the measurement, objectives and usefulness of the available techniques. In some cases of industrial fermentations which contain complex media, indirect methods for estimation need to be used however, no matter what method is used, considerable care is required in interpreting the results.
Bacterial growth can be measured either by:
(i) Colony counting or cell counting,
(ii) By weighing the cell i.e. cell mass measurement or
(iii) By cell activity (turbidity method) measurement.
Parameters of Growth:
(i) Cell Counting by Direct Microscopic Count Method:
Direct microscopic counts are rapid but limited for their inability to distinguish between the living and dead cells unless differentiated by use of a vital staining technique. Bacterial cells can be accurately counted by using Petroff- Hausser counting chamber (the chamber includes a glass slide, a cover slip which is framed and kept 1/50 mm above the slide so that bacterial suspension is present in each ruled square of the slide.
The area of square is 1/400 mm2; glass cover slip rests 1/50 mm above the slide hence volume over a square is 1/20,000 mm3 or 1/20,000,000 cm3, for example – if in one square, an average of five bacteria is present, then these are 5 × 20,000,000, or 108 bacteria per ml.
(ii) Colony Counting (Plate-Counting Technique):
This method is based on the fact that one viable cell gives rise to one colony. Therefore, a colony count on an agar plate reveals the viable microbial population. For carrying out this, a measured amount of the sample of bacterial suspension is mixed in the agar medium (when it is in liquid form at 40-45±°C).
It is plated after mixing thoroughly. Each organism grows, reproduces and forms a visible mass in the form of colony. These are counted either by using colony counter or with the aid of large magnifying lens. If too many colonies are appearing and overlapping each other, the sample is diluted so that the colonies are accurately counted.
This method is called pour plate method. The plate count is also performed by spread plate method. In this method 0.1 ml sample containing bacteria is spread over the surface of an agar plate using a sterile glass spreader.
In both pour plate and spread plate methods the plates containing bacterial suspension are incubated until the colonies appear and the colonies are counted. To obtain the appropriate colony number, the sample must be diluted.
Serial dilutions of the sample are usually adopted. To make a 10-fold (10-1) dilution, 10 ml sample is mixed with 90 ml diluent. Serial dilution of soil sample is shown in Fig. 3.5. In most cases, serial dilutions are needed to obtain final dilution.
Demerits of the above methods are not only the suitability of the culture medium and incubation conditions but sometimes bacterial cells are deposited on the plate, does not show their visibility in the form of colony if incubation period is short.
Further, viable counts, preparations of dilutions of the sample also give wrong information’s. To get correct information’s, viable counts are often expressed as the number of colony forming units (CFU) per millilitre rather than number of viable cells. This method is adopted in counting microorganisms in soil.
(iii) Measurement of Cell Mass and Turbidity:
Cell mass is directly proportional to cell number. This can be obtained after centrifugation of a known volume of culture and weighing the pellet obtained. This is called fresh weight but dry weight of cells is obtained by drying the pelleted cells at 90-110°C overnight.
(iv) Turbidity Measurement by Optical Density Method:
The cell mass and number is also obtained by using optical density method. Turbidity is developed in the liquid medium due to the presence of cells which make cloudy appearance to the eyes. Measurement of microbial growth is given in Fig. 3.7.
When sample is more turbid it means that more cells are present. Hence more light is scattered. Turbidity can be measured with a photometer or a spectrophotometer device that detects the amount of un-scattered light recorded in photometer unit (for example “klett units” or optical density (OD) as shown in Fig. 3.8.
The method is indirect; hence some direct measurements of cell number should also be determined. Since OD is proportional to cell mass and thus also to cell number, therefore, turbidity reading acts as an estimate of cell number or cell mass.
The plotting between semi-logarithmic versus time, growth rate of microbial cultures is obtained and used to calculate the generation time of the growing culture. Such a curve can contain data for both cell number and cell mass, allowing for an estimate of both parameters from a single turbidity reading.
If concentration of the cell in the sample is high, light scattered away from the detecting unit by one to one cell can be re-scattered back by another. Hence, the one to one correspondence between cell number and turbidity does not follow linearity as shown. Secondly, dead cells also interfere during measurement. Hence, this method is reasonably accurate only for measurement of microbial growth till early log phase.
Growth in Continuous Culture:
When growth occurs in a fixed volume of a culture medium, it is called batch culture. When culture becomes old, the composition of the culture medium is drastically changed. To keep the cultures in constant environments for longer durations continuous culture method is adopted.
A continuous culture essentially requires a flow of constant volume to which medium is added continuously and from which continuous removal of medium along with culture can occur. When such a system is in equilibrium, cell number and nutrient status remain constant and system is in steady state.
The fresh culture medium flows into a growth chamber at a carefully controlled rate, the volume of culture is maintained by controlling the rates of inflow and out flow, the rate of growth is then controlled by regulating the inflows rates.
The rate of loss of cells through overflow can be expressed as:
dM/ dt = F/V = DM
Where flow rate F is measured in the culture volume V/hr. The expression F/VM is called the dilution rate (D). In a continuous culture, KM = DM
K = D, i.e. growth rate (K), will equal the dilution rate in a stabilized continuous culture. Under such conditions growth of the culture is linear rather than exponential, since the amount of mass/ unit volume (growth rate) remains constant. The continuous culture does not represent synchronous growth since these do not contain cells that are physiologically identical.
(i) Chemostat for Measurement of Growth:
Chemostat (kee-mo-stat, kem. o. stat) means a device for maintaining organisms in continuous culture; it regulates the growth rate of the organism by regulating the concentration of an essential nutrient. The growth rate and growth yield can be controlled independently on each other by adjusting the dilution rate and by varying the concentration of nutrients present in a limiting amount.
The high dilution rate does not allow the organism to grow fast enough to keep up with its dilution rates, a large fraction of the cells may die from starvation due to non-availability of nutrients required for maintenance of cell metabolism (Fig. 3.9). Hence, a minimum amount of energy is required to maintain cell structure. Such energy is called maintenance energy.
The number of cells/ml is called cell density which is controlled in the chemostat by the nutrients. If the concentration of the nutrient in the incoming medium is raised with the dilution rate remaining constant, the cell density will increase although growth rate will remain the same.
(ii) Water Availability:
The water availability is one of the factors affecting the growth of microorganisms in nature. Water availability depends upon the water content present in the environment and water soluble salts, sugars and other substances. Actually, dissolved substances have an ability for water which make the water associated with solutes unavailable to organisms.
Water availability in physical sense is called as water activity denoted by aw, which is a ratio between vapour pressure of the air in equilibrium with a substance or solution to the vapour pressure at the same temperature of pure water.
Most of the agricultural soil has 0.9-1.0 water activities. If the soil is saline, it makes the dry environment. It means cells are in the habitat of high salt concentration. Such organism is called halophiles such as Pseudomonas spp. and Vibrio sp.
Hetero-tolerant organisms can tolerate some reduction in water activity (aw) and grow best in the absence of added solute. The organisms capable to grow at high concentration of salt is called extreme halophiles for example Halo-bacterium and Halo-coccus spp..
These organisms generally require 15-30% sodium chloride for their optimum growth. Similar to these, there are certain organisms, that require high sugar percentage, are called osmophiles such as Saccharomyces, Bacilli and Penicillium spp. Lack of water supports the Xerophiles for example Xeromyces bisporus.
There are occasions as the dissolved salt concentration increases in water; it becomes unavailable to the microorganism. To overcome this situation, organisms start producing intracellular compatible solutes (solute present in the cell adjust cytoplasmic water activity which is non- inhibitory to biochemical processes of the cell), which make the cell in equilibrium state and positive water balance (Table 3.1).
Growth in Batch Culture:
A batch culture is that in which growth of microbes occurs in a limited volume of liquid medium. During growth in liquid medium of unicellular microorganisms, the increase in cell number is logarithmic (exponential) for some time.
Since each cell gives rise to two cells which in turn gives rise to four cells and so on. When the logarithmic of cell number in the population is plotted against time, a straight line is obtained to suggest that there is an equal percentage increase in the number of cells during a constant time interval.
There are four general patterns of microbial growth. Bacteria divide by binary fission. During the growth cycle cells double its mass and also the amount of all the cell constituents. Prior to division, cell wall and membrane is synthesized and the parent cell divides into two identical cells, each of which grows exactly as the original cell. Doubling time for most bacteria is reported to be as fast as 15-20 minutes under optimal conditions.
Yeasts divide by budding (exceptions to include yeast that grow by fission or by forming hyphae). Budding involves the formation of a bud on the mother cell. The bud grows until it becomes same in the size of the mother. At this point the daughter cell separates. Under optimal conditions, yeast may divide in as little as 45 minutes however, 90-120 minutes is optimum.
Molds grow by chain elongation and branching. Growth proceeds from the tip of the mycelium by forming septa between the cells. Depending upon the physiological environment, the mycelium may be long or diffuse, short and highly branched, or a mixture of two.
Typically molds have optimal mass doubling time of 4- 8 h although, some are reported to double as little as 60-90 min. Actinomyces and Streptomyces are classes of organisms closely related to bacteria that are extremely important industrially. Both classes also grow as mycelial organisms.
Microbial viruses or phages do not follow the normal growth patterns. They require a host to multiply. So they must infect a cell and utilised the cell protein and nucleic acid synthesizing machinery to produce new viral material. A virus inside the cell can replicate itself 50-300 times before the cell bursts. The growth is exponential but the exponent is much greater than two.
When bacteria are transferred from a slant culture to a known volume of liquid medium the population undergoes a characteristic sequence in its rate of increase in cell number. Four recognizable phases are seen when the increase in cell number is determined in relation to time. In lag phase, there is no increase in the number of viable cells. However, cell growth occurs as indicated by the increase in cell mass.
During this period the cells increase in size as a result of extensive macromolecule synthesis. This stage represents a period of active growth without cell division and the cells prepare for division.
The length of the lag phase depends upon a variety of factors such as the age of the inoculum, the composition of the growth medium and the environmental factors such as temperature, pH, aeration, etc. The lag phase is then followed by the phase of exponential growth (log phase) (Fig. 3.10).
In the log phase, the cell population increases logarithmically and the cells divide at the maximum rate permitted by the composition of medium and environmental conditions.
The cell number during this period increases as a function of the exponent (21, 22, 23, 24, …… 2n). Growth rates are measured during this period since growth occurs at a maximal rate. The growth rate can be expressed in time. For example, a culture which has a double time of 60 minutes will give a growth rate of one.
The rate of growth is influenced by a variety of factors such as the composition of medium, environmental and the inherent properties of the culture. The length of log period is determined mainly by the composition of the medium and the rate of accumulation of inhibitory products.
Although in the log phase, the growth is maximum, a culture in logarithmic phase represents a population of cells of different ages, some have just divided and others are in the intermediate stages of the division cycle. Therefore, in physiological terms the population is not homogeneous.
The period of exponential growth is followed by stationary phase in which the total number of viable cells remain constant. In fact, we can say that the stationary phase is reached when the viable cell number does not increase.
The duration of stationary phase varies with the organism and environmental conditions. Some organisms may display long stationary phase lasting for several days while others may show a very short stationary phase of only few hours before the next phase begins.
The death phase (the phase of decline) is characterized by an exponential decrease m number of the viable cells. A cell is considered to have died or to have to become non-viable when it is no longer capable of multiplying. The phase of decline is seen generally in bacteria, which also be rapid if cell lysis occurs.
Some bacteria such as the sporulating bacteria may form endospores as they reach the stationary phase of growth and these would be resistant to lysis or death. In such cases the number of viable cells will remain constant after attaining the stationary phase and the phase of decline may not be seen.
When a population of cell from a stationary phase or death phase is used to inoculate fresh growth medium, the cells will not continue to die but re-enter the lag phase and initiate new growth. Cells from the death phase may, however, show a longer lag in contrast to cells transferred from either the stationary phase or the logarithmic phase.
All the four phases are applied for population growth.
Mathematics of bacterial growth:
Bacterial cells divide by binary fission, hence their increase in cell number is a function of the exponent (21, 22, 23, 24, …… 2n). where n = exponent, number of cell division if M1 is the number of cells at T1 time and M2 ………… at T2 time
M2 = M1 2n
By taking log on both sides
log M2 = log M1 n log2
if equation is simplified:
log M2/M1= n log 2;
or n = log M2 – log M1/log2 growth equation
If t is the mean generation time. Then,
n x t = T2 – T1 or n = T2 – T1/t
where n = number of cell division or generations that the population has undergone during an interval of time (T2-T1).
Hence, log M2/M1 = T2 – T1/t × log2
The plot of log cell number against time will therefore be a straight line.
If a bacterial culture contains 105 cells/ml at t0 and 1010 cells/ml after 4 hours, calculate its specific growth rate and doubling time.
Specific growth rate (m)
µ= 2.303 log10 (t-t0)/time interval
= 2.303 log10 (1010 – 105)/4
= 2.303 5/4
Doubling time (td) of bacterial cells
= 0.693/µ = 0.693/2.88 = 0.240h × 60 = 14.43 minutes
When the cultures in log phase are analyzed, cells are present in various stages and division cycle. Analysis of such population therefore, yields only average value of any parameter. To understand the properties of individual cell, during the course of its division cycle, it is necessary to analyze each cell, which is practically not possible. A system that closely resembles and amplifies the behaviour of single cell is a synchronous culture, which contains cells that are physiologically identical and are in the stage of division cycle.
A synchronous population can be generated either by physically separating the cells in the same stage of division or by forcing a cell population to attain an identical physiological condition by a change in the environment. To obtain synchronized population, cells may be inoculated into a medium at a sub-optimal temperature. After sometime, they will metabolize slowly but will not divide.
When the temperature is raised to optimum, the cells will undergo a synchronized division. In the synchronized culture, the cells are physiologically identical, cell division occurs periodically at constant intervals. The dry mass of the cell, optical density, total proteins, or RNA contents per cell increase at a constant rate. The amount per cell will increase in population to the cell number.
On the other hand, the pattern of DNA synthesis can be either periodic or continuous depending upon how fast the culture is growing. E. coli grows in a medium with a generation time greater than 40 min, will show a period when no DNA synthesis occurs.
Recently, to obtain synchronous culture, the exponentially grown culture is centrifuged either in sucrose, glycerol or sorbitol gradients in order to separate cells based on their densities which is directly related to their age. Such fractions provided the same results as by using synchronous culture.
In a culture medium containing two carbon sources, bacteria such as E.coli displays a growth curve, called diauxic (Fig. 3.11). Under this conditions, if glucose and lactose are supplemented in medium having E. coli. First E. coli will utilize glucose and after it is exhausted lactose will be utilized. In between a short lag period is there. This led us to conclude that E. coli preferentially utilizes certain carbon sources.