Food and Industrial Microbiology

Module 2. Microorganisms and food materials

Lesson 3
MICROBIAL GROWTH AND ITS QUANTIFICATION

3.1 Introduction

Growth is an orderly increase in the quantity of cellular constituents. It depends upon the ability of the cell to form new protoplasm from nutrients available in the environment. In most bacteria, growth involves increase in cell mass and number of ribosomes, duplication of the bacterial chromosome, synthesis of new cell wall and plasma membrane, partitioning of the two chromosomes, septum formation, and cell division. This asexual process of reproduction is called binary fission. For unicellular organisms such as bacteria, growth can be measured in terms of two different parameters: changes in cell mass and changes in cell numbers.

3.2 Methods for Measurement of Cell Biomass

Methods for the measurement of the cell mass involve both direct and indirect techniques.

i. Direct physical measurement of dry weight, wet weight, or volume of cells after centrifugation.

ii. Direct chemical measurement of some chemical component of the cells such as total N, total protein, or total DNA contents.

iii. Indirect measurement of chemical activity such as rate of O₂ production or consumption, CO₂ production or consumption, etc.

iv. Turbidity measurements employ a variety of instruments to determine the amount of light scattered by a suspension of cells. Particulate objects such as bacteria scatter light in proportion to their numbers. The turbidity or optical density of a suspension of cells is directly related to cell mass or cell number. The method is simple and nondestructive, but the sensitivity is limited to about 10⁷ cells per ml for most bacteria.

3.3 Methods for Measurement of Cell Numbers

Measuring techniques involve direct counts, visually or instrumentally, and indirect viable cell counts.

3.3.1 Direct microscopic counts (DMC)

DMC are possible using special slides known as counting chambers. Dead cells cannot be distinguished from living ones. Only dense suspensions can be counted (>10⁷ cells per ml), but samples can be concentrated by centrifugation or filtration to increase sensitivity.

A variation of the direct microscopic count has been used to observe and measure growth of bacteria in natural environments. In order to detect and prove that thermophilic bacteria were growing in boiling hot springs, T.D. Brock immersed microscope slides in the springs and withdrew them periodically for microscopic observation. The bacteria in the boiling water attached to the glass slides naturally and grew as micro-colonies on the surface.

3.3.2 Electronic counting chambers

This is done to measure size distribution of cells. F or cells size of the bacteria, the suspending medium must be very clean. Such electronic devices are more often used to count eucaryotic cells such as blood cells.

3.3.3 Indirect viable cell counts

This is also called plate counts, involve plating out (spreading) a sample of a culture on a nutrient agar surface. The sample or cell suspension can be diluted in a nontoxic diluent (e.g. water or saline) before plating. If plated on a suitable medium, each viable unit grows and forms a colony. Each colony that can be counted is called a colony forming unit (cfu) and the number of cfu's is related to the viable number of bacteria in the sample.

Advantages of the technique are its sensitivity (theoretically, a single cell can be detected), and it allows for inspection and positive identification of the organism counted. Disadvantages are (1) only living cells develop colonies that are counted; (2) clumps or chains of cells develop into a single colony; (3) colonies develop only from those organisms for which the cultural conditions are suitable for growth. The latter makes the technique virtually useless to characterize or count the total number of bacteria in complex microbial ecosystems such as soil or the animal rumen or gastrointestinal tract. Genetic probes can be used to demonstrate the diversity and relative abundance of procaryotes in such an environment, but many species identified by genetic techniques have so far proven unculturable.

3.4 The Bacterial Growth Curve

In the laboratory, under favorable conditions, a growing bacterial population doubles at regular intervals. Growth is by geometric progression: 1, 2, 4, 8, etc. or 2⁰, 2¹, 2², 2³.........2ⁿ exponential growth. In reality, exponential growth is only part of the bacterial life cycle, and not representative of the normal pattern of growth of bacteria in nature. (where n = the number of generations). This is called

When a fresh medium is inoculated with a given number of cells, and the population growth is monitored over a period of time, plotting the data will yield a typical bacterial growth curve (Figure 3.1).

Fig. 3.1 The typical bacterial growth curve

When bacteria are grown in a closed system (also called a batch culture), like a test tube, the population of cells almost always exhibits these growth dynamics: cells initially adjust to the new medium (lag phase) until they can start dividing regularly by the process of binary fission (exponential phase). When their growth becomes limited, the cells stop dividing (stationary phase), until eventually they show loss of viability (death phase). Note the parameters of the x and y axes. Growth is expressed as change in the number viable cells vs time. Generation times are calculated during the exponential phase of growth. Time measurements are in hours for bacteria with short generation times.

3.4.1 Four phases of the growth cycle

3.4.1.1 Lag phase

Immediately after inoculation of the cells into fresh medium, the population remains temporarily unchanged. Although there is no apparent cell division occurring, the cells may be growing in volume or mass, synthesizing enzymes, proteins, RNA, etc., and increasing in metabolic activity.

The length of the lag phase is apparently dependent on a wide variety of factors including the size of the inoculum; time necessary to recover from physical damage or shock in the transfer; time required for synthesis of essential coenzymes or division factors; and time required for synthesis of new (inducible) enzymes that are necessary to metabolize the substrates present in the medium.

3.4.1.2 Exponential (log) phase

The exponential phase of growth is a pattern of balanced growth wherein all the cells are dividing regularly by binary fission, and are growing by geometric progression. The cells divide at a constant rate depending upon the composition of the growth medium and the conditions of incubation. The rate of exponential growth of a bacterial culture is expressed as generation time, also the doubling time of the bacterial population. Generation time (G) is defined as the time (t) per generation (n = number of generations). Hence, G=t/n is the equation from which calculations of generation time derive.

3.4.1.3 Stationary phase

Exponential growth cannot be continued forever in a batch culture (e.g. a closed system such as a test tube or flask). Population growth is limited by one of the three factors viz., 1. exhaustion of available nutrients; 2. accumulation of inhibitory metabolites or end products; 3. exhaustion of space, in this case called a lack of "biological space".

During the stationary phase, if viable cells are being counted, it cannot be determined whether some cells are dying and an equal number of cells are dividing, or the population of cells has simply stopped growing and dividing. The stationary phase, like the lag phase, is not necessarily a period of quiescence. Bacteria that produce secondary metabolites, such as antibiotics, do so during the stationary phase of the growth cycle (Secondary metabolites are defined as metabolites produced after the active stage of growth). It is during the stationary phase that spore-forming bacteria have to induce or unmask the activity of dozens of genes that may be involved in sporulation process.

3.4.1.4 Death phase

If incubation continues after the population reaches stationary phase, a death phase follows, in which the viable cell population declines. However, if counting is done by turbidimetric measurements or microscopic counts, the death phase cannot be observed. During the death phase, the number of viable cells decreases geometrically (exponentially), essentially the reverse of growth during the log phase.

3.5 Growth Rate and Generation Time

As mentioned above, bacterial growth rates during the phase of exponential growth, under standard nutritional conditions (culture medium, temperature, pH, etc.), define the bacterium's generation time. Generation times for bacteria vary from about 12 minutes to 24 hours or more. The generation time for E. coli in the laboratory is 15-20 minutes, but in the intestinal tract, the coliform's generation time is estimated to be 12-24 hours. For most known bacteria that can be cultured, generation times range from about 15 minutes to 1 hour. Symbionts such as Rhizobium tend to have longer generation times. Many lithotrophs, such as the nitrifying bacteria, also have long generation times. Some bacteria that are pathogens, such as Mycobacterium tuberculosis and Treponema pallidum, have especially long generation times, and this is thought to be an advantage in their virulence. Generation times for a few bacteria are shown in Table 3.1.

Table 3.1 Generation times for some common bacteria under optimal conditions of growth

3.5.1 Calculation of generation time

When growing exponentially by binary fission, the increase in a bacterial population is by geometric progression. If we start with one cell, when it divides, there are 2 cells in the first generation, 4 cells in the second generation, and 8 cells in the third generation, and so on. The generation time is the time interval required for the cells (or population) to divide.

G (generation time) = (time, in minutes or hours)/n(number of generations)

G = t/n

t = time interval in hours or minutes

B = number of bacteria at the beginning of a time interval

b = number of bacteria at the end of the time interval

n = number of generations (number of times the cell population doubles during the time interval)

b = B x 2ⁿ (This equation is an expression of growth by binary fission)

Solve for n:

logb = logB + nlog2

n = logb - logB
log2

n = logb - logB
.301

n = 3.3 logb / B

G = t/n

Solve for G

G = t
3.3 log b/B

3.6 Continuous Culture of Bacteria

The cultures so far discussed for growth of bacterial populations are called batch cultures. Since the nutrients are not renewed, exponential growth is limited to a few generations. Bacterial cultures can be maintained in a state of exponential growth over long periods of time using a system of continuous culture (Figure 2.2), designed to relieve the conditions that stop exponential growth in batch cultures. Continuous culture, in a device called a chemostat or turbidostat that can be used to maintain a bacterial population at a constant density, a situation that is, in many ways, more similar to bacterial growth in natural environments.

Chemostat is a device for the continuous culture of bacteria. The chemostat relieves the environmental conditions that restrict growth by continuously supplying nutrients to cells and removing waste substances and spent cells from the culture medium.

In a chemostat, the growth chamber is connected to a reservoir of sterile medium. Once growth is initiated, fresh medium is continuously supplied from the reservoir. The volume of fluid in the growth chamber is maintained at a constant level by some sort of overflow drain. Fresh medium is allowed to enter into the growth chamber at a rate that limits the growth of the bacteria. The bacteria grow (cells are formed) at the same rate that bacterial cells (and spent medium) are removed by the overflow. The rate of addition of the fresh medium determines the rate of growth because the fresh medium always contains a limiting amount of an essential nutrient. Thus, the chemostat relieves the insufficiency of nutrients, the accumulation of toxic substances, and the accumulation of excess cells in the culture, which are the parameters that initiate the stationary phase of the growth cycle. The bacterial culture can be grown and maintained at relatively constant conditions, depending on the flow rate of the nutrients.

Fig. 3.2 Schematic diagram of a chemostat

3.6.1 Synchronous growth of bacteria

Studying the growth of bacterial populations in batch or continuous cultures does not permit any conclusions about the growth behavior of individual cells, because the distribution of cell size (and hence cell age) among the members of the population is completely random. Information about the growth behavior of individual bacteria, however, is obtained by the study of synchronous cultures. Synchronized cultures must be composed of cells which are all at the same stage of the bacterial cell cycle. Measurements made on synchronized cultures are equivalent to measurements made on individual cells.

Synchronous growth of a population of bacterial cells is illustrated in Figure 2.3. Synchronous cultures rapidly lose synchrony because not all cells in the population divide at exactly the same size, age or time.

Fig. 3.3 The synchronous growth of a bacterial population.
By careful selection of cells that have just divided, a bacterial population can be synchrinized in the bacterial cell division cycle.
Synchrony can be maintained for only a few generations