In this lecture we will discuss microbial growth and its relationship to macromolecular synthesis, growth occurring in the absence of cell division usually results in an increase in the size and weight of the cell. In many organisms growth is followed by cell division, this results in an increase in cell numbers, the new cell formed eventually attain the same size as the original cell. Growth of a cell results in an increase in the number of cells as a consequence of cell growth and cell division. The study of cell growth of microbes is difficult because of the small size of bacteria, therefore growth studies in microbiology deal with population.
Since bacterial growth is determined by population lecture 6 is concerned with methods for the measurement of a population of bacteria. This will include total counts as determined by a direct or a viable count. Counts of a population of bacteria by direct microscopic examination or by a mechanical apparatus only. A direct count of a bacterial population only detects the total number of organisms and include both dead and living organisms while a viable count with the use of a pour plate culture procedure estimates the number of living bacteria in a population. This is significant because one would wish to have an estimate of the number of bacteria in a population when dealing with estimating the virulence of an organism (pathogenicity) or the use of a challenge dose when developing vaccine.
To estimate how rapidly bacteria multiple a discussion of growth curves are often utilized. The rate of growth of a bacterial culture is population to the number of bacteria at that moment. Growth of bacteria is usually expressed as generation time ( time required for the doubling of the population at a given time) for organisms which reproduce by binary fission, a generation is defined as a doubling of cell numbers. Methods of calculation of the generation time of a bacteria as well as the different growth curves will be discussed.
6.2.1 Definition of Growth:
Growth of an organism is thought to be the ordinary increase in all of the components. Thus, the intake of water or deposits of lipid which may increase cell size is not true growth. True growth may occur either in size or in numbers. The individual cell may get progressively larger, or it may divide and give rise to two daughter cells.
6.2.2 Measurement of Growth:
Growth may be estimated by counting the number of bacteria, by measuring their size, or by combining the two so as to calculate the total volume of bacterial substance produced. Growth may also be measured using bacterial dry weight, nitrogen content, etc.
6.2.3 Measurement of Size of Bacteria:
The dimensions of bacteria in stained, fixed preparations may be measured by the use of an eyepiece micrometer having movable cross-threads. Measurement may also be made on bacteria which have been fixed but not dried, by photomicography with phase-contrast, on darkground illumination, and subsequently measuring on a photographic enlargement. Bacteria may also be measured using electron micrographs.
6.2.4 Measurement of Number of Bacteria:
Bacteria may be counted in such a way as to obtain an estimate either of the total number of organisms alive and dead, or of the number of living organisms only. The first is referred to as the TOTAL COUNT, the second as the VIABLE COUNT.
6.3.1 DIRECT COUNTING
under the microscope of a standard preparation on a slide: In this method, a drop of known specimen (usually 0.01 ml) is spread over a known area on a glass slide, dried, fixed, stained, and examined under the microscope. The average number of stained organisms per field is determined and from this and the area of the field the total number of bacteria in the original volume is calculated. However, since it is impractical to count all the organisms on a smear accurately, it is customary to count the number of bacteria in several microscopic fields and determine the number per milliliter of the original sample from the average of the counts. The calculations required to make this conversion, i.e., the average number of bacteria per microscopic field computed as the number of bacteria per milliliter of original material, are the following:a. The area of the microscope field =_r2. The diameter of the radius of the microscopic field can be measured using a stage micrometer.
b. The area of the smear in ml divided by the area of one microscopic field contained in the 1 sq cm over which the specimen was spread.
c. The number of microscopic fields in the film times the average number of bacteria per field times 100 (since only 0.01 ml was spread on the film) equals the number of bacteria per ml. This method, though much used in the past, especially in the Breed method for counting bacteria in milk, is open to several objections.
Microscopic Examination of Organisms in a COUNTING CHAMBER: In this method a hemocytometer may be used with the bacteria counted when stained with a suitable dye. Special counting chambers such as the Petroff-Hausser bacteria counting chamber are also used for making direct counts. The bacterial suspension is placed in a groove-ruled chamber, the dimensions of which are known, and covered with a coverslip. The examination can be made more accurately with a dark-field or phase-contrast microscope if the cells are not stained and hence not conspicuous by light microscopic examination. The volume above each ruled line can be measured since the counting chamber is marked off into definite areas and since the depth of the suspension in the chamber is known. All that is required, therefore, is to count the number of organisms in certain areas, obtain an average figure for the count per area, and then multiply this average by an appropriate factor to convert the count to number of bacteria per ml.
6.3.2 MICROSCOPIC EXAMINATION OF BACTERIA CONCENTRATED BY MEMBRANE FILTER:
This method is particularly useful when the bacterial count is low. When the suspension is free of other particulate matter, counts of total bacteria can be obtained by filtering a known volume through a membrane filter, staining the bacteria deposited on the upper surface of the membrane, and counting the number per field by microscopy.
6.3.3 MECHANICAL ESTIMATION OF PARTICLE CONCENTRATION:
A Coulter Counter may be used to estimate the population of bacteria in a given sample. In the Coulter Counter, a standard volume of a suitable dilution of a suspension of cells or bacteria or other particles are automatically driven at high speed through an aperture in a glass membrane. The passage of the particle through the aperture causes an alteration in the electrical resistance across it, which is recorded as an impulse and counted by a suitable circuit. The number of impulses registered shows the number particles in the standard volume. The aperture gives satisfactory results with particles of the size of mammalian cells and has been adapted for counting and sizing bacteria.
Probably the most convenient methods currently used are as follows:
6.4.1 Colony Count Method
220.127.116.11 USING POUR PLATE:
This method involves preliminary dilution when necessary of the suspension, the plating out of unit quantities of suitable dilutions into a suitable solid media, the counting of colonies that develop after incubation. The average number of colonies per plate multiplied by the reciprocal of the dilution affords and estimation of the living bacteria contained in the original suspension.
18.104.22.168 USING SURFACE PLATE INOCULATION BY DROPS:
The bacterial suspension, instead of being mixed with the melted agar, is deposited in the form of a drop on the surface of solid media. The count is estimated from the number of colonies that develop. The method is particularly useful in making counts of an organism that grows best on opaque medium, such as blood.
22.214.171.124 USING SURFACE PLATE INOCULATION BY A SPREADER:
The plate is inoculated with 0.1 ml of the suspension, which is then distributed as uniformly as possible over the surface with a glass spreader. The colonies are then counted in the usual way.
126.96.36.199 USING MEMBRANE FILTER:
When only a small number of bacteria are in the material to be tested, i.e., in the examination of water, the bacteria may be concentrated by filtration of the sample through a bacteria-proof membrane. If the membrane is then incubated on the surface of a pad soaked in a suitable liquid medium or nutrient agar, the bacteria produced on the upper surface of the medium can be counted, under magnification if necessary.
It must be remembered that certain limitations should be considered in the interpretation of viable counts of bacteria by any of these methods:
First, one method estimates the concentration of viable particles in a suspension of bacteria. If the suspension contains clumps, filaments or chains of bacteria, the concentration of cells may appear to be less than that of individual bacteria.
Second, the count of viable bacteria, even of a pure culture, on a given medium at a given temperature of incubation under given atmosphere may be much lower than the count on some other media, or at a different temperature or atmospheric condition. In essence, "viable" has no absolute meaning; what is meant is the number of bacteria able to form colonies or produce turbidity under a specified condition.
Third, the theoretical limits to precision of both total and viable counts which arise from sampling error must be considered.
6.5.1 THE TURBIDIMETRIC AND NEPHELOMETRIC METHODS:
The turbidity or opacity of a bacterial suspension results mainly from the scattering of light at the interfaces between the optically dense bacteria and the media. The determination of the intensity of a beam of light of suitable wavelength after it has transferred a known thickness of suspension may be measured and the "OPTICAL DENSITY" of the suspension calculated; within a certain range, bacterial suspensions obey Beer's Law. The intensity of the scattered light may be measured in a NEPHELOMETER, Figure 19 (Handout).
The viable cell count is usually considered the measure of cell concentration. However, the general practice is to measure the light absorption or light scattering of a culture by photoelectric means and to relate these viable counts to a standard curve. By means of a standard curve, all further optical readings can be converted to cell concentration. However, it is essential that a separate standard curve be determined for each stage in the growth of the culture so that differences in the average cell size can be taken into account. Since it is technically difficult to perform a large number of dry weight measurements and since such measurements are adequate only with relatively large numbers of cells, various indirect measurements are used. These include photoelectric measurements, nitrogen determination, and centrifugation in special vessels. It is therefore necessary in each case to construct a standard curve equating the measurements (values) with known dry weights.
6.5.2 Exponential Growth (Growth Constant)
Since the two new cells produced by the growth and division of a single cell are each capable of growing at the same rate as the parent cell, the number of cells in a culture increases with time as a geometric progression, i.e., exponentially.
The rate of growth of a culture at a given time is directly proportional to the number of cells present at that moment. This relationship is given by the following equation:
dN = KN 1
Integration of the above expression gives:
N = N0ekt 2
Where N0 is the number of cells at time zero and N is the number of cells at any later time, t.
In Eq. 2 above, k is the growth constant. Solving the equation for k gives:
k = 1n(N/N0) 3
Thus, k represents the rate at which the natural logarithm of cell numbers increase with time, and can be determined graphically with time as shown on next page.
Log of 8 Slope:InN-InNo = k
1 2 3 4 5 6
The above graph represents the rate at which the natural logarithm of cell number increases with time.
In bacteriology it is generally customary to express the growth of bacteria or microbial cultures in terms of generation hours. For an organism which reproduces by binary fission, a generation is defined as a doubling of cell numbers. The number of cells (N) increases with the generation (g) as follows:
Not all bacteria have the same generation time. For some, such as Escherichia coli, it may be 15 to 20 minutes; for others, it may be several hours. Similarly, the generation time is not the same for a particular bacterium under all conditions. The amount and kinds of nutrients in the medium and the specific physical environment cause variation in the generation time.
6.7 Growth Curve of Bacterial Populations
If a liquid medium is inoculated with microbial cells taken from a culture which has previously been grown to saturation and if the number of viable cells per ml are determined periodically and plotted, a curve of the type shown in Figure 18 is usually obtained.
The curve may be discussed in terms of six phases, represented by the letters A - F on Figure 18.
__________________________________________________________Section of Curve Phase Growth Rate
__________________________________________________________A Lag Zero
B Acceleration Increasing
C Exponential Constant
D Retardation Decreasing
E Maximum Stationary Zero
F Decline Negative (death)
6.7.1 The Lag Phase
This growth phase represents a period during which the cells, depleted of metabolites and enzymes as a result of unfavorable conditions obtained at the end of their previous culture history, adapt to their new environment. Enzymes and intermediates form and accumulate until they are present in concentrations permitting growth to resume. In such cases when cells are taken from an entirely different medium, it often happens that they are genetically incapable of growth in the new medium. In such instances, the lag represents the time necessary for a few mutants in the inoculum to multiply sufficiently for a net increase in the cell number to be apparent.
6.7.2 The Exponential or Logarithmic Phase
During the exponential phase the cells are in a steady state condition, the mathematics of which have already been discussed. As we have already seen in a previous illustration, the log of the number of cells plotted against time is a straight line. Under appropriate conditions, the growth rate is maximal at this phase. New cell material is being synthesized at a constant rate, but the new material is itself catalytic, and the mass increases in an exponential manner. This continues until one of two things happens: either one or more nutrients in the medium becomes exhausted, or toxic metabolic products accumulate and inhibit growth. For aerobic organisms, the nutrient which becomes limited is usually oxygen; when the cell concentration exceeds 1 x 107/ml (in the case of bacteria), the growth rate will decrease unless oxygen is forced into the medium by agitation or by bubbling in air. When the cell concentration reaches 4-5 x 108/ml, the rate of oxygen diffusion cannot meet the demand, even in the aerated medium, and thus growth is progressively slowed.
6.7.3 The Stationary Phase
Eventually the exhaustion of nutrients or the accumulation of toxic products causes growth to cease completely. In most cases, however, cell turnover takes place in the stationary phase; there is very little loss of cells through death, which is just balanced by the formation of new cells through growth and division.
6.7.4 The Death Phase (Phase of Decline)
After the stationary period, the bacteria may die at a rate which exceeds the rate of production of new cells, if indeed some cells are still capable of reproducing. The actual cause of death during this phase of the culture is undoubtedly a variety of conditions, and may vary with each bacterial type. Generally, an accumulation of products to an inhibitory level (e.g., acid) and/or a depletion of essential nutrients are sufficient to account for this development. The immediate transition from the stationary phase to the area of a diminishing viable population is followed in some instances by a death rate that is the logarithmic inverse of the log phase. The death rate may continue for a period of many days, or all cells may die after 2 to 3 days, depending on the particular type of bacteria. Some bacteria may remain viable in a culture medium for months or even years.
6.7.5 Transitional Periods between Growth Phases
Cultures truly proceed gradually from one phase of growth to another as outlined above. This indicates that not all the cells are in exactly identical physiological conditions toward the end of a given phase of growth. Time is required for some to catch up with others. An appreciation of the pattern of normal growth for microorganisms is of great importance for many academic and practical reasons. For example, one can reasonably predict the approximate time required for a particular bacterium to attain its maximal population under special conditions of cultivation. In terms of physiological conditions, the growth curve includes young, actively metabolizing cells, cells in the process of dying, and cells in-between these extremes. For studies of the metabolism of organisms, it may be desirable to use cells from the logarithmic phase of growth. The effect of chemical substances and physical conditions on an organism is not the same during all the phases of growth. Thus for many reasons the study and use of microorganisms necessitates familiarity with the population changes that occur during the growth of a culture.
Study Questions:1. In what stage of the bacterial growth curve are the cells most uniform in their morphology? Increase markedly in size? Explain.
2. When appropriate data is plotted for a bacterial growth curve, why is a curved line obtained instead of an abrupt straight line?
3. Compare the direct and indirect methods for estimating bacterial populations as to advantage, limitation of use and practical application.
4. What is the significance and practical advantage of knowing the generation time of bacteria? Growth curve?
5. In the calculation of generation time for a particular species of bacteria, what specific data is required?
6. What is the generation time of Escherichia coli? How does this compare with the generation time of other bacteria?
7. What is the relationship between protoplasts, spheroplasts and L forms of bacteria?
8. What structures of the bacterial cell are likely to contain significant amounts of diaminopimelic acid? calcium dipicolinate? DNA, teicholic acid?
9. Why is the significance of the position and size of the endospore in a vegetative cell of value for the identification of a bacterium? Supplement your answers with specific examples.
10. Is endospore formation in bacteria a method of reproduction or a means of multiplication? Explain.
11. Define the following terms: optical density, virulence, phase- contrast coulter counter, exponential growth.
12. Compare a viable count and a total count of a bacterial population.
13. Renew the various stages of bacterial growth.