FUNDAMENTALS OF MICROBIOLOGY

Lesson 16. QUANTITATIVE MEASUREMENT OF GROWTH

Module 5. Microbial growth and nutrition

Lesson 16

QUANTITATIVE MEASUREMENT OF GROWTH

16.1 Introduction

As stated in Lesson 15, growth in a biological system is an orderly increase in the quantity of cellular constituents and which 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 (binary fission).

Methods for measurement of the cell mass involve both direct and indirect techniques: (Table 16.1)

• Direct physical measurement of dry weight, wet weight, or volume of cells after centrifugation.
• Direct chemical measurement of some chemical component of the cells such as total N, total protein, or total DNA content.
• Indirect measurement of chemical activity such as rate of O₂ production or consumption, CO₂ production or consumption, etc.
• 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, after construction and calibration of a standard curve. The method is simple and nondestructive, but the sensitivity is limited to about 107 cells per ml for most bacteria. Measuring techniques involve direct counts, visually or instrumentally, and indirect viable cell counts.

16.2 Methods for Measurement of Cell Mass

16.2.1 Direct microscopic count

In the direct microscopic count, a counting chamber consisting of a ruled slide and a coverslip is employed. It is constructed in such a manner that the coverslip, slide, and ruled lines delimit a known volume. The number of bacteria in a small known volume is directly counted microscopically and the number of bacteria in the larger original sample is determined by extrapolation.

The Petroff-Hausser counting chamber (commonly used in dairy industry) for example, (Figure 16. 1) has small etched squares 1/20 of a millimeter (mm) by 1/20 of a mm and is 1/50 of a mm deep. The volume of one small square therefore is 1/20,000 of a cubic mm or 1/20,000,000 of a cubic centimeter (cc). There are 16 small squares in the large double-lined squares that are actually counted, making the volume of a large double-lined square 1/1,250,000 cc. The normal procedure is to count the number of bacteria in five large double-lined squares and divide by five to get the average number of bacteria per large square. This number is then multiplied by 1,250,000 since the square holds a volume of 1/1,250,000 cc, to find the total number of organisms per cc in the original sample. If the bacteria are diluted, such as by mixing the bacteria with dye before being placed in the counting chamber, then this dilution must also be considered in the final calculations.

The formula used for the direct microscopic count is

The number of bacteria per cc = The average number of bacteria per large double-lined square X The dilution factor of the large square (1,250,000) X The dilution factor of any dilutions made prior to placing the sample in the counting chamber, e.g., mixing

Fig. 16.1 Direct microscopic count method

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 microcolonies on the surface.

16.2.2 Electronic enumeration of cells

A Coulter counter (Fig.16.2) is an apparatus for counting and sizing particles and cells. It is used, for example, for bacteria and air quality particle size distributions. The counter detects change in electrical conductance of a small aperture as fluid containing cells is drawn through. Cells, being non-conducting particles, alter the effective cross-section of the conductive channel.

It was an American inventor named Wallace H. Coulter who was responsible for the theory and design of the Coulter Counter. He first devised the theory behind its operation in 1947 while experimenting with electronics. Coulter determined that electrical charge could be used to determine the size and number of microscopic particles in a solution. This phenomenon is now known as the Coulter Principle. A typical Coulter counter has one or more microchannels that separate two chambers containing electrolyte solutions. When a particle flows through one of the microchannels, it results in the electrical resistance change of the liquid filled microchannel. This resistance change can be recorded as electric current or voltage pulses, which can be correlated to size, mobility, surface charge and concentration of the particles.

Fig. 16.2 Coulter counter

16.2.3 Plate count method

Standard Plate Count (SPC) is a techniques under this category which is commonly employed in microbiological laboratories for enumeration of bacteria .The SPC is the number of bacterial colonies that develop on a medium in a petri dish seeded with a known amount of inoculum. The number of bacteria in a given sample is usually too great to be counted directly. However, if the sample is serially diluted (Fig.16.3) and then plated out on an agar surface in such a manner that single isolated bacteria form visible isolated colonies , the number of colonies can be used as a measure of the number of viable (living) cells in that known dilution. However, keep in mind that if the organism normally forms multiple cell arrangements, such as chains, the colony-forming unit may consist of a chain of bacteria rather than a single bacterium. In addition, some of the bacteria may be clumped together. Therefore, when doing the plate count technique, we generally say we are determining the number of Colony-Forming Units (CFUs) in that known dilution. By extrapolation, this number can in turn be used to calculate the number of CFUs in the original sample.

Normally, the bacterial sample is diluted by factors of 10 and plated on agar either by pour plate or spread plate technique. After incubation, the number of colonies on a dilution plate showing between 30 and 300 colonies (Fig. 16.4 and Fig. 16.5) is determined. A plate having 30-300 colonies is chosen because this range is considered statistically significant. If there are less than 30 colonies on the plate, small errors in dilution technique or the presence of a few contaminants will have a drastic effect on the final count. Likewise, if there are more than 300 colonies on the plate, there will be poor isolation and colonies will have grown together.

Generally, one wants to determine the number of CFUs per milliliter (ml) of sample. To find this, the number of colonies (on a plate having 30-300 colonies) is multiplied by the number of times the original ml of bacteria was diluted (the dilution factor of the plate counted). For example, if a plate containing a 1/1,000 dilution of the original ml of sample shows 159 colonies, then 159 represents 1/1,000 the number of CFUs present in the original ml. Therefore the number of CFUs per ml in the original sample is found by multiplying 159 x 103 (or preferably represented as 1.59 x 105) as shown in the formula below:

Fig 16.4 Pour plate and spread plate techniques

Fig.16.5 Pour plate and spread plate methods

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

• Only living cells develop colonies that are counted;
• Clumps or chains of cells develop into a single colony;
• 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.

Table 16.1 Comparison of various methods of measurement of bacterial growth

16.2.4 Membrane filter count method

This method is suitable for liquid or semi-liquid samples (e.g. water) and commonly used for enumeration of Coliform and Staphylococcus spp. Membrane filtration method is used with relatively low numbers. A known volume of liquid passed through membrane filter. Filter pore size retains organism. It filters microorganism of size more than 0.45 micrometer (Fig.16.6). Filter is placed on appropriate growth medium and incubated and cells are counted (Fig. 16.7).

Fig. 16.6 Membrane filtration method

Fig. 16.7 Membrane filter enumeration method

(A. Cells on filter as viewed by scanning electron microscopy; b. Colonies on filter)

16.2.5 Turbidity measurement methods

When bacteria growing in a liquid medium are mixed, the culture appears turbid. This is because a bacterial culture acts as a colloidal suspension that blocks and reflects light passing through the culture. Within limits, the light absorbed by the bacterial suspension will be directly proportional to the concentration of cells in the culture. By measuring the amount of light absorbed by a bacterial suspension, one can estimate and compare the number of bacteria present. The instrument used to measure turbidity is a spectrophotometer (Fig. 16.8). It consists of a light source, a filter which allows only a single wavelength of light to pass through, the sample tube containing the bacterial suspension, and a photocell that compares the amount of light coming through the tube with the total light entering the tube. The ability of the culture to block the light can be expressed as either percent of light transmitted through the tube or the amount of light absorbed in the tube. The percent of light transmitted is inversely proportional to the bacterial concentration. The absorbance (or optical density) is directly proportional to the cell concentration.

Fig. 16.8 Turbidity measurement using spectrophotometer

Turbidimetric measurement is often correlated with some other method of cell count, such as the direct microscopic method or the plate count. In this way, turbidity can be used as an indirect measurement of the cell count. For example:

• Several dilutions can be made of a bacterial stock.
• A Petroff-Hausser counter can then be used to perform a direct microscopic count on each dilution.
• Then a spectrophotometer can be used to measure the absorbance of each dilution tube.
• A standard curve comparing absorbance to the number of bacteria can be made by plotting absorbance versus the number of bacteria per cc.
• Once the standard curve is completed, any dilution tube of that organism can be placed in a spectrophotometer and its absorbance read. Once the absorbance is determined, the standard curve can be used to determine the corresponding number of bacteria per cc.

16.2.6 Determination of nitrogen content

The major constituent of cell material is protein, and since nitrogen is a characteristic part of proteins, one can measure a bacterial population or cell crop in terms of bacterial nitrogen. Bacteria average approximately 14 percent nitrogen on a dry weight basis, although this figure is subject to some variation introduced by changes in culture conditions or differences between species. To measure growth by this technique, you must first harvest the cells and wash them free of medium and then perform a quantitative chemical analysis for nitrogen. Bacterial nitrogen determinations are somewhat laborious and can be performed only on specimens free of all other sources of nitrogen. Furthermore, the method is applicable only for concentrated populations. For these and other reasons, this procedure is used primarily in research.

16.2.7 Determination of dry weight

This is one of the simplest indirect methods in situations where determining the number of microorganisms is difficult or undesirable for other reasons. These methods measure some quantifiable cell property that increases as a direct result of microbial growth. The simplest technique of this sort is to measure the weight of cells in a sample. Portions of a culture can be taken at particular intervals and centrifuged at high speed to sediment bacterial cells to the bottom of a vessel. The sedimented cells (called a cell pellet) are then washed to remove contaminating salt, and dried in an oven at 100-105°C to remove all water, leaving only the mass of components that make up the population of cells. An increase in the dry weight of the cells correlates closely with cell growth. However, this method will count dead as well as living cells. There might also be conditions where the dry weight per cell changes over time or under different conditions. For example, some bacteria that excrete polysaccharides will have a much higher dry weight per cell when growing on high sugar levels (when polysaccharides are produced) than on low. If the species under study forms large clumps of cells such as those that grow filamentously, dry weight is a better measurement of the cell population than is a viable plate count.

It is also possible to follow the change in the amount of a cellular component instead of the entire mass of the cell. This method may be chosen because determining dry weights is difficult or when the total weight of the cell is not giving an accurate picture of the number of individuals in a population. In this case, only one component of the cell is followed such as total protein or total DNA. This has some of the same advantages and disadvantages listed above for dry weight. Additionally, the measurement of a cellular component is more labor-intensive than previously mentioned methods since the component of interest has to be partially purified and then subjected to an analysis designed to measure the desired molecule. The assumption in choosing a single component such as DNA is that that component will be relatively constant per cell. This assumption has a problem when growth rates are different because cells growing at high rates actually have more DNA per cell because of multiple initiations of replication.

16.2.8 Measurement of specific chemical changes

The bacterial growth can be indirectly estimated by detecting specific changes caused in growth medium as a result of activity and multiplication of bacterial cells. It includes detecting activity cell products such as acid and gas production. The dye reduction tests such as methylene blue and resazurin reduction tests is based on the fact that the color imparted to milk by the addition of a dye such as methylene blue will disappear more or less quickly. The removal of the oxygen from milk and the formation of reducing substances during bacterial metabolism cause the color to disappear. The agencies responsible for the oxygen consumption are the bacteria. Though certain species of bacteria have considerably more influence than others, it is generally assumed that the greater the number of bacteria in milk, the quicker will the oxygen be consumed, and in turn the sooner will the color disappear. Thus, the time of reduction is taken as a measure of the number of organisms in milk although actually it is likely that it is more truly a measure of the total metabolic reactions proceeding at the cell surface of the bacteria. Gas production by bacteria is another major activity which can be taken up as an index of bacterial growth. Detection of gas production using Durham tube and change in color of the growth medium due to reduction of pH sensitive ingredients present in medium are commonly used for detection of acid and gas producing coliforms and yeasts. An apparatus for measuring CO₂ production is depicted in (Fig.16.9)

Fig. 16.9 Apparatus for measuring the carbon di oxide during fermentation