6.Microbial Grow 117 Table 6.2 rglas mfor Selected 525050 4m0 6.2 Measurement of Microbial Growth e 6.5 The Petroff-Hausser Cou e.(e)An creases in both.The most commonly vt×400 dthe dvantage late the concer nofcd nental situation bacteria/mm(bacteria/square)(5). Measurement of Cell Numbers The most obvious way to determine microbial numbers is bacteria/cm'=(28 bacteria)(25 squares(501(10'=3.5 x 10? of microrPctrotl-H counting chambe can used for counting procaryotes:;he are stained,or when a phase-contrast ora fluorescence micro
Prescott−Harley−Klein: Microbiology, Fifth Edition II. Microbial Nutrition, Growth, and Control 6. Microbial Growth © The McGraw−Hill Companies, 2002 6.2 Measurement of Microbial Growth 117 Table 6.2 Generation Times for Selected Microorganisms Temperature Generation Time Microorganism (°C) (Hours) Bacteria Beneckea natriegens 37 0.16 Escherichia coli 40 0.35 Bacillus subtilis 40 0.43 Staphylococcus aureus 37 0.47 Pseudomonas aeruginosa 37 0.58 Clostridium botulinum 37 0.58 Rhodospirillum rubrum 25 4.6–5.3 Anabaena cylindrica 25 10.6 Mycobacterium tuberculosis 37 12 Treponema pallidum 37 33 Algae Scenedesmus quadricauda 25 5.9 Chlorella pyrenoidosa 25 7.75 Asterionella formosa 20 9.6 Euglena gracilis 25 10.9 Ceratium tripos 20 82.8 Protozoa Tetrahymena geleii 24 2.2–4.2 Leishmania donovani 26 10–12 Paramecium caudatum 26 10.4 Acanthamoeba castellanii 30 11–12 Giardia lamblia 37 18 Fungi Saccharomyces cerevisiae 30 2 Monilinia fructicola 25 30 6.2 Measurement of Microbial Growth There are many ways to measure microbial growth to determine growth rates and generation times. Either population mass or number may be followed because growth leads to increases in both. The most commonly employed techniques for growth measurement are examined briefly and the advantages and disadvantages of each noted. No single technique is always best; the most appropriate approach will depend on the experimental situation. Measurement of Cell Numbers The most obvious way to determine microbial numbers is through direct counting. Using a counting chamber is easy, inexpensive, and relatively quick; it also gives information about the size and morphology of microorganisms. Petroff-Hausser counting chambers can be used for counting procaryotes; hemocytometers can be used for both procaryotes and eucaryotes. Procaryotes are more easily counted in these chambers if they are stained, or when a phase-contrast or a fluorescence microCover glass Chamber holding (a) bacteria (b) (c) Figure 6.5 The Petroff-Hausser Counting Chamber. (a) Side view of the chamber showing the cover glass and the space beneath it that holds a bacterial suspension. (b) A top view of the chamber. The grid is located in the center of the slide. (c) An enlarged view of the grid. The bacteria in several of the central squares are counted, usually at 400 to 500 magnification. The average number of bacteria in these squares is used to calculate the concentration of cells in the original sample. Since there are 25 squares covering an area of 1 mm2 , the total number of bacteria in 1 mm2 of the chamber is (number/square)(25 squares). The chamber is 0.02 mm deep and therefore, bacteria/mm3 = (bacteria/square)(25 squares)(50). The number of bacteria per cm3 is 103 times this value. For example, suppose the average count per square is 28 bacteria: bacteria/cm3 = (28 bacteria) (25 squares)(50)(103 ) = 3.5 × 107 . scope is employed. These specially designed slides have chambers of known depth with an etched grid on the chamber bottom (figure 6.5). The number of microorganisms in a sample can be calculated by taking into account the chamber’s volume and any
w2 6.Microbial Grewth Chapter 6 Microbial Growtl ints.Low counts will result if clumps of cells are not bro ken upand cult to distinguish hetween living and dead cells in counting dividual cell.the results are often expressed in terms of colom chambers without special techniques unted with electronic coun sults.Of course the counts ill also be low if the agar medium em al Co he microbial su le m organisms pr h the cell is counted.The and other problems separate colony.A colony count gives the number of micro and gaims in the pcinTcdacanbcuscdt In mos counting proced res.ad ute used to count bac surface.Each micro ate memb acridine orange or DAPI and observed microscopically.Acridin in a sample. ely used for v o stain live and dead cells differe ntly are now microorganisms in a sample ()
Prescott−Harley−Klein: Microbiology, Fifth Edition II. Microbial Nutrition, Growth, and Control 6. Microbial Growth © The McGraw−Hill Companies, 2002 sample dilutions required. There are some disadvantages to the technique. The microbial population must be fairly large for accuracy because such a small volume is sampled. It is also difficult to distinguish between living and dead cells in counting chambers without special techniques. Larger microorganisms such as protozoa, algae, and nonfilamentous yeasts can be directly counted with electronic counters such as the Coulter Counter. The microbial suspension is forced through a small hole or orifice. An electrical current flows through the hole, and electrodes placed on both sides of the orifice measure its electrical resistance. Every time a microbial cell passes through the orifice, electrical resistance increases (or the conductivity drops) and the cell is counted. The Coulter Counter gives accurate results with larger cells and is extensively used in hospital laboratories to count red and white blood cells. It is not as useful in counting bacteria because of interference by small debris particles, the formation of filaments, and other problems. Counting chambers and electronic counters yield counts of all cells, whether alive or dead. There are also several viable counting techniques, procedures specific for cells able to grow and reproduce. In most viable counting procedures, a diluted sample of bacteria or other microorganisms is dispersed over a solid agar surface. Each microorganism or group of microorganisms develops into a distinct colony. The original number of viable microorganisms in the sample can be calculated from the number of colonies formed and the sample dilution. For example, if 1.0 ml of a 1 106 dilution yielded 150 colonies, the original sample contained around 1.5 108 cells per ml. Usually the count is made more accurate by use of a special colony counter. In this way the spread-plate and pour-plate techniques may be used to find the number of microorganisms in a sample. Plating techniques are simple, sensitive, and widely used for viable counts of bacteria and other microorganisms in samples of food, water, and soil. Several problems, however, can lead to inaccurate counts. Low counts will result if clumps of cells are not broken up and the microorganisms well dispersed. Because it is not possible to be absolutely certain that each colony arose from an individual cell, the results are often expressed in terms of colony forming units (CFU) rather than the number of microorganisms. The samples should yield between 30 and 300 colonies for best results. Of course the counts will also be low if the agar medium employed cannot support growth of all the viable microorganisms present. The hot agar used in the pour-plate technique may injure or kill sensitive cells; thus spread plates sometimes give higher counts than pour plates. Spread-plate and pour-plate techniques (pp. 106–8) Microbial numbers are frequently determined from counts of colonies growing on special membrane filters having pores small enough to trap bacteria. In the membrane filter technique, a sample is drawn through a special membrane filter (figure 6.6). The filter is then placed on an agar medium or on a pad soaked with liquid media and incubated until each cell forms a separate colony. A colony count gives the number of microorganisms in the filtered sample, and special media can be used to select for specific microorganisms (figure 6.7). This technique is especially useful in analyzing aquatic samples. Analysis of water purity (pp. 653–57) Membrane filters also are used to count bacteria directly. The sample is first filtered through a black polycarbonate membrane filter to provide a good background for observing fluorescent objects. The bacteria then are stained with a fluorescent dye such as acridine orange or DAPI and observed microscopically. Acridine orange–stained microorganisms glow orange or green and are easily counted with an epifluorescence microscope (see section 2.2). Usually the counts obtained with this approach are much higher than those from culture techniques because some of the bacteria are dead. Commercial kits that use fluorescent reagents to stain live and dead cells differently are now available. This makes it possible to directly count the number of live and dead microorganisms in a sample (see figure 2.13d). 118 Chapter 6 Microbial Growth Membrane filter removed and placed in plate containing the appropriate medium Water sample filtered through membrane filter (0.45 µm) Membrane filter on a filter support Incubation for 24 hours Typical colonies Figure 6.6 The Membrane Filtration Procedure. Membranes with different pore sizes are used to trap different microorganisms. Incubation times for membranes also vary with the medium and microorganism
6.Microbial Growh 119 Figure 6.7 Colonie variety of media Measurement of Cell Mass in a population are of roughly constant size.the amount of scat teria r about cells(1)per ml,the mediumap slightly clou centr d,dried in an oven,and weighed.This is an especially use so little,it may be necessary several hundred milli- growth can be e the puthe am crobial cells scatter light striking them.Because microbial cells tal quantity of that cell constituent is directly related to the total
Prescott−Harley−Klein: Microbiology, Fifth Edition II. Microbial Nutrition, Growth, and Control 6. Microbial Growth © The McGraw−Hill Companies, 2002 Measurement of Cell Mass Increases in the total cell mass, as well as in cell numbers, accompany population growth. Therefore techniques for measuring changes in cell mass can be used in following growth. The most direct approach is the determination of microbial dry weight. Cells growing in liquid medium are collected by centrifugation, washed, dried in an oven, and weighed. This is an especially useful technique for measuring the growth of fungi. It is time consuming, however, and not very sensitive. Because bacteria weigh so little, it may be necessary to centrifuge several hundred milliliters of culture to collect a sufficient quantity. More rapid, sensitive techniques depend on the fact that microbial cells scatter light striking them. Because microbial cells in a population are of roughly constant size, the amount of scattering is directly proportional to the biomass of cells present and indirectly related to cell number. When the concentration of bacteria reaches about 10 million cells (107 ) per ml, the medium appears slightly cloudy or turbid. Further increases in concentration result in greater turbidity and less light is transmitted through the medium. The extent of light scattering can be measured by a spectrophotometer and is almost linearly related to bacterial concentration at low absorbance levels (figure 6.8). Thus population growth can be easily measured spectrophotometrically as long as the population is high enough to give detectable turbidity. If the amount of a substance in each cell is constant, the total quantity of that cell constituent is directly related to the total 6.2 Measurement of Microbial Growth 119 Figure 6.7 Colonies on Membrane Filters. Membrane-filtered samples grown on a variety of media. (a) Standard nutrient media for a total bacterial count. An indicator colors colonies red for easy counting. (b) Fecal coliform medium for detecting fecal coliforms that form blue colonies. (c) m-Endo agar for detecting E. coli and other coliforms that produce colonies with a green sheen. (d) Wort agar for the culture of yeasts and molds. (a) (b) (c) (d) Spectrophotometer meter Photocell or detector Tube of bacterial suspension Lamp 10 9 8 7 4 5 6 3 2 1 0 0 .0 .1 .6 .5 .4 .3 .2 .8 .7 1.9 1. 10 9 8 7 4 5 6 3 2 1 0 0 .0 .1 .6 .5 .4 .3 .2 .8 .7 1.9 1. Figure 6.8 Turbidity and Microbial Mass Measurement. Determination of microbial mass by measurement of light absorption. As the population and turbidity increase, more light is scattered and the absorbance reading given by the spectrophotometer increases. The spectrophotometer meter has two scales. The bottom scale displays absorbance and the top scale, percent transmittance. Absorbance increases as percent transmittance decreases