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5-5 Growth of microbes in batch culture

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The Phases of Growth

Growth in the bacterial context is normally described as an increase in cell number. Microorganisms, depending upon the specific species, increase their numbers by binary fission, budding or by filamentous growth. Binary fission is the separation of the initial cell, the mother cell, into two daughter cells of approximately equal size. This is a very common method of multiplication and most of the organisms we will investigate divide in this manner.

Figure 5-8 The various forms of division

Cells can increase in number by binary fission, budding or filamentous growth. This animation demonstrates each of these processes.

Budding division involves the asymmetric creation of a growing bud, on the mother cell. The bud increases in size and eventually is severed from the parental cell. After division is complete, the mother cell reinitiates the process by growing another bud. Yeast and some bacteria (Caulobacter is one example) use this form of division.

Figure 5-9 Budding cells

A picture of Histoplasma capsulatum var. duboisii cells, a yeast that causes histoplasmosis, in the process of budding. Picture courtesy of Dr. Libero Ajello and the Centers for Disease Control and Prevention.

Filamentous growth is characterized by the formation of long, branching, non-divided filaments, containing multiple chromosomes. Sometimes the filaments will have cross walls separating chromosomes. As growth proceeds, the filaments increase in length and number. Under nutrient limiting conditions, some filamentous microorganisms will go through developmental changes, with a fraction of the filaments differentiating to form spores. The structures and mechanisms used to form these spores can be spectacular. Streptomyces species and many molds grow in this manner.

Figure 5-10 Filamentous growth

The bacteria Nocardia asteroides. Note the long branching cells and the circular spores in the picture.

The best characterized type of growth is binary fission and this is what we will focus on in this experiment. When grown in liquid medium, bacterial cultures progress through several distinguishable phases, which can be characterized by plotting the log of the cell number vs. time. Figure 5-12 shows an example of a typical growth curve with the 4 phases of growth, lag phase, exponential growth phase (also termed balanced growth), stationary phase and death phase.

Figure 5-11 Binary fission

As a cell divides by binary fission, a cross-wall is formed to separate the daughter cells. In this EM of Staphylococcus aureus, the cross-wall is already formed to divide the cells.

Figure 5-12 A bacterial growth curve

A bacterial growth curve generated from actual data obtained in the teaching laboratories at the University of Wisconsin-Madison. The four phases of growth (lag (1), exponential (2), stationary (3) and death phase(4)) are labeled.

When an organism is inoculated into a fresh medium, it needs to adapt to the new nutrients available, synthesize RNA, protein and finally replicate its DNA before starting division. These processes take time and there is no net increase in cell numbers, thus a lag phase is observed.

In the subsequent discussion, the numbers refer to the figure above. Once the appropriate enzymes for growth in a particular medium have been expressed cells begin to multiply. This period of maximal division can last for several hours or days, depending upon the organism, and is called the log or exponential growth phase (2).

Eventually the increase in cell number ceases, either because cells stop dividing or the rate of division equals the rate of cell death, resulting in a stationary phase (3). This is usually caused by limitation of a nutrient or the accumulation of a toxic waste product. Depending on the bacterium, stationary phase can last for several hours to many days.

The final chapter of a growth curve is the death phase (4). An exponential decrease in the number of organisms due to cell death occurs during this phase. Some microorganisms never experience a death phase or it is greatly delayed due to their ability to survive for long periods without nutrients.

Measurement of Bacterial Growth

Growth of microorganisms can be measured by following the increase in cell number or the increase in a cellular macromolecule such as DNA or protein. In most cases, the increase in cell number is determined. Cell numbers can be measured in a variety of ways. For this experiment we will use the viable plate count, which you are familiar with, and turbidometric measurement, which is was explained in the chapter on quantification of microorganisms.

So which wavelength of light do we use to measure cell numbers? Typically the shorter the wavelength the greater the sensitivity of the measurement. Some wavelengths cannot be used, however, because cellular constituents absorb light, not scatter them. For example proteins absorb light at 280 nm. Likewise the color of the medium affects absorbance. For this experiment we will be measuring the growth of E. coli in either a yellow or a clear medium, 600 nm works well. Beware, however, that the choice of wavelength must be considered for each experimental condition.

The sample is placed in a sample chamber. The chamber will contain a holder, an entrance for the selected wavelength, and an exit, leading to the detector. A critical piece of equipment used in the sample chamber is the cuvette. A cuvette is a special test tube that holds the sample. Cuvettes must be clean and free of aberrations, both of which could scatter light, resulting in inaccurate readings. Most good cuvettes are expensive and must be treated with care!

The actual operation of a spectrophotometer is much simpler than understanding its parts. There are a few general, rules of thumb, when using any spectrophotometer.

  1. Before you begin, be sure the wavelength selector is set for the wavelength of light you are going to use. This involves setting the correct wavelength on the selector, making sure the correct lamp is on for the wavelength you have selected and that you are using the correct cuvette.
  2. Make sure to zero the spectrophotometer. When this is performed you are adjusting the machine to 100% transmittance. This is done by using a sample that contains all components of a mixture except the component to be measured. For example, if you are measuring the growth of B. cereus in nutrient broth, the machine would be blanked with sterile, uninoculated nutrient broth. Realize that the machine is being blanked to read 100% T (or 0 absorbance) with a specific solution in a specific cuvette, with the cuvette in a specific orientation. This last consideration, nullifies any aberrations in the cuvette.
  3. With proper technique, nothing should be spilled into the spectrophotometer. If it is, clean it up and immediately notify an instructor. This type of spillage usually happens if cuvettes are filled over a spectrophotometer, a very bad habit to form!
  4. Clean the cuvette after each use by rinsing with distilled water and allowing to dry upside down in a test tube rack. For more stubborn stains use a dilute solution containing a mild soap (like Ivory). In rare cases, difficult blemishes can be removed with dilute acid (1-5%) or with ethanol. Never let a sample dry in a cuvette! Protein and nucleic acids form a strong bond with glass and can be impossible to remove.
  5. When measuring the turbidity of a cell suspension, absorbance values in the range of 0.1--0.8 are acceptable. Readings below 0.1 push the limits of the spectrophotometer, since it is not sensitive enough in that range. Absorbance values above 0.8 result in microorganisms casting shadows onto one another and not being seen by the spectrophotometer. If a reading is above this range, the culture must be diluted with sterile medium. If a reading is below this range, concentrate your culture using a centrifuge.
  6. When graphing growth versus time, you must plot absorbance on a log scale. Even though absorbance is a logarithmic unit, absorbance readings are proportional to cell mass which is increasing exponentially during growth.
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