Microbiology

Chapter 8

INTRODUCTION
 In certain locations, such as within microbiology laboratories, the growth of microbes is encouraged; in other
words, scientists want them to grow.
 In other locations such as on hospital wards, in intensive care units, in operating rooms, in kitchens, bathrooms,
and restaurants-it is necessary or desirable to inhibit the growth of microbes.
 Both concepts, encouraging and inhibiting the in vitro growth of microbes, are discussed in this chapter. (Recall
from Chapter 1 that, as used in this book, in vitro refers to events that occur outside the body, whereas in vivo
refers to events that occur inside the body.)
 Before discussing these concepts, however, various factors that affect the growth of microbes are examined.

PART 1: FACTORS THAT AFFECT MICROBIAL GROWTH

 Microbial growth is affected by many different environ mental factors, including the availability of nutrients and
moisture, temperature, pH, osmotic pressure, barometric pressure, and composition of the atmosphere.
 These environmental factors affect microorganisms in our daily lives and play important roles in the control of
microorganisms in laboratory, industrial, and hospital settings.
 Whether scientists wish to encourage or inhibit the growth of microorganisms, they must first understand the
fundamental needs of microbes.

Availability of Nutrients
 As discussed in Chapter 7, all living organisms require nutrients the various chemical compounds that organisms
use to sustain life.
 Therefore, to survive in a particular environment, appropriate nutrients must be available. Many nutrients are
energy sources; organisms will obtain energy from these chemicals by breaking chemical bonds. Nutrients also
serve as sources of carbon, oxygen, hydrogen, nitrogen, phosphorus, and sulfur as well as other elements (e.g.,
sodium, potassium, chlorine, magnesium, calcium, and trace elements such as iron, iodine, and zinc) that are
usually required in lesser amounts. About two dozen of the approximately 92 naturally occurring elements are
essential to life.
Moisture
 On Earth, water is essential for life, as we know it. Cells consist of anywhere between 70% and 95% water. All
living organisms require water to carry out their normal metabolic processes, and most will die in environments
containing too little moisture.
 There are certain microbial stages (e.g., bacterial endospores and protozoan cysts), however, that can survive
the complete drying process (desiccation). The organisms contained within the spores and cysts are in a
dormant or resting state; if they are placed in a moist, nutrient-rich environment, they will grow and reproduce
normally.

Temperature
 Every microorganism has an optimum growth temperature. The temperature at which the organism grows best.
 Every microorganism also has a minimum growth temperature, below which it ceases to grow, and a maximum
growth temperature, above which it dies.
 The temperature range (i.e., the range of temperatures from the minimum growth temperature to the
maximum growth temperature) at which an organism grows can differ greatly from one microbe to another.
 To a large extent, the temperature and pH ranges over which an organism grows best are determined by the
enzymes present within the organism.
 As discussed in Chapter 7, enzymes have optimum temperature and pH ranges at which they operate at peak
efficiency.
 If an organism's enzymes are operating at peak efficiency, the organism will be metabolizing and growing at its
maximum rate. Table 8-1 contains information regarding temperatures at which various organisms live in bodies
of water at Yellowstone National Park.
 Microorganisms that grow best at high temperatures are called thermophiles (i.e., organisms that love heat).
  Thermophiles can be found in hot springs, compost pits, and silage as well as in and near hydrothermal vents at
the bottom of the ocean (check the Point for "A Closer Look at Hydrothermal Vents").
 Thermophilic cyanobacteria, certain other types of bacteria, and algae cause many of the colors observed in the
near-boiling hot springs found in Yellowstone National Park (Fig. 8-1).
 Organisms that favor temperatures above 100°C are referred to as hyperthermophiles (or extreme
thermophiles). The highest temperature at which a bacterium has been found living is around 113°C; it was an
archaeon named Pyrolobus fumarii.
 Microbes that grow best at moderate temperatures are called mesophiles. This group includes most of the
species that grow on plants and animals and in warm soil and water. Most pathogens and members of the
indigenous microbiota are mesophilic because they grow best at normal body temperature (37°C).
 Psychrophiles prefer cold temperatures. They thrive in cold ocean water. At high altitudes, algae (often pink)
can be seen living on snow. Biologists studying microbial life in the Antarctic have reported finding bacteria in a
lake that has been iced over for at least 2,000 years. These microbes thrive in an environment that is -13°C, has
20% salinity, and contains high concentrations of ammonia and sulfur.
 Ironically, the optimum growth temperature of one group of psychrophiles (called psychrotrophs) is
refrigerator temperature (4°C); perhaps you encountered some of these microbes (e.g., bread moulds) the last
time you cleaned out your refrigerator. Microorganisms that prefer warmer temperatures, but can tolerate or
endure very cold temperatures and can be preserved in the frozen state, are known as psychroduric organisms.
Refer to Table 8-2 for the temperature ranges of psychrophilic, mesophilic, and thermophilic bacteria.

pH
 The term "pH" refers to the hydrogen ion concentration of a solution and thus the acidity or alkalinity of the
solution (see Appendix 3 on the Point: "Basic Chemistry Concepts"). Most microorganisms prefer a neutral or
slightly alkaline growth medium (pH 7.0-7.4), but acidophilic microbes (acidophiles), such as those that can live
in the human stomach and in pickled foods, prefer a pH of 2 to 5.
 Fungi prefer acidic environments. Acidophiles thrive in highly acidic environments, such as those created by the
production of sulfurous gases in hydrothermal vents and hot springs as well as in the debris produced from coal
mining. Alkaliphiles prefer an alkaline environment (pH > 8.5), such as that found inside the intestine (pH ~9),
in soils laden with carbonate, and in the so-called soda lakes.
 Vibrio cholerae-the bacterium that causes cholera-is the only human pathogen that grows well above pH 8.

Osmotic Pressure and Salinity

 Osmotic pressure is the pressure exerted on a cell mem brane by solutions both inside and outside the cell.
 When cells are suspended in a solution, the ideal situation is that the pressure inside the cell is equal to the
pressure of the solution outside the cell.
 Substances dissolved in liquids are referred to as solutes. When the concentration of solutes in the environment
outside a cell is greater than the concentration of solutes inside the cell, the solution in which the cell is
suspended is said to be hypertonic.
 In such a situation, whenever possible, water leaves the cell by osmosis in an attempt to equalize the two
concentrations.
 Osmosis is defined as the movement of a solvent (e.g., water), through a permeable membrane, from a solution
having a lower concentration of solute to a solution having a higher concentration of solute.
 If the cell is a human cell, such as a red blood cell (erythrocyte), the loss of water causes the cell to shrink; this
shrinkage is called crenation, and the cell is said to be crenated (Fig. 8-2).
 If the cell is a bacterial cell, having a rigid cell wall, the cell does not shrink.
 Instead, the cell membrane and cytoplasm shrink away from the cell wall. This condition, known as plasmolysis,
inhibits bacterial cell growth and multiplication.
 Salts and sugars are added to certain foods as a way of preserving them.
 Bacteria that enter such hypertonic environments will die as a result of loss of water and desiccation.
 When the concentration of solutes outside a cell is less than the concentration of solutes inside the cell, the
solution in which the cell is suspended is said to be hypotonic.
  In such a situation, whenever possible, water enters the cell in an attempt to equalize the two concentrations. If
the cell is a human cell, such as an erythrocyte, the increased water within the cell causes the cell to swell.
 If sufficient water enters, the cell will burst (lyse). In the case of erythrocytes, this bursting is called hemolysis. If
a bacterial cell is placed in a hypotonic solution (such as distilled water), the cell may not burst (because of the
rigid cell wall), but the fluid pressure within the cell increases greatly.
 This increased pressure occurs in cells having rigid cell walls, such as plant cells and bacteria.
 If the pressure becomes so great that the cell ruptures, the escape of cytoplasm from the cell is referred to as
plasmoptysis.
 When the concentration of solutes outside a cell equals the concentration of solutes inside the cell, the solution
is said to be isotonic.
o In an isotonic environment, excess water neither leaves nor enters the cell, and thus, no plasmolysis or
plasmoptysis occurs; the cell has normal turgor (fullness). Refer to Figure 8-3 for a comparison of the
effects of various solution concentrations on bacterial cells and red blood cells.
 Sugar solutions for jellies and pickling brines (salt solutions) for meats preserve these foods by inhibiting the
growth of most microorganisms.
o However, some types of moulds and bacteria can survive and even grow in a salty environment.
 Those microbes that actually prefer salty environments (such as the concentrated salt water found in the Great
Salt Lake and solar salt evaporation ponds) are called halophilic organisms;
o halo referring to "salt" and philic meaning "to love."
o Microbes that live in the ocean, such as V. cholerae (mentioned earlier) and other Vibrio species, are
halophilic.
 Organisms that do not prefer to live in salty environments but are capable of surviving there (such as
Staphylococcus aureus) are referred to as haloduric organisms.

Barometric Pressure
 Most bacteria are not affected by minor changes in barometric pressure.
 Some thrive at normal atmospheric pressure (about 14.7 psi).
 Others, known as piezophiles, thrive deep in the ocean and in oil wells, where the atmospheric pressure is very
high. Some archaea, for example, are piezophiles, capable of living in the deepest parts of the ocean (check
thePoint for "A Closer Look at Barometric Pressure").

Gaseous Atmosphere
 As discussed in Chapter 4, microorganisms vary with respect to the type of gaseous atmosphere that they
require. For example, some microbes (obligate aerobes) prefer the same atmosphere that humans do (i.e.,
about 20%-21% oxygen and 78% -79% nitrogen, with all other atmospheric gases combined representing <1%).
 Although microaerophiles also require oxygen, they require reduced concentrations of oxygen (around 5%).
 Obligate anaerobes are killed by the presence of oxygen. Thus, in nature, the types and concentrations of gases
present in a particular environment determine which species of microbes are able to live there.
 To grow a particular microorganism in the laboratory, it is necessary to provide the atmosphere that it requires.
For example, to obtain maximum growth in the laboratory, capnophiles require increased concentrations of
carbon dioxide (usually from 5% to 10%).

PART 2: ENCOURAGING THE GROWTH OF MICROBES IN VITRO

 There are many reasons why the growth of microbes is encouraged in microbiology laboratories. For example,
technologists and technicians who work in clinical microbiology laboratories must be able to isolate
microorganisms from clinical specimens and grow them on culture media so that they can then gather
information that will enable identification of any pathogens that are present. In mi crobiology research
laboratories, scientists must culture microbes so that they can learn more about them, harvest antibiotics and
other microbial products, test new antimi crobial agents, and produce vaccines. Microbes must also be cultured
in genetic engineering laboratories and in the laboratories of certain food and beverage companies, as well as
other industries.
  Many different types of microbes can be cultured (grown) in vitro, including viruses, bacteria, fungi, and
protozoa. In this chapter, emphasis is placed on culturing bacteria. Culturing other types of microbes is
mentioned only briefly.

Culturing Bacteria in the Laboratory

 In many ways, modern microbiology laboratories resemble those of 50, 100, or even 150 years ago.
 Today's laboratories still use many of the same basic tools that were used in the past.
 For example, microbiologists still use compound light microscopes, Petri dishes containing solid culture media,
tubes containing liquid culture media, wire or plastic inoculating loops, bottles of staining reagents, and
incubators.
 However, a closer inspection will reveal many modern, commercially available products and instruments that
would have been inconceivable in the days of Louis Pasteur and Robert Koch.

Bacterial Growth
 With respect to humans, the term growth refers to an increase in size, for example, going from a tiny newborn
baby to a large adult. Although bacteria do increase in size before cell division, bacterial growth refers to an
increase in the number of organisms rather than an increase in their size.
 Thus, with respect to bacteria, growth refers to their proliferation or multiplication. When each bacterial cell
reaches its optimum size, it divides by binary fission (bi meaning "two") into two daughter cells (i.e., each
bacterium simply splits in half to become two identical cells).
 (Recall from Chapter 3 that DNA replication must occur before binary fission occurs, so that each daughter cell
has exactly the same genetic makeup as the parent cell.)
 On solid medium, binary fission continues through many generations until a colony is produced.
 A bacterial colony is a mound or pile of bacteria containing millions of cells (Fig. 8-4).
 Binary fission continues for as long as the nutrient supply, water, and space allow and ends when the nutrients
are depleted or the concentration of cellular waste products reaches a toxic level. The division of staphylococci
by binary fission was shown in Figure 2-12.
 The time taken for one cell to become two cells by binary fission is called the generation time.
 The generation time varies from one bacterial species to another. In the laboratory, under ideal growth
conditions, Escherichia coli, V. cholerae, Staph ylococcus spp., and Streptococcus spp. all have a generation time
of about 20 minutes, whereas some Pseudomonas and Clostridium spp. may divide every 10 minutes, and
Mycobacterium tuberculosis may divide only every 18 to 24 hours.
 Bacteria with short generation times are referred to as rapid growers, whereas those with long generation
times as slow growers.
 The growth of microorganisms in the body, in nature, or in the laboratory is greatly influenced by temperature,
pH, moisture content, available nutrients, and the characteristics of other organisms present.
 Therefore, the number of bacteria in nature fluctuates unpredictably because these factors vary with the
seasons, rainfall, temperature, and time of day.
 In the laboratory, however, a pure culture of a single species of bacteria can usually be maintained if the
appropriate growth medium and environmental conditions are provided.
 The temperature, pH, and proper atmosphere are quite easily controlled to provide the optimum conditions for
growth.
 Appropriate nutrients must be provided in the growth medium, including an appropriate energy and carbon
source.
 Some bacteria, described as being fastidious, have complex nutritional requirements.
 Often, special mixtures of vitamins and amino acids must be added to the medium to culture these fastidious
organisms.
 Some organisms will not grow at all on artificial culture media, including obligate intracellular pathogens, such as
viruses, rickett sias, and chlamydias.
 To propagate obligate intracellular pathogens in the laboratory, they must be inoculated into live animals,
embryonated chicken eggs, or cell cultures.
  Other microorganisms that will not grow on artificial media include Treponema pallidum (the bacterium that
causes syphilis) and Mycobacterium leprae (the bacterium that causes leprosy).

Culture Media
 The media (sing., medium) that are used in microbiology laboratories to culture bacteria are referred to as
artificial media or synthetic media because they do not occur naturally; rather, they are prepared in the
laboratory.
 There are a number of ways of categorizing the media that are used to culture bacteria.
 One way to classify culture media is based on whether the exact contents of the media are known.
 A chemically defined medium is one in which all the ingredients are known; this is because the medium was
prepared in the laboratory by adding a certain number of grams of each of the components (e.g., carbohydrates,
amino acids, and salts).
 A complex medium is one in which the exact contents are not known.
 Complex media contain ground-up or digested extracts from animal organs (e.g., hearts, livers, and brains),
fish, yeasts, and plants, which provide the necessary nutrients, vitamins, and minerals.
 Culture media can also be categorized as liquid or solid (Fig. 8-5).
o Liquid media (also known as broths) are contained in tubes and are thus often referred to as tubed
media.
o Solid media are prepared by adding agar to liquid media and then pouring the media into tubes or Petri
dishes, where the media solidifies.
o Bacteria are then grown on the surface of the agar-containing solid media.
o Agar is a complex polysaccharide that is obtained from a red marine alga; it is used as a solidifying agent,
much like gelatin is used as a solidifying agent in the kitchen.
 An enriched medium is a broth or solid medium. containing a rich supply of special nutrients that promotes the
growth of fastidious organisms. It is usually prepared by adding extra nutrients to a medium called nutrient agar.
o Blood agar (nutrient agar plus 5% sheep red blood cells) and
o chocolate agar (nutrient agar plus powdered hemoglobin) are examples of solid enriched media that are
used routinely in the clinical bacteriology laboratory.
o Blood agar is bright red, whereas chocolate agar is brown (the color of chocolate). Although both of
these media contain hemoglobin,
o chocolate agar is considered to be more enriched than blood agar because the hemoglobin is more
readily accessible in chocolate Chocolate agar.
o Agar is used to culture important, fastidious, bacterial pathogens, such as Neisseria gonorrhoeae and
Haemophilus influenzae, which will not grow on blood agar.
o A selective medium has added inhibitors that discourage the growth of certain organisms without
inhibiting the growth of the organism being sought.
 For example, MacConkey agar inhibits the growth of Gram-positive bacteria and thus is
selective for Gram-negative bacteria.
 Phenylethyl alcohol (PEA) agar and colistin-nalidixic acid (CNA) agar inhibit the growth of
Gram-negative bacteria and are thus selective for Gram-positive bacteria.
 Thayer-Martin agar and Martin-Lewis agar (chocolate agars containing extra nutrients plus
several antimicrobial agents) are selective for N. gonorrhoeae.
 Only salt-tolerant (haloduric) bacteria can grow on mannitol salt agar (MSA).
 A differential medium permits the differentiation of organisms that grow on the medium.
o For example, MacConkey agar is frequently used to differentiate among various Gram-negative bacilli
that are isolated from fecal specimens.
 Gram-negative bacteria capable of fermenting lactose (an ingredient of MacConkey agar)
produce pink colonies, whereas those that are unable to ferment lactose produce colorless
colonies (Fig. 8-6).
 Thus, MacConkey agar differentiates between lactose-fermenting and nonlac tose-fermenting
Gram-negative bacteria.
 MSA is used to screen for S. aureus.
 o S. aureus not only will grow on MSA but also turns the originally pink medium to yellow because of its
ability to ferment mannitol (Fig. 8-7).
o In a sense, blood agar is also a differential medium because it is used to determine the type of
hemolysis (alteration or destruction of red blood cells) that the bacterial isolate produces (Fig. 8-8).
o Any bacteria capable of growing in a 7.5% sodium chloride concentration will grow on this medium, but
S. aureus will turn the medium yellow because of its ability to ferment the mannitol in the medium.
o The organism growing on the upper section of the plate is unable to ferment mannitol, but the organism
growing on the lower section is a mannitol fermenter. (From Koneman E, et al. Color Atlas and Textbook
of Diagnostic Microbiology. 5th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 1997.)
