This document discusses the nutritional classifications and requirements of microorganisms. It describes how bacteria can be classified based on their carbon source and energy requirements as either autotrophs or heterotrophs. Autotrophs use inorganic carbon sources while heterotrophs require organic carbon. Heterotrophs are further divided into photoheterotrophs and chemoheterotrophs based on their energy source. The document also discusses the use of defined and complex media to culture bacteria based on their nutritional needs and fastidiousness. Selective, differential, and selective-differential media are described which allow isolation and identification of bacteria based on their growth characteristics.

1. MEDIA CLASSIFICATION AND BACTERIAL NUTRITIONAL
REQUIREMENTS
Microorganisms are extraordinarily diverse in their requirements for growth. As
you have learned in the lectures, microorganisms are greatly affected by environmental
conditions and will grow in accordance to how these environmental niches support their
individual needs. Factors that affect microbial growth include but are not limited to, pH,
osmolarity, water activity, temperature and oxygen levels. There is a great deal of
nutritional diversity among microorganisms; therefore, microbial growth is greatly
affected by the nutrients that are available in their environment.
There are several nutritional classifications for bacteria. Autotrophs are
organisms that are able to use inorganic carbon dioxide as their sole carbon source for the
biosynthesis of macromolecules. Heterotrophs, require organic carbon for biosynthesis.
Autotrophs can be further broken down into two categories: the chemoautotrophs
derive energy from the oxidation of inorganic compounds such as iron, hydrogen sulfide
and hydrogen gas. Photoautotrophs, such as the cyanobacteria, convert light energy
into chemical energy. Heterotrophic organisms can also be divided into two major
subgroups. Photoheterotrophs, use organic carbon sources for biosynthesis but use light
energy to produce ATP (photosynthesis). Chemoheterotrophs, use organic compounds
such as sugars, proteins and lipids as their source of energy. As chemoheterotrophs are
more abundant and easier to work with, one usually works with these organisms in a
typical teaching laboratory setting. Such bacteria include but are not limited to Bacillus
subtilis, Escherichia coli and Staphylococcus aureus. Organisms of this classification
that one might encounter in a hospital setting include, the Clostridium spp. that cause
tetanus, botulism and gas gangrene, Vibrio cholerae and Streptococcus pyogenes (the
causative agent of strept throat, impetigo, scarlet fever and cellulitis, i.e. the “flesh eating
bacteria”).
The specific nutritional requirements of heterotrophic bacteria can also be quite
diverse. All microorganisms are made up of four biochemical molecules: proteins, lipids,
carbohydrates and nucleic acids; however, these organisms may differ in their individual
ability to synthesize these molecules. Some heterotrophs are metabolically flexible and
require only a few organic compounds for energy production and biosynthesis of cellular
components. Other heterotrophs require greater numbers of different organic compounds
from their environment. Organisms that fall into the latter class are called fastidious
organisms. The successful cultivation of microbes in a laboratory setting requires that
one understands the nutritional needs of an individual organism, as the absence of a
single required nutrient would prevent growth. Fastidious organisms tend to require
more ingredients in their growth media than less fastidious organisms.
The media that one uses in a laboratory setting may be classified as defined or
complex (undefined). Complex media are composed of extracts from plants, animals or
yeast and therefore are rich in nutrients. Such media is complex because the precise
individual components of these media are unknown; however, as these media contain a
wide range of nutrients that are well above the minimal nutritional requirements of the
organism being cultured, these media support the growth of a wide range of organisms.
A defined media is a media in which all of the constituents and the amounts of these
constituents are known. Defined media typically supports a narrower range of
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2. heterotrophic microorganisms. Defined media typically consist of salts and a carbon
source in the form of glucose. Depending on the fastidiousness of an organism, these
media can be supplemented with vitamins, nucleic acids, cofactors and amino acids.
Using Selective, Differential and Selective-Differential
Media in the isolation and identification of individual
bacteria
Bacteria must be isolated from their natural environments before they can be
characterized. As bacteria exist in mixed populations in the soil, water, food and within
or on the human body, specialized media are often used to isolate individual bacteria
from these populations and to characterize the isolated bacteria.
Selective media are used to select for the growth of some bacteria while
inhibiting the growth of others. Such media take advantage of the differences in the
nutritional needs of individual bacteria or exploit the ability of some organisms to grow
in the presence of a noxious compound. For example, Pseudomonas aeruginosa is able
to grow in the presence of a variety of antibiotics (compounds that inhibit bacterial
growth or kill bacteria) while organisms like Staphylococcus aureus or Escherichia coli
are sensitive to these antibiotics.
Differential media allow a variety of organism to grow but contain substances
that allow the student to distinguish between the different types of bacteria growing on
the media.
Selective-Differential Media have characteristics of both selective media
and differential media. These media only allow a subset of bacteria to grow and allow
the student to distinguish between the different types of bacteria that are able to grow on
these media. For example, one can distinguish between bacteria that ferment lactose and
those that do not in MacConkey Agar by adding a carbon source (lactose) and a pH
indicator (methyl red)—bacteria that ferment lactose produce acids that imparts a color
change to the media surrounding the individual bacterial colony.
In the following lab exercise, each student will examine the growth characteristics
of different bacterial species on Starch Agar, MacConkey Agar, Mannitol Salt Agar
and Hektoen Enteric Agar in terms of whether the media support the growth of these
bacteria and in terms of the differences in the metabolic requirements of these bacteria.
Starch Agar: This is a differential medium used to determine whether a
given bacterium is able to use starch as a carbon source and an energy source. Starch is a
polymer (polysaccharide) of repeating glucose monomers and is too large to be
transported into the cell. Organisms that use starch produce an exoenzyme called alpha-
amylase that breaks the bonds between the glucose monomers such that the glucose
monomers can be taken up into the cell and catabolized. Alpha-amylase is secreted
outside of the cell but is still active at this location. To determine if the organism
produces this exoenzyme, one adds Gram’s Iodine to the plate that turns the intact starch
in the medium purple. If the organism utilizes starch, the area around the colony will be
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3. clear because, the starch has been broken down into glucose monomers (starch positive).
If the organism does not utilize starch then the media surrounding the organism will be
dark purple (starch negative.
MacConkey Lactose Agar: This is a selective-differential media that
selects for the growth of enteric bacteria. Enteric bacteria are gram-negative rods that
are facultatively anaerobic. Enterics are most commonly found in the gastro-intestinal
tract of humans and include E. coli, Serratia spp. and Salmonella spp. Enterics grow on
MacConkey media because the bile salts that are present in the media inhibit the
growth of non-enteric organisms while the crystal violet inhibits the growth of Gram
positive organisms that might otherwise grow on this media. MacConkey media also
differentiates between the noncoliforms and the coliforms that also grow on this
medium. Enteric bacteria can be coliforms or noncoliforms. Enteric bacteria that
ferment lactose to form acid and gas are coliforms, enteric bacteria that do not ferment
lactose are noncoliforms. Coliforms will produce bright pink colonies on MacConkey
lactose agar because a pH indicator (methyl red) is present in the media. The non-
coliforms will grow on the media but the colonies will appear either light yellow or
colorless.
Hektoen Enteric Agar: This is also a selective-differential media that
contains bile salts to select for enteric organisms but distinguishes between these enterics
by virtue of their abilities to ferment lactose, salicin or sucrose and to reduce sulfur to
hydrogen sulfide gas. In addition to these fermentable sugars, H-E Agar contains ferric
ammonium citrate that reacts with the hydrogen sulfide to form a black precipitate and
the dyes acid fuchsin and bromothymol blue that are color indicators. This media is often
used to isolate Salmonella and Shigella spp from other enterics. The enterics that ferment
the sugars produce acids and form yellow to pink colonies on this medium. Shigella and
Salmonella spp.do not ferment these sugars and produce blue colonies. Salmonella spp.
(but not Shigella spp.) reduce sulfur to hydrogen sulfide forming colonies containing a
black precipitate.
Exercise 1: Each student will be given an organism to analyze on the aforementioned
media: The organisms in question are Staphylococcus aureus, Bacillus subtilis, Bacillus
cereus, Enterobacter aerogenes, Serratia marcescens, Shigella flexneri, Pseudomonas
aeruginosa, Klebsiella pneumoniae and Citrobacter freundii.
To analyze the organism in question streak the organism on the plates as described by the
instructor. At the next lab, record whether the organism grew and the growth behavior
of the organism. Record the results of your classmates!
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4. Mannitol Salt Agar: Is another example of a selective differential media.
The media contains a high percentage of NaCl (7.5% w/v) to select for organisms that
prefer or can tolerate high salt conditions. Furthermore this media contains the sugar
mannitol and the pH indicator phenol red to identify the organisms that can ferment this
sugar. If the organism is able to ferment mannitol to produce acid then the pH of the
media will drop around the colonies and cause the phenol red to turn yellow. This media
is often used to isolate Staphylococcus aureus since this organism is halotolerant and able
to use mannitol as a carbon source.
Exercise 2: In this exercise each student will examine the growth characteristics of
Staphylococcus epidermidis (StE), Staphylococcus aureus (StA), Bacillus subtilis (BS)
and an isolate from the body. To this end each student will need test tubes containing one
of the three organisms and a cotton swab to isolate bacteria from their nose or other body
parts.
Divide the plate into four sectors, as shown by your instructor. Label each sector with the
organism you will inoculate into that sector. Using a loop sample each of the bacteria
individually and inoculate the bacteria into the corresponding sector. Finally, using a
sterile cotton swab, sample bacteria from your external nares and inoculate the isolate
into the fourth sector.
StE
StA
BS
Nose
sampl
e
Fill
entire
grid
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5. Growth Characteristics of Bacteria on various
media
Starch Agar:
Staphylococcus aureus,
Bacillus subtilis,
Bacillus cereus
Enterobacter aerogenes,
Proteus mirabilis,
Serratia marcescens,
Shigella flexneri,
Pseudomonas aeruginosa,
Klebsiella pneumoniae
Citrobacter freundii
MacConkey Lactose:
Staphylococcus aureus,
Bacillus subtilis,
Enterobacter aerogenes,
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6. Proteus mirabilis,
Serratia marcescens,
Shigella flexneri,
Pseudomonas aeruginosa,
Klebsiella pneumoniae
Citrobacter freundii
Hektoen Enteric Agar:
Staphylococcus aureus,
Bacillus subtilis,
Enterobacter aerogenes,
Proteus mirabilis,
Serratia marcescens,
Shigella flexneri,
Pseudomonas aeruginosa,
Klebsiella pneumoniae
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7. Citrobacter freundii
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