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Microbial Growth Requirements

Microbial growth requires suitable environmental conditions, a source of energy, and nourishment. These requirements can be
divided into two categories, physical and chemical.
Chemical Factors
Table of the elements required for microbial growth as found in nature compared to the chemical forms supplied to
microbiological media.

Requirements for Form usually found in Chemical Form commonly


Growth Nature added to Microbiological Media

Carbon Carbon dioxide (CO2), HCO3- Organic; simple sugars e.g.


organic compounds glucose, acetate or pyruvate; extracts such as peptone,
tryptone, yeast extract etc.
Inorganic; carbon dioxide (CO2)
or hydrogen carbonate salts (HCO3-)*

Hydrogen Water (H2O)


organic compounds
Oxygen Water (H2O), oxygen gas (O2),
organic compounds
Nitrogen Ammonia (NH3), nitrate (NO3-) Organic; amino acids,
organic compounds nitrogenous bases
e.g. amino acids Inorganic; NH4CI, (NH4)2S04, KNO3, and for dinitrogen fixers
nitrogen gas (N2)
Phosphorus Phosphate (PO43-) KH2PO4, Na2HPO4*

Sulphur Hydrogen sulphide(H2S), Na2SO4, H2S


sulphate (SO42-),
organic compounds
e.g cysteine
Potassium K+ KCI, K2HPO4*

Magnesium Mg2+ MgCI2, MgSO4

Calcium Ca2+ CaCI2, Ca(HC03)2*

Sodium Na+ NaCI

Iron Fe3+ organic iron complexes FeCI3, Fe(NH4)(SO4)2, Fe-chelates1)

Trace elements Usually present at very low CoCI2, ZnCI2, Na2MoO4, CuCI2, MnSO4, NiCI2, Na2SeO4, Na2W
concentrations Na2VO4

Organic growth factors Usually present at very low Vitamins, amino acids, purines, pyrimidines
concentrations

*also act as buffers


1)To facilitate the solubilisation or retention of iron in solution, complexing agents such as EDTA or citrate may be added to the med

Physical / Environmental Factors


Temperature
Most microorganisms grow well at the normal temp.s favoured by man, higher plants and animals. However, certain bacteria grow at
temperatures (extreme heat or cold) at which few higher organisms can survive. Depending on their preferred temperature range, bac
divided into three groups: Psychrophiles (cold-loving microorganisms) found mostly in the depths of the oceans, in ice and snow and
arctic regions, have an optimum growth temperature between 0°C and 15°C and a maximum growth temperature of not more than 20
Mesophiles (moderate-temperature-loving bacteria) found in water, soil and in higher organisms, are the most common type of micro
Their optimum growth tempe. ranges between 25°C and 40°C. The optimum temperature for many pathogenic bacteria is 37°C, thus
mesophiles constitute most of our common spoilage and disease microbes. Thermophiles (heat-loving microbes) are capable of grow
temperatures with an optimum above 60°C. Some organisms grow at temperatures near the boiling point of water and even above 10
under pressure. Most thermophiles cannot grow below 45°C.

pH
Most bacteria grow best in an environment with a narrow pH range near neutrality between pH 6.5 and 7.5. Those that grow at extrem
are classed as acidophiles (acid-loving) or alkalinophiles (base-loving). Acidophiles grow at pH values below 4 with some bacteria st
at a pH of 1. Alkalinophilic bacteria prefer pH values of 9-10 and most cannot grow in solutions with a pH at or below neutral. Often
bacterial growth, organic acids are released into the medium, which lower its pH and so interfere with or totally inhibit further growth
Although common media ingredients such as peptones and amino acids have a small buffering effect, an external buffer is needed in
bacteriological media to neutralise the acids and maintain the correct pH. Phosphate salts are the most commonly used buffers becaus
buffer in the growth range of most bacteria, are non-toxic and provide a source of phosphorus, an essential nutrient element. High ph
concentration has the disadvantage, however, that it can result in a severe nutrient limitation caused by the precipitation of insoluble m
phosphates (such as iron) in the medium.

Osmotic Pressure
Microbes contain approximately 80-90% water and if placed in a solution with a higher solute concentration will lose water which ca
shrinkage of the cell (plasmolysis). However, some bacteria have adapted so well to high salt concentrations that they actually require
growth. These bacteria are called halophiles (salt-loving) and are found in salterns or in areas such as the Dead Sea.
Factor Class of Organism Minimum Optimum Maximum Example

Temperature (0C) extreme psychrophile -2 5 10 Raphidonema nivale


(snow algae)
psychrophile 0 15 20 Vibrio marinus
mesophile 10-15 24-40 35-45 Escherichia coli
facultative 37 45-55 70 Bacillus stearothermophilus
thermophile
obligate thermophile 45 70-75 85-90 Thermus aquaticus
extreme thermophile 60 75-80 85-110 Sulfolobus acidocaldarius

pH acidophile 0.8 2-3 5 Thiobacillus thiooxidans


alkal(in)ophile ca 7 9-10.5 11-11.5 Bacillus alcalophilus

Osmotic pressure halophile 0.5 1-2 4-4.5 Vibrio costicola


(Molar salt conc) extreme halophile 3 35 5.2 Halobacterium halobium
Oxygen

Microbes that use oxygen for energy-yielding purposes are called aerobes, if they require oxygen for their metabolism they are called
obligate aerobes. Obligate aerobes are at a disadvantage because oxygen is poorly soluble in water and much of the environment is
acking in this necessary element. Often, aerobic bacteria have retained the ability to grow without oxygen; these are called facultative
anaerobes. Those bacteria that are unable to use oxygen and in fact may be harmed by it are known as obligate anaerobes. Further
groups include: the microaerophiles which are aerobic microbes that tolerate only a narrow band of oxygen concentrations usually lower
han that of the atmosphere and are therefore often difficult to cultivate in the laboratory, and aerotolerant bacteria that grow in the
presence of oxygen but do not require it.
Water
n contrast to higher organisms, the metabolism of microorgansims is dependent on the presence of liquid water. The requirements of
microorganisms with respect to available water differ widely. In order to compare the available water content of solids and solutions,
water activity or relative humidity are useful parameters.
Carbon Dioxide
n autotrophic metabolisms, microbes tap various sources of energy and reducing power, which they use to reduce CO2 to organic
compounds. Sodium hydrogencarbonate is usually added to the culture media if autotrophic CO2-fixing microorganisms are to be grown
and incubation is performed in a carbon dioxide-containing atmosphere in closed vessels or, alternatively, air or carbon dioxide-enriched
air is circulated through the vessel. While some chemoautotrophs are aerobic, using oxygen as the ultimate electron acceptor and
deriving energy from the respiration of various inorganic electron donors, other microorganisms engage in anaerobic respiration, using
an inorganic terminal electron acceptor other than oxygen. Heterotrophic (= assimilating organic carbon sources) microorganisms
equire carbon dioxide as well. Many bacteria living in blood, tissue or in the intestinal tract are adapted to a carbon dioxide content
higher than that of normal air. These bacteria are therefore incubated in an atmosphere containing 10%(vol) carbon dioxide.
Phototrophic bacteria are obligate anaerobes and use energy from light for a succession of reactions that convert carbon dioxide to
riosephosphate and other cell constituents. Even though carbon dioxide is recycled rather than assimilated, nearly all growing cells have
an absolute requirement for an adequate pCO2. It is therefore important to note that the removal of carbon dioxide e.g. by KOH-
absorption, inhibits the growth of nearly all bacteria.
Microbiological Culture Methods I
Taxonomy and Identification of Microorganisms
Taxonomy is the theory and practice of the classification of individuals into groups. There are three groups of taxonomic
methods:
Numerical Taxonomy
This is defined as "the grouping by numerical methods of taxonomic units into taxa on the basis of their characteristics". This
involves studying all the physiological characters of bacteria using a series of biochemical and culture tests such as: the variety of
organic compounds degraded, the requirement for various vitamins or co-enzymes, staining reactions, and the inbitition of growth
by antibiotics. The results are coded on a computer and the relationships between individuals expressed as a dendrogram. This
form of taxonomic classification makes no reference to the evolutionary relationship between the bacterial strains. Kits containing
many of these tests are now commercially available facilitating the identification of several groups of bacteria.
Chemical Taxonomy
Here, the grouping of individuals is carried out depending on a set of characters presumed to be inherited from a common
ancestor. In the case of chemical taxonomy, bacteria are clustered according to the chemical similarity between structural
components of the bacteria. The most commonly used materials are proteins, which are molecules that are well preserved during
evolution. To establish common ancestry, chemotaxonomists commonly analyse the primary structure of enzymes, peptidogylcan,
the cytoplasmic membrane and its fatty acid composition, the outer membrane and the end products of metabolism.
Molecular Taxonomy
This is the comparison of the genetic sequences of chromosomal DNA or ribosomal RNA to establish similarity patterns and the
phylogenetic evolution of a group. Although the DNA content in purine (G, guanine; A, adenine) and pyrimidine (C, cytosine; T,
thymine) bases vary from one individual to another, they remain constant within a given species. The G+C content can therefore
be used to establish taxonomic relationships.

Similarities between the sequences of 16S or 23S ribosomal RNA are also compared in order to study the phylogeny of a bacterial
group.
Antimicrobial Sensitivity Testing
The antimicrobial activity of a compound is usually determined by measuring the lowest concentration of the compound which is
needed to inhibit growth of the test microorganism (MlC-minimum inhibitory concentration). The tests rely on the diffusion of the
antibiotic through the microbial medium to inhibit the growth of the susceptible organism growing in it or on it. The zones of
inhibition are taken to be representative of the susceptibility of the microbe. Antibiotic sensitivity has been used for many years as
a characteristic for classification and identification.
Anaerobic Growth
The cultivation of strict anaerobic bacteria poses a special problem because these bacteria may be killed by exposure to air.
Dissolved oxygen in the medium forms toxic free radicals and hydrogen peroxide in the presence of metabolic electrons. Obligate
anaerobes are incapable of detoxifying these active forms of oxygen. To grow non stringent anaerobes on solid media, anaerobic
jars are used together with gas generating "Gas-Paks", which release both CO2 and H2. The hydrogen reacts with oxygen in the
presence of a palladium catalyst to produce water, thus removing oxygen from the jar. Completely anaerobic chambers equipped
with air locks and filled with inert gases used for the cultivation of strict (obligate) anaerobes are commercially available.
Redox Potential (O/R Potential)
This is the proportion of oxidized to reduced molecules in a medium: when oxygen dissolves in a medium, for instance, the
organic compounds present become more oxidized and the medium exhibits a positive redox potential. As microbial growth
consumes the oxygen, the medium moves towards a more negative redox potential. Strict anaerobes require the medium to be
kept at a very low (negative) potential during growth. To achieve this, reducing agents are added to the media prior to
autoclaving. Commonly used reducing agents are sodium thioglycolate (HS-CH2COONa) or sodium dithionite, which easily
donate protons to other compounds. The relationship between redox potential, pH and microbial growth is illustrated below.
Eh and pH ranges for microbial growth (adapted from Zajic 1969): the figure has been compiled from reports where both pH and
Eh were given for growth behaviour. It is very probable that the growth ranges of the groups extend beyond the boundaries shown
in the figure.
Monitoring Microbial Growth

Serial Dilutions
The inoculum is diluted out in a series of dilution tubes which are plated out. The number of colonies on the plate are counted and
corrected for the dilution to calculate the number of organisms in the original inoculum.
Most Probable Number Method
A statistical method estimates the most probable number of bacteria present in an inoculum which has been used to make a
dilution series. Several series are made with different initial volumes of inoculum; the results are recorded as a series of positives,
i.e. growth in the tube, which can then be calculated to give an MPN. The result is the probable number of microorganisms that
would be expected to yield this result.
Direct Microscope Observation
Specially constructed microscope slides are used which have a shallow well of known volume and a grid etched into the glass.
The well is filled with the bacterial suspension and the average number of bacteria in each of the grid squares is determined and
then multiplied by a factor to give the counts per millilitre. Selective staining (employing fluorescent dyes) is used to differentiate
bacteria from non-living material in environmental samples. Electronic cell counters are also available which automatically count
the number of cells in a measured volume of liquid.
Turbidity (Optical Density)
The turbidity of a liquid medium increases as bacteria multiply and can be measured on a spectrophotometer. The amount of light
reaching the detector is inversely proportional to the number of bacteria under standardized conditions. The absorbency of the
sample (optical density) is dependent on the number of cells, their size and shape, and is used to plot bacterial growth. If