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Microbiology Introduction
Read more about
Microbial Growth Requirements
Physical / Environmental Factors
Taxonomy and Identification of Microorganisms
Monitoring Microbial Growth
Preserving Bacterial Cultures
References & Literature
An initial aim of all microbiologists is the reproducible growth of their microbial cultures, no matter
whether the microorganisms are of natural origin or have been genetically engineered by man.
Reproducible growth requires defined environmental conditions with respect to energy source,
temperature, pH and nutrients (Microbial Growth Requirements). With this in mind, we supply a
range of products and services (see list of culture collections, comparison of media etc.) designed to
meet the needs of general microbiologists and specialists alike.
Microorganisms
In the group of organisms classified as microorganisms, there are simple unicellular forms (cocci,
bacilli, virio and spirillae) as well as multicellular forms (filaments and sheaths). The group includes
the blue green algae (cyanobacteria), fungi, protozoans and bacteria.
In order to survive and grow, microorganisms require a source of energy and nourishment. Bacteria
are the most primitive forms of microorganisms but are composed of a great variety of simple and
complex molecules and are able to carry out a wide range of chemical transformations. Depending
on their requirements and the source of energy used they are classified into different nutritional
groups.
Figure 1.
The size of microorganisms varies from a fraction of a μM for viruses, which can only be seen in the
electron microscope, to several cm for filamentous algae or fungi, for example:
Organisms Size range (μM) Example (size in μM)
Prokaryotes
Bacterium: typical rod 1.0-0.5 x 1.0-10 Pseudomonas aeruginosa (1.5 x 0.5)
Bacterium: typical sphere 1.0 diameter Bacillus megaterium (7.6 x 2.4)
Eukaryotes
Fungi: filamentous 8-15 x 4-8 Mucor hiemalis (8 diameter)
3
Fungi: yeast cell Saccharomyces cerevisiae (29-49.1 μM )
Alga 28-32 x 8-12 Chlamydomonas
Viruses
Virus 0.015 x 0.3 Poliovirus (0.03 x 0.03)
Tobacco mosaic (0.02 x 0.3)
Table 1.
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
Organic; simple sugars e.g.
glucose, acetate or pyruvate; extracts such as
Carbon dioxide (CO 2 ),
- peptone,
Carbon HCO 3
tryptone, yeast extract etc.
organic compounds
Inorganic; carbon dioxide (CO 2 )
-
or hydrogen carbonate salts (HCO 3 )*
Water (H 2 O)
Hydrogen
organic compounds
Water (H 2 O), oxygen gas
Oxygen (O 2 ),
organic compounds
Ammonia (NH 3 ), nitrate
- Organic; amino acids,
(NO 3 )
nitrogenous bases
Nitrogen organic compounds
Inorganic; NH 4 CI, (NH 4 ) 2 S0 4, KNO 3 , and for
e.g. amino acids
dinitrogen fixers N 2
nitrogen gas (N 2 )
3-
Phosphorus Phosphate (PO 4 ) KH 2 PO 4 , Na 2 HPO 4 *
Sulphur Hydrogen sulphide(H 2 S), Na 2 SO 4 , H 2 S
2-
sulphate (SO 4 ),
organic compounds
e.g cysteine
+
Potassium K KCI, K 2 HPO 4 *
2+
Magnesium Mg MgCI 2 , MgSO 4
2+
Calcium Ca CaCI 2 , Ca(HC0 3 ) 2 *
+
Sodium Na NaCI
3+
Fe organic iron 1)
Iron FeCI 3 , Fe(NH 4 )(SO 4 ) 2 , Fe-chelates
complexes
Usually present at very low CoCI 2 , ZnCI 2 , Na 2 MoO 4 , CuCI 2 , MnSO 4 ,
Trace elements
concentrations NiCI 2 , Na 2 SeO 4 , Na 2 WO 4 , Na 2 VO 4
Organic growth Usually present at very low
Vitamins, amino acids, purines, pyrimidines
factors concentrations
Table 2.
*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 medium.
PHYSICAL / ENVIRONMENTAL FACTORS
Temperature
Most microorganisms grow well at the normal temperatures 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, bacteria are divided into
three groups: Psychrophiles (cold-loving microorganisms) found mostly in the depths of the oceans,
in ice and snow and in the arctic regions, have an optimum growth temperature between 0 °C and
15 °C and a maximum growth temperature of not more than 20 °C. Mesophiles (moderate-
temperature-loving bacteria) found in water, soil and in higher organisms, are the most common
type of microbes studied. Their optimum growth temperature ranges between 25 °C and 40 °C. The
optimum temperature for many pathogenic bacteria is 37 °C, thus the mesophiles constitute most
of our common spoilage and disease microbes. Thermophiles (heat-loving microbes) are capable of
growth at high temperatures with an optimum above 60 °C. Some organisms grow at temperatures
near the boiling point of water and even above 100 °C when 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 extremes of pH are classed as acidophiles (acid-loving) or alkalinophiles
(base-loving). Acidophiles grow at pH values below 4 with some bacteria still active 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 during 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 most bacteriological media to neutralise the acids and maintain the correct pH.
Phosphate salts are the most commonly used buffers because they buffer in the growth range of
most bacteria, are non-toxic and provide a source of phosphorus, an essential nutrient element.
High phosphate concentration has the disadvantage, however, that it can result in a severe nutrient
limitation caused by the precipitation of insoluble metal 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 causes shrinkage of the cell (plasmolysis). However, some
bacteria have adapted so well to high salt concentrations that they actually require them for
growth. These bacteria are called halophiles (salt-loving) and are found in salterns or in areas such
as the Dead Sea.
Class of
Factor Minimum Optimum Maximum Example
Organism
Temperature extreme -2 5 10 Raphidonema
(°C) psychrophile nivale
(snow algae)
psychrophile 0 15 20 Vibrio marinus
mesophile 10-15 24-40 35-45 Escherichia coli
facultative 37 45-55 70 Bacillus
thermophile stearothermophilus
obligate 45 70-75 85-90 Thermus aquaticus
thermophile
extreme 60 75-80 85-110 Sulfolobus
thermophile acidocaldarius
pH acidophile 0.8 2-3 5 Thiobacillus
thiooxidans
alkal(in)ophile ca 7 9-10.5 11-11.5 Bacillus
alcalophilus
Osmotic halophile 0.5 1-2 4-4.5 Vibrio costicola
pressure
(Molar salt extreme 3 35 5.2 Halobacterium
conc) halophile halobium
Table 3.
Oxygen
Microbes that use oxygen for energy-yielding purposes are called aerobes, if they require oxygen
Figure 2:
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 lacking 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 than 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
In contrast to higher organisms, the metabolism of microorgansims is dependent on the presence of
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
In autotrophic metabolisms, microbes trap various sources of energy and reducing power, which
they use to reduce CO 2 to organic compounds. Sodium hydrogen carbonate is usually added to the
culture media if autotrophic CO 2 -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 require 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 triose-phosphate and other cell constituents. Even though
carbon dioxide is recycled rather than assimilated, nearly all growing cells have an absolute
requirement for an adequate pCO 2 . 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 & ISOLATING
MICROORGANISMS FROM NATURE
Microorganisms can be isolated from their natural environments by a variety of techniques. If
microbial populations are frequent, dense or large enough, they can be sampled directIy with a
sterile swab or loop and inoculated into a suitable liquid medium or streaked out on an agar plate.
This applies especially to medical samples in which organisms are present in large numbers and in
localised areas. Environmental samples with large microbial populations, e.g. soil, may also be added
directly to a suitable medium. Where microorganisms are infrequent, a pre-enrichment stop is
necessary. This is achieved by filtering the samples and incubating the filters in a suitable medium.
Filtration is commonly applied to liquid samples (e.g. river and sea water) and when sampling air. In-
situ sampling of environmental samples involves techniques such as the buried slide or buried
capillary methods, in which microscope slides or capillary tubes coated with a suitable medium are
buried in the natural environment (soil or sediment) and only retrieved after a certain period of
time. The slides or capillaries are then added to fresh media and the organisms sub-cultured. These
methods are applied when organisms are slow growing, require special conditions; or when
minimum disturbance of the environment is called for.
All samples can be sub-cultured with the use of any of the microbiological culture methods
illustrated.
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
Figure 3.
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 method 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 CO 2 and H 2 . 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-CH 2 COONa) or
sodium dithionite, which easily donate protons to other compounds. The relationship between
redox potential, pH and microbial growth is illustrated below.
Figure 4.
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
Figure 5
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
absorbency readings are matched with a direct count of the same culture, its protein content or dry
mass, the correlation can be used in a future estimate of bacterial numbers or biomass based
directly on turbidity measurements.
Metabolic Activity
Another indirect way to estimate bacterial numbers is to use the metabolic activity of the
population. The amount of a metabolic product is measured and assumed to be proportional to the
number of bacteria present. Examples of metabolic products include CO 2 and organic acids.
Oxygen uptake can also be measured with a reduction test, for example the use of the methylene
blue dye, which changes colour from blue in the presence of oxygen to colourless in its absence.
PRESERVING BACTERIAL CULTURES
Refrigeration
Can be used for short-term storage. Cultures streaked on agar slants or stab cultures may be viable
over several months when stored at 4 °C . Plates have to be sealed to prevent their tendency to dry
out. To preserve cultures for longer periods of time, two methods are commonly employed:
Deep Freezing
A pure culture of bacteria is suspended in a liquid and quick-frozen (often with liquid nitrogen) at
temperatures between -50 °C and -95 °C. Sensitive microorganisms require the presence of
glycerol (end concentration 15-20 %), which acts as an "antifreeze", or extra protein (skimmed milk
powder) to protect them. Cultures can be thawed and used up to several years later.
Lyophilization
A suspension of bacteria is quickly frozen and the water removed by means of a high vacuum. The
microbes survive in these powder like residue for several years and can be revived at any time by
rehydration of the culture in a nutrient medium. Bacterial strains ordered from strain collections are
usually delivered in this form.
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