Microbial Nutrition and Cultivation

Question 1

We introduced the major groups of macromolecules found in living cells. The raw materials from which these are synthesised are ultimately derived from the
organism’s environment in the form of nutrients. These can be conveniently
divided into those required in large quantities* (macronutrients) and those which are needed only in trace amounts (micronutrients or trace elements). Give examples.

Answer 1

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Note that Micronutrients are all metal ions, and frequently serve as cofactors for enzymes.

Question 2

Carbon

Answer 2

Carbon is the central component of the biological macromolecule. Carbon incorporated into biosynthetic pathways may be derived from organic or inorganic sources; some organisms can derive it from CO2, while others require their carbon in ‘ready-made’, organic form.

Question 3

Hydrogen

Answer 3

Hydrogen is also a key component of macromolecules, and participates in energy generation processes in most microorganisms. In autotrophs, hydrogen is required to reduce carbon dioxide in the synthesis of macromolecules.

Question 4

Oxygen

Answer 4

Oxygen is of central importance to the respiration of many microorganisms, but in
its molecular form (O2), it can be toxic to some forms. These obtain the oxygen they need for the synthesis of macromolecules from water.

Question 5

Nitrogen

Answer 5

Nitrogen is needed for the synthesis of proteins and nucleic acids, as well as for
important molecules such as ATP. Microorganisms range in their demands for nitrogen from those that are able to assimilate (‘fix’) gaseous nitrogen (N2) to those that require all 20 amino acids to be provided preformed. Between these two extremes come species that are able to assimilate nitrogen from an inorganic source such as nitrate, and those that utilise ammonium salts or urea as a nitrogen source.

Question 6

Sulphur

Answer 6

Sulphur is required for the synthesis of proteins and vitamins, and in some types is
involved in cellular respiration and photosynthesis. It may be derived from sulphurcontaining amino acids (methionine, cysteine), sulphates and sulphides.

Question 7

Phosphorus

Answer 7

Phosphorus is taken up as inorganic phosphate, and is incorporated in this form into nucleic acids and phospholipids, as well as other molecules such as ATP.

Question 8

Metals such as copper, iron and magnesium

Answer 8

Are required as cofactors in enzyme reactions.
A cofactor is a nonprotein
component of an enzyme (often a
metal ion) essential for its normal functioning.

Question 9

Growth factors

Answer 9

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Many microorganisms are unable to synthesise certain organic compounds necessary for growth and must therefore be provided with them in their growth medium. These are termed growth factors, of which three main groups can be identified: amino acids, purines and pyrimidines (required for nucleic acid synthesis) and vitamins. Vitamins are complex organic compounds required in very small amounts for the cell’s normal functioning. They are often either coenzymes or their precursors. Microorganisms vary greatly in their vitamin requirements. Many bacteria are completely self-sufficient, while protozoans, for example, generally need to be supplied with a wide range of these dietary supplements. A vitamin requirement may be absolute or partial; an organism
may be able, for example, to synthesise enough of a vitamin to survive, but grow more vigorously if an additional supply is made available to it.

Question 10

Nutritional categories - introduction

Answer 10

Microorganisms can be categorised according to how they obtain their carbon and energy. As we have seen, carbon is the most abundant component of the microbial cell, and most microorganisms obtain their carbon in the form of organic molecules, derived directly or indirectly from other organisms. This mode of nutrition is the one that is familiar to us as humans (and all other animals); all the food we eat is derived as complex organic molecules from plants and other animals (and even some representatives of the microbial world such as mushrooms!).

Question 11

Heterotrophs

Answer 11

A heterotroph must use one or more organic compounds as its source
of carbon. Include all the fungi and protozoans as well as most types of bacteria. Microorganisms as a group are able to incorporate the carbon from an incredibly wide range of organic
compounds into cellular material. In fact there is hardly any such compound occurring in nature that cannot be metabolised by some microorganism or other, explaining in part why microbial life is to be found thriving in the most unlikely habitats. Many synthetic materials can also serve as carbon sources for some microorganisms, which can have considerable economic significance.

Question 12

Autotrophs

Answer 12

An autotroph can derive its carbon from carbon dioxide.
A significant number of bacteria and all of the algae do not, however, take up their carbon preformed as organic molecules in this way, but derive it instead from carbon dioxide. These organisms are called autotrophs, and again we can draw a parallel with higher organisms, where all members of the plant kingdom obtain
their carbon in a similar fashion.

We can also categorise microorganisms nutritionally by the way they derive the energy they require to carry out essential cellular reactions.
Autotrophs thus fall into two categories. Chemoautotrophs obtain their energy
as well as their carbon from inorganic sources; they do this by the oxidation of inorganic molecules such as sulphur or nitrite.
Photoautotrophs have photosynthetic
pigments enabling them to convert light energy into chemical energy.

Question 13

Chemoautotrophs

Answer 13

A chemotroph obtains its energy from chemical compounds.

Question 14

Phototrophs

Answer 14

A phototroph uses light as its
source of energy.

Question 15

Chemoheterotrophs

Answer 15

The great majority of heterotrophs obtain energy as well as carbon from the
same organic source. Such organisms release energy by the chemical oxidation of organic nutrient molecules, and are therefore termed chemoheterotrophs.

Question 16

Photoheterotrophs

Answer 16

Those few heterotrophs which do not follow this mode of nutrition include the green and purple non-sulphur bacteria. These are able to carry out photosynthesis and are known as photoheterotrophs.

Question 17

Lithotrophs and Organotrophs

Answer 17

A lithotroph is an organism that uses inorganic molecules as a source of electrons.
An organotroph uses organic molecules for the same purpose.

Whether organisms are chemotrophs or phototrophs, they need a molecule to act as a source of electrons (reducing power) to drive their energygenerating
systems. Those able to use an inorganic electron donor such as H2O, H2S or ammonia are called lithotrophs, while those requiring an organic molecule to fulfil the role are organotrophs. Most (but
not all) microorganisms are either photolithotrophic autotrophs (algae, blue-greens) or chemo-organotrophic
heterotrophs (most bacteria). For the latter category, a single organic compound can often act as the provider of carbon, energy and reducing power. The substance used
by chemotrophs as an energy source may be organic (chemoorganotrophs) or inorganic (chemolithotrophs).

Question 18

Having found a source of a given nutrient, a microorganism must:

Answer 18

• have some means of taking it up from the environment
• possess the appropriate enzyme systems to utilise it.

Question 19

The plasma membrane represents a selective barrier, allowing into the cell only those substances it is able to utilise. This selectivity is due in large part to …

Answer 19

This selectivity is due in large part to the hydrophobic nature of the lipid bilayer.

Question 20

A substance can be transported across the cell membrane in one of three ways, known as:

Answer 20

simple diffusion

facilitated diffusion

active transport

Question 21

Simple diffusion

Answer 21

In simple diffusion, small molecules move across the membrane in response to a
concentration gradient (from high to low), until concentrations on either side of the
membrane are in equilibrium. The ability to do this depends on being small (H2O,
Na+, Cl−) or soluble in the lipid component of the membrane (non-polar gases such as
O2 and CO2).

Question 22

Facilitated diffusion

Answer 22

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Larger polar molecules such as glucose and amino acids are unable to enter the cell
unless assisted by membrane-spanning transport proteins by the process of facilitated diffusion. Like enzymes, these proteins are specific for a single/small number of related solutes;
another parallel is that they too can become saturated by too much ‘substrate’. As with simple diffusion, there is no expenditure of cellular energy, and an inward concentration gradient is required. The transported substance tends to be metabolised rapidly once inside the cell, thus maintaining the concentration gradient from outside to inside.

Question 23

Active transport

Answer 23

Diffusion is only an effective method of internalising substances when their concentrations are greater outside the cell than inside. Generally, however, microorganisms find themselves in very dilute environments; hence the concentration gradient runs in the other direction, and diffusion into the cell is not possible. Active transport enables the cell to overcome this unfavourable gradient. Here, regardless of the direction of the gradient, transport takes place in one direction only, into the cell. Energy, derived from hydrolysis of adenosine triphosphate is required to achieve this, and again specific transmembrane proteins are involved. They bind the solute molecule with high affinity outside of the cell, and then undergo a conformational change that causes them to be released into the interior.

Question 24

Active transport in Procaryotic cells

Answer 24

Procaryotic cells can carry out a specialised form of active transport called group translocation, whereby the solute is chemically modified as it crosses the membrane, preventing its escape. A well-studied example of this is the phosphorylation of glucose in E. coli by the phosphotransferase system. Glucose
present in very low concentrations outside the cell can be concentrated within it by this
mechanism. Glucose is unable to pass back across the membrane in its phosphorylated
form (glucose-6-phosphate), however it can be utilised in metabolic pathways in this form.

Question 25

Culturing techniques enable…

Answer 25

Critical to the development of microbiology during its ‘golden age’ was the advance in
culturing techniques, enabling the isolation and pure culture of specific microorganisms.
The study of pure cultures made it possible to determine the properties of a specific
organism such as its metabolic characteristics or its ability to cause a particular disease.
It also opened up the possibility of classifying microorganisms, on the basis of the characteristics they display in pure culture.

Question 26

The artificial culture of any organism requires…

Answer 26

The artificial culture of any organism requires a supply of the necessary nutrients, together with the provision of appropriate conditions such as temperature, pH and oxygen concentration. The nutrients and conditions provided in the laboratory are usually a reflection of those found in the organism’s natural habitat. It is also essential that appropriate steps are taken to avoid contamination.

Question 27

Agar

Answer 27

AGAR is a complex polysaccharide derived from seaweeds, and was suggested by the wife of one of Koch’s colleagues, who had used it as a setting agent in jam making. Agar does not melt until near boiling point; this means that cultures can be incubated at 37 ◦C or above without the medium melting. Moreover, when it cools, agar remains molten until just over 40 ◦C, allowing heat-sensitive media components
such as blood to be added. In addition, most bacteria can tolerate a short exposure to temperatures in this range, so they too can be inoculated into molten agar. Crucially, agar is more or less inert nutritionally; only a very few organisms are known that are able to use agar as a food source; consequently, it is the near ideal setting agent, resisting both thermal and microbial breakdown. Agar soon became the setting agent of choice, and has remained so ever since; shortly afterwards, Richard Petri developed the two-part culture dish that was named after him, and which could be sterilised separately from the medium and provided protection from contamination by means of its lid,. This again is still standard equipment today,
although the original glass has been largely replaced by presterilised, disposable plastic.

Question 28

Streak plate technique

Answer 28

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The standard method of obtaining a pure bacterial culture is the creation of a streak
plate. A wire inoculating loop is used to spread out a drop of bacterial suspension on an agar plate in such a way that it becomes progressively more dilute;eventually, individual cells will be deposited on the agar surface. Following incubation at an appropriate temperature, a succession of cell divisions occurs, resulting in the formation of a bacterial colony, visible to the naked eye. Colonies arise because movement is not possible on the solid surface and all the progeny stay in the same place. A colony represents, in theory at least, the offspring of a single cell and its members are therefore genetically identical. (In reality, a clump of cells may be deposited together and give rise to a colony; this problem can be overcome by repeated isolation and restreaking of single colonies.)

Question 29

Pour plate technique

Answer 29

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An alternative method for the isolation of pure cultures is the pour plate.
In this method, a dilute suspension of bacteria is mixed with warm molten agar, and poured into an empty petri plate. As the agar sets, cells are immobilised, and once again their progeny are all kept together, often within, as well as on, the agar. This method is especially useful for the isolation of bacteria that are unable to tolerate atmospheric levels of oxygen.

Question 30

Growth media for the cultivation of bacteria

Answer 30

A synthetic growth medium may be defined, that is, its exact chemical composition is known, or undefined.
A defined growth medium may have few or many constituents, depending on the nutritional requirements of the organism in question. An undefined or complex medium may have a variable composition due to the inclusion of a component such as blood, yeast extract or tap water.
Peptones are also commonly found in
complex media; these are the products of partially digesting protein sources such as beef or casein. The exact composition of a complex medium is neither known nor critically important. A medium of this
type would generally be chosen for the cultivation of fastidious bacteria such as Neisseria gonorrhoeae (the causative agent of gonorrhoea); it is easier and less expensive to supply the many nutrients required by such an organism in this form rather than supplying them all individually.
Bacteria whose specific nutrient requirements are not known are also grown on complex media.
Whilst media such as nutrient agar are used to support the growth of a wide range of organisms, others are specifically designed for the isolation and identification of particular types. Selective media such as bismuth sulphite medium preferentially support the growth of particular bacteria. The bismuth ion inhibits the growth of
Gram-positive organisms as well as many Gram-negative types; this medium is used for the isolation of the pathogenic bacterium Salmonella typhi, one of the few organisms that can tolerate the bismuth.

Question 31

Specific media differential

Answer 31

Specific media called differential media can be used to distinguish between
organisms whose growth they support, usually by means of a coloured indicator.
Ex MacConkey agar

Question 32

MacConkey agar

Answer 32

MacConkey agar contains lactose and a pH indicator, allowing the differentiation between lactose fermenters (red colonies) and nonlactose fermenters (white/pale pink colonies). MacConkey agar also contains bile salts and the dye crystal violet, both of which serve to inhibit the growth
of unwanted Gram-positive bacteria.

Question 33

Mannitol salt agar

Answer 33

Mannitol salt agar is also both selective and differential. The high (7.5 per cent) salt content suppresses growth of most bacteria, whilst a combination of mannitol and an indicator permits the detection of mannitol fermenters in a similar fashion to that just described.

Question 34

Enrichment media

Answer 34

Sometimes, it is desirable to isolate an organism that is present in small numbers in a large mixed population (e.g. faeces or soil).
Enrichment media provide conditions that selectively encourage the growth of these organisms; the use of blood agar in the isolation of streptococci provides an example of such a medium.
Blood agar can act as a differential medium, in allowing the user to distinguish between haemolytic and nonhaemolytic bacteria.

Question 35

Preservation of microbial cultures

Answer 35

Microbial cultures are preserved by storage at low temperatures, in order to suspend
growth processes. For short periods, most organisms can be kept at refrigerator temperature (around 4 ◦C), but for longer-term storage, more specialised treatment is necessary. Using deep freezing or freeze-drying, cultures can be kept for many years, and then resurrected and re-cultured. Deep freezing requires rapid freezing to −70 ◦C to −95 ◦C, while freeze-drying (lyophilisation) involves freezing at slightly less extreme temperatures and removing the water content under vacuum. Long-term storage may be desirable to avoid the development of mutations or loss of cell viability.

Question 36

Estimation of microbial numbers methods

Answer 36

Total cell counts

Viable cell count

Question 37

Total cell counts

Answer 37

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Total cell counts are generally done by direct microscopic examination. A specialised glass slide is employed, which carries an etched grid of known area. The
depth of the liquid sample is also known, so by counting the number of cells visible in
the field of view, the number of cells per unit volume can be determined. The method may be made more accurate by the use of a fluorescent dye such as acridine orange, which binds to DNA, and hence avoids confusion with non-cellular debris. However, such methods cannot differentiate between living and non-living cells. Their usefulness is further limited by the fact that the smallest bacteria are difficult to resolve as individual cells by light microscopy. Other total cell count methods use cell-sorting devices, originally developed for separating blood cells in medical research. These pass the cell suspension through an extremely fine nozzle, and a detector registers the conductivity change each time a particle passes it. Again, no distinction can be made between viable
and non-viable cells.

Question 38

Viable cell count

Answer 38

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A viable cell count is a measure of the number of living cells in a sample, or more specifically those capable of multiplying and producing a visible colony of cells. It is most commonly estimated by spreading a known volume of cell suspension onto an agar plate, and counting the number of colonies that arise after a period of incubation. The method is based on the premise that each visible colony has derived from the repeated divisions of a single cell. In reality, it is accepted that this is not always the case, and so viable counts are expressed in colony-forming units (cfu), rather than cells, per unit volume. It is generally necessary to dilute the suspension before plating out, otherwise the resulting colonies will be too numerous to count. In order to improve statistical reliability, plates are inoculated in duplicate or triplicate, and the mean value is taken.

Question 39

Viable cell counts can also be made using liquid media, in the Most Probable Number
(MPN) technique

Answer 39

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A series of tubes containing a broth are inoculated with a sample of a progressively more dilute cell suspension, incubated, and examined for growth. The method is based on the statistical probability of each sample containing viable cells. It is well suited to the testing of drinking water, where low bacterial densities are to be expected.

Question 40

Membrane filter test

Answer 40

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Another method employed for the enumeration of bacteria in water is the membrane filter test. Here, a large volume of water is passed through a membrane filter with a pore size (0.45μm) suitable for trapping bacteria. The filter is placed on an
appropriate solid growth medium and colonies allowed to develop.

Question 41

None of the methods described above provides a particularly rapid result, yet sometime it is desirable to have an estimate of bacterial numbers immediately. A useful method for doing this is based on how cloudy or turbid the liquid growth medium becomes due to bacterial growth.

Turbidimetric methods

Answer 41

Turbidimetric methods measure the change in optical density or absorbance of the medium, that is, how much a beam of light is scattered by the suspended particulate matter. They can be carried out very quickly
by placing a sample in a spectrophotometer. Values of optical density can be directly related to bacterial numbers or mass by reference to a standard calibration curve. Thus, an estimate of bacterial numbers, albeit a fairly approximate one, can be obtained almost instantaneously during an experimental procedure. Other indirect methods of measuring cell density include wet and dry weight estimations, and the measurement of cell components such as total nitrogen, protein or nucleic acid.

Question 42

Factors affecting microbial growth

Answer 42

nutrient requirements of microorganisms

temperature

pH

oxygen

carbon dioxide

osmotic pressure

light

Question 43

Factors affecting microbial growth: temperature