Microbiology Introduction

• 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:

Table 1.

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)
Fungi: yeast cell		Saccharomyces cerevisiae (29-49.1 μM3)
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)

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.

Table 2.

Requirements for growth	Form usually found in nature	Chemical form commonly added to microbiological media
Carbon	Carbon dioxide (CO2), HCO3- organic compounds	Organic; simple sugars e.g. 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 compounds e.g. amino acids nitrogen gas (N2)	Organic; amino acids, nitrogenous bases Inorganic; NH4CI, (NH4)2S04, KNO3, and for dinitrogen fixers N2
Phosphorus	Phosphate (PO43-)	KH2PO4, Na2HPO4*
Sulphur	Hydrogen sulphide(H2S), sulphate (SO42-), organic compounds e.g cysteine	Na2SO4, H2S
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 concentrations	CoCI2, ZnCI2, Na2MoO4, CuCI2, MnSO4, NiCI2, Na2SeO4, Na2WO4, Na2VO4
Organic growth factors	Usually present at very low concentrations	Vitamins, amino acids, purines, pyrimidines

*Also act as buffers.
¹To 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.

Table 3.

Factor	Class of Organism	Minimum	Optimum	Maximum	Example
Temperature (°C)	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 thermophile	37	45-55	70	Bacillus stearothermophilus
	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	3 5	5.2	Halobacterium halobium

Figure 2:

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 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 CO₂ to organic compounds. Sodium hydrogen carbonate 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 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 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 & 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.

Figure 3.

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 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 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.

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 CO₂ 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.