Microbial Growth Requirements

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 Growth

Form usually found in Nature

Chemical Form commonly added to Microbiological Media

Carbon

Carbon dioxide (CO2), HCO3organic 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 Water (H2O), oxygen gas (O2), organic compounds Ammonia (NH3), nitrate (NO3-) organic compounds e.g. amino acids nitrogen gas (N2)

Oxygen Nitrogen

Organic; amino acids, nitrogenous bases Inorganic; NH4CI, (NH4)2S04, KNO3, and for dinitrogen fixers

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, Na2W Na2VO4

Organic growth factors

Usually present at very low concentrations

Vitamins, amino acids, purines, pyrimidines

*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

psychrophile mesophile facultative thermophile obligate thermophile extreme thermophile

0 10-15 37

15 24-40 45-55

20 35-45 70

Raphidonema nivale (snow algae) Vibrio marinus Escherichia coli Bacillus stearothermophilus

45 60

70-75 75-80

85-90 85-110

Thermus aquaticus Sulfolobus acidocaldarius

pH

acidophile alkal(in)ophile

0.8 ca 7

2-3 9-10.5

5 11-11.5

Thiobacillus thiooxidans Bacillus alcalophilus

Osmotic pressure (Molar salt conc)

halophile extreme halophile

0.5 3

1-2 35

4-4.5 5.2

Vibrio costicola 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 KOHabsorption, 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