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Microbiology

Microbiology Introduction

Introduction

An initial aim of all microbiologis ts is the reproducible growth of their microbial cultures , no matter whether the microorganis ms a re ofnatural origin or have been genetically engineered by man. Reproducible growth requires defined environmental conditions with respect toenergy source, temperature, pH and nutrients (s ee chapter Microbial Growth Requirements). With this in mind Fluka supplies a range ofproducts and services (see lis t of culture collections, comparison of media etc.) designed to meet the needs of general microbiologis ts andspecialists alike.

Microorganisms

In the group of organisms class ified as microorganism s, there are simple unicellular forms (cocci, bacilli, virio and spirillae) as well asmulticellular forms (filaments and s heaths). The group includes the blue green algae (cyanobacteria), fungi, protozoans and bacteria.

In order to survive and grow, microorganism s require a source of energy and nourishment. Bacteria are the mos t primitive forms ofmicroorganism s but are composed of a great variety of sim ple and complex molecules and are ab le to carry out a wide range of chemicaltransformations. Depending on their requirements and the source of energy used they are class ified into different nutritional groups .

The size of microorganisms varies from a fraction of a m for viruses, which can only be seen in the electron microscope, to several cm forfilamentous algae or fungi, for example:

Organisms Size range (m) Example (size in m)

Prokaryotes

Bacterium: typical rod 1.0-0.5 x 1.0-10 Pseudomonas aeruginosa (1.5 x 0.5)

Bacillus megaterium (7.6 x 2.4)

Bacterium: typical sphere 1.0 diam

Eukaryotes

Fungi: filamentous 8-15 x 4-8 Mucor hiemalis (8 diam)

Fungi: yeast cell Saccharomyces cerevisiae (29-49.1 m3)

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 intotwo categories, physical and chem ical.

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), 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-)*

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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 (SO4

2-),

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 buffers1)To facilitate the solubilis ation or retention of iron in solution, complexing agents s uch 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 attemperatures (extreme heat or cold) at which few higher organis ms can survive. Depending on their preferred temperature range, bacteriaare divided into three groups: Psychrophiles (cold-loving microorganisms) found mostly in the depths of the oceans, in ice and snow and inthe arctic regions, have an optimum growth temperature between 0C and 15C and a maximum growth temperature of not more than20C. Mesophiles (moderate-temperature-loving bacteria) found in water, soil and in higher organis ms, are the mos t common type ofmicrobe s tudied. Their optimum growth temperature ranges between 25C and 40C. The optimum temperature for many pathogenic

bacteria is 37C, thus the mesophiles constitute most of our common spoilage and dis ease microbes . Thermophiles (heat-lovingmicrobes) are capable of growth at high temperatures with an optimum above 60C. Some organis ms grow at temperatures near theboiling point of water and even above 100C when unde r pressure. Most thermophiles cannot grow below 45C.

pH

Most bacteria grow bes t in an environment with a narrow pH range near neutrality between pH 6.5 and 7.5. Those that grow at extremes ofpH are class ed as acidophiles (acid-loving) or alkalinophiles (base-loving). Acidophiles grow at pH values below 4 with som e bacteria stillactive at a pH of 1. Alkalinophilic bacteria prefer pH values of 9-10 and mos t cannot grow in solutions with a pH at or below neutral. Oftenduring bacterial growth, organic acids are releas ed into the medium , which lower its pH and s o interfere with or totally inhibit further growth.

Although comm on media ingredients s uch as peptones and amino acids have a small buffering effect, an external buffer is needed in mos tbacteriological media to neutralise the acids and maintain the correct pH. Phosphate salts a re the most comm only used buffers becausethey buffer in the growth range of mos t bacteria, are non-toxic and provide a s ource of phosphorus , an ess ential nutrient element. Highphosphate concentration has the disadvantage, however, that it can result in a s evere nutrient limitation caused by the precipitation ofinsoluble 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 themfor 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

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

Oxygen

Literature & Resources

Supelco 2012 Catalog,Interactive PDF
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Microbes that use oxygen for energy-yielding purposes are called ae robes, if they require oxygen for their metabolism they are calledobligate 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 facultativeanaerobes. Those bacteria that are unable to use oxygen and in fact may be harmed by it are known as obligate anaerobes. Further groupsinclude: the microaerophiles which are aerobic m icrobes that tolerate only a narrow band of oxygen concentrations usually lower than thatof the atmosphere and are therefore often difficult to cultivate in the laboratory, and aerotolerant bacteria that grow in the presence of oxygenbut do not require it.

Water

In contrast to higher organisms , the metabolism of microorgansims is dependent on the presence of liquid water. The requirements ofmicroorganism s w ith respect to available water differ widely. In order to compare the available water content of solids and solutions, wateractivity or relative humidity are useful parameters.

Carbon Dioxide

In autotrophic metabolism s, microbes tap various s ources 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 microorganism s are to be grown,

and incubation is performed in a carbon dioxide-containing atmosphere in clos ed vessels or, alternatively, air or carbon dioxide-enrichedair is circulated through the vess el. While some chemoautotrophs are aerobic, us ing oxygen as the ultimate electron acceptor and derivingenergy from the respiration of various inorganic electron donors, other microorganisms engage in anaerobic res piration, using aninorganic terminal electron acceptor other than oxygen. Heterotrophic (= assim ilating organic carbon sources) m icroorganisms requirecarbon dioxide as well. Many bacteria living in blood, tissue or in the intestinal tract are adapted to a carbon dioxide content higher than thatof normal air. These bacteria are therefore incubated in an atmosphere containing 10%(vol) carbon dioxide. Phototrophic bacteria areobligate anaerobes and use energy from light for a success ion of reactions that convert carbon dioxide to triosephosphate and other cellconstituents. Even though carbon dioxide is recycled rather than ass imilated, nearly all growing cells have an absolute requirem ent for anadequate 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


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Isolating Microorganisms from Nature

Microorganisms can be is olated from their natural environments by a variety of techniques. If microbial populations are frequent, dense orlarge enough, they can be sampled directIy with a sterile swab or loop and inoculated into a suitable liquid medium or streaked out on anagar plate. This applies es pecially to medical samples in which organisms are present in large numbers and in localised areas .Environmental sam ples with large m icrobial populations, e.g. soil, may also be added directly to a suitable medium. Wheremicroorganism s are infrequent, a pre-enrichment stop is neces sary This is achievd by filtering the samp les and incubating the filters in asuitable medium . Filtration is commonly applied to liquid sam ples (e.g. river and sea water) and when sam pling air. In-situ sam pling ofenvironmental samples i nvolves techniques s uch as the buried slide or buried capillary methods, in which microscope s lides or capillarytubes coated with a suitable m edium are buried i n the natural environment (soil or s ediment) and only retrieved after a certain period oftime. The slides or capillaries are then added to fresh media and the organism s s ub-cultured. These methods are applied whenorganism s are s low 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 microbio logica l culture methods ill ustrated.

Microbiological Culture Methods II


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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 bas is o f their characteristics". This involvesstudying all the physiological characters of bacteria using a series of biochem ical and culture tests such as : the variety of organiccompounds degraded, the requirement for various vitamins o r 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 taxonomicclass ification makes no reference to the evolutionary relationship between the bacterial strains. Kits containing many of these tests are nowcommercially available facilitating the identification of several groups of bacteria.

Chemical Taxonomy

Here, the grouping of individuals is carried out depending on a s et of characters presumed to be inherited from a com mon ances tor. In thecase of chemical taxonomy, bacteria are clus tered according to the chemical sim ilarity between s tructural componen ts of the bacteria. Themos t commonly used m aterials are proteins, which are molecules that are well preserved during evolution. To establish com mon ances try,chemotaxonomists com monly analyse the prim ary structure of enzymes, peptidogylcan, the cytoplasm ic mem brane and its fatty acidcompos ition, the outer membrane and the end products of metabolism.

Molecular Taxonomy

This is the comparis on of the genetic sequences of chromosomal DNA or ribosomal RNA to establish sim ilarity patterns and thephylogenetic 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 cons tant within a g iven species. The G+C content can therefore be us ed to establishtaxonomic relationships .

Similarities between the sequences of 16S or 23S ribosomal RNA are also compared in order to s tudy the phylogeny of a bacterial group.

Antimicrobial Sensitivity Testing

The antimicrobial activity of a compound is usually determined by measuring the lowes t concentration of the compound which is needed toinhibit growth of the test m icroorganism (MlC-minimum inhibitory concentration). The tests rely on the diffusion of the antibiotic through themicrobial medium to inhibit the growth of the sus ceptible organism growing in it or on it. The zones of inhibition are taken to berepresentative of the s usceptibility of the mi crobe. Antibiotic sensitivity has been used for many years as a characteristic for class ificationand identification.

Anaerobic Growth

The cultivation of strict anaerobic bacteria poses a special problem because these bacteria may be killed by exposure to air. Diss olvedoxygen in the m edium forms toxic free radicals and hydrogen peroxide in the pres ence of metabolic electrons. Obligate anaerobes areincapable of detoxifying these active forms of oxygen. To grow non stringent anaerobes on solid media, anaerobic jars are used togetherwith gas generating "Gas-Paks", which release both CO2 and H2. The hydrogen reacts with oxygen in the pres ence of a palladium catalystto produce water, thus removing oxygen from the jar. Completely anaerobic chambers equipped with air locks and filled with inert gasesused for the cultivation of strict (obligate) anaerobes are comm ercially available.

Redox Potential (O/R Potential)

This is the proportion of oxidized to reduced molecules in a medium : when oxygen dissolves in a m edium, for instance, the organiccompounds 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 m edium to be kept at a very low (negative)potential during growth. To achieve this, reducing agents are added to the media prior to autoclaving. Common ly used reducing agents aresodium thioglycolate (HS-CH2COONa) or sodium dithionite, which eas ily donate protons to other compounds. The relationship betweenredox potential, pH and m icrobial growth is illustrated below.


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Eh and pH ranges for microbial growth (adapted from Zajic 1969): the figure has been compiled from reports where both pH and Eh weregiven 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 andcorrected for the dilution to calculate the number of organism s in the original inoculum.

Most Probable Number Method

A statis tical method es timates the mos t probable num ber of bacteria pres ent in an inoculum which has been used to make a dilu tionseries . Several series are made with different initial volumes of inoculum; the results are recorded as a series of positives, i.e. growth in thetube, which can then be calculated to give an MPN. The result is the probable number of microorganism s that would be expected to yieldthis result.

Direct Microscope Observation

Specially constructed microscope s lides are used which have a shallow well of known volume and a grid etched into the glass. The well isfilled with the bacterial sus pension and the average number o f bacteria in each of the grid squares is determined and then multiplied by afactor to give the counts per millilitre. Selective s taining (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 m easuredvolume of liquid.

Turbidity (Optical Density)

The turbidity of a liquid medium increas es as bacteria multiply and can be m easured on a spectrophotometer. The amount of light reachingthe detector is inversely proportional to the number of bacteria under standardized conditions. The absorbency of the sample (opticaldensity) is dependent on the number of cells , their size and s hape, and is us ed to plot bacterial growth. If absorbency readings arematched with a direct count of the sam e culture, its protein content or dry mass, the correlation can be us ed in a future estimate of bacterialnumbers or biom ass based directly on turbidity measurements.

Metabolic Activity

Another indirect way to estim ate bacterial num bers i s to use the metabolic activity of the population. The amount of a metabo lic product ismeasured and assum ed to be proportional to the number of bacteria present. Examples of metabolic products include CO2 and organic

acids. Oxygen uptake can als o be m easured with a reduction test, for example the use of the methylene blue dye, which changes colourfrom blue in the presence of oxygen to colourless in its absence.

Preserving Bacter ial Cultures

Refrigeration

Can be us ed for short-term storage. Cultures s treaked on agar slants or s tab cultures m ay be viable over several months when s tored at4C. Plates have to be s ealed to prevent their tendency to dry out. To preserve cultures for longer periods of time, two methods arecommonly employed:

Deep Freezing

A pure culture of bacteria is sus pended in a liquid and quick-frozen (often with liquid nitrogen) at temperatures between -50C and-95C.Sensitive microorganisms require the presence of glycerol (end concentration 15-20 %), which acts as an "antifreeze", or extra protein(skimm ed milk powder) to protect them. Cultures can be thawed and used up to several years later.


7/7



Lyophilization

A sus pens ion of bacteria is quickly frozen and the water removed by means of a high vacuum. The microbes s urvive in thes powderlikeresidue for several years and can be revived at any time by rehydration of the culture in a nutrient medium. Bacterial strains ordered fromstrain collections are usually delivered in this form.

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