Media for cultivation of bacteria. Type and mechanism of bacteria nutrition.

Types of bacterial respiration. The methods of creation of anaerobic conditions. Growth and multiplication of bacteria.

Metabolism refers to all the biochemical reactions that occur in a cell or organism. The study of bacterial metabolism focuses on the chemical diversity of substrate oxidations and dissimilation reactions (reactions by which substrate molecules are broken down), which normally function in bacteria to generate energy. Also within the scope of bacterial metabolism is the study of the uptake and utilization of the inorganic or organic compounds required for growth and maintenance of a cellular steady state (assimilation reactions). These respective exergonic (energy-yielding) and endergonic (energy-requiring) reactions are catalyzed within the living bacterial cell by integrated enzyme systems, the end result being self-replication of the cell. The capability of microbial cells to live, function, and replicate in an appropriate chemical milieu (such as a bacterial culture medium) and the chemical changes that result during this transformation constitute the scope of bacterial metabolism.

The bacterial cell is a highly specialized energy transformer. Chemical energy generated by substrate oxidations is conserved by formation of high-energy compounds such as adenosine diphosphate (ADP) and adenosine triphosphate (ATP) or compounds containing the thioester bond

O

║

(R –C ~ S – R), such as acetyl ~ S-coenzyme A

(acetyl ~ SCoA) or succinyl ~ SCoA. ADP and ATP represent adenosine monophosphate (AMP) plus one and two high-energy phosphates (AMP ~ P and AMP ~ P~ P, respectively); the energy is stored in these compounds as high-energy phosphate bonds. In the presence of proper enzyme systems, these compounds can be used as energy sources to synthesize the new complex organic compounds needed by the cell. All living cells must maintain steady-state biochemical reactions for the formation and use of such high-energy compounds.

From a nutritional, or metabolic, viewpoint, three major physiologic types of bacteria exist: the heterotrophs (or chemoorganotrophs), the autotrophs (or chemolithotrophs), and the photosynthetic bacteria (or phototrophs) (Table 1). These are discussed below.

Heterotrophic Metabolism

Heterotrophic bacteria, which include all pathogens, obtain energy from oxidation of organic compounds. Carbohydrates (particularly glucose), lipids, and protein are the most commonly oxidized compounds. Biologic oxidation of these organic compounds by bacteria results in synthesis of ATP as the chemical energy source. This process also permits generation of simpler organic compounds (precursor molecules) needed by the bacteria cell for biosynthetic or assimilatory reactions.

Table 1. Nutritional Diversity Exhibited Physiologycally Different Bacteria

Required Components for Bacterial Growth
Physiologic Type	Carbon Source	Nitrogen Source^a Source^b	Energy Source	Hydrogen Source
Heterotrophic (chemoorganotrophic)	Organic	Organic or inorganic	Oxidation of organic compounds	–
Autotrophic ^achemolithotrophic)	CO2	Inorganic	Oxidation of inorganic compounds	–
Photosynthetic Photolithotrophic^b (Bacteria)	CO2	Inorganic	Sunlight	H2S or H2
Cyanobacteria	CO2	Inorganic	Sunlight	Photolysis of H2O^c
Photoorganotrophic (Bacteria)	CO2	Inorganic	Sunlight	Organic compounds^d

^a Common inorganic nitrogen sources are NO₃^- or NH₄⁺ ions; nitrogen fixers can use N₂;

^b Many prototrophs and chemotrophs are nitrogen-fixing organisms;

^c Results in O₂ evolution (or oxygenic photosynthesis) as commonly occurs in plants;

^d Organic acids such as formate, acetate, and succinate can serve as hydrigen donors.

The Krebs cycle intermediate compounds serve as precursor molecules (building blocks) for the energy-requiring biosynthesis of complex organic compounds in bacteria. Degradation reactions that simultaneously produce energy and generate precursor molecules for the biosynthesis of new cellular constituents are called amphibolic.

All heterotrophic bacteria require preformed organic compounds. These carbon- and nitrogen-containing compounds are growth substrates, which are used aerobically or anaerobically to generate reducing equivalents (e.g., reduced nicotinamide adenine dinucleotide; NADH + H⁺); these reducing equivalents in turn are chemical energy sources for all biologic oxidative and fermentative systems. Heterotrophs are the most commonly studied bacteria; they grow readily in media containing carbohydrates, proteins, or other complex nutrients such as blood. Also, growth media may be enriched by the addition of other naturally occurring compounds such as milk (to study lactic acid bacteria) or hydrocarbons (to study hydrocarbon-oxidizing organisms).

Respiration

Glucose is the most common substrate used for studying heterotrophic metabolism. Most aerobic organisms oxidize glucose completely by the following reaction equation:

C₆ H₁₂O₆ + 6O₂ ® 6CO₂ + 6H₂O + energy

This equation expresses the cellular oxidation process called respiration. Respiration occurs within the cells of plants and animals, normally generating 38 ATP molecules (as energy) from the oxidation of 1 molecule of glucose. This yields approximately 380,000 calories (cal) per mode of glucose (ATP ~ 10,000 cal/mole). Thermodynamically, the complete oxidation of one mole of glucose should yield approximately 688,000 cal; the energy that is not conserved biologically as chemical energy (or ATP formation) is liberated as heat (308,000 cal). Thus, the cellular respiratory process is at best about 55% efficient.

Glucose oxidation is the most commonly studied dissimilatory reaction leading to energy production or ATP synthesis. The complete oxidation of glucose may involve three fundamental biochemical pathways. The first is the glycolytic or Embden- Meyerhof-Parnas pathway, the second is the Krebs cycle (also called the citric acid cycle or tricarboxylic acid cycle), and the third is the series of membrane-bound electron transport oxidations coupled to oxidative phosphorylation.

Respiration takes place when any organic compound (usually carbohydrate) is oxidized completely to CO₂ and H₂O. In aerobic respiration, molecular O₂ serves as the terminal acceptor of electrons. For anaerobic respiration, NO₃-, SO₄^2-, CO₂, or fumarate can serve as terminal electron acceptors (rather than 0₂), depending on the bacterium studied. The end result of the respiratory process is the complete oxidation of the organic substrate molecule, and the end products formed are primarily CO₂ and H₂O. Ammonia is formed also if protein (or amino acid) is the substrate oxidized.

Metabolically, bacteria are unlike cyanobacteria (blue-green algae) and eukaryotes in that glucose oxidation may occur by more than one pathway. In bacteria, glycolysis represents one of several pathways by which bacteria can catabolically attack glucose. The glycolytic pathway is most commonly associated with anaerobic or fermentative metabolism in bacteria and yeasts. In bacteria, other minor heterofermentative pathways, such as the phosphoketolase pathway, also exist.

Fermentation

Fermentation, another example of heterotrophic metabolism, requires an organic compound as a terminal electron (or hydrogen) acceptor. In fermentations, simple organic end products are formed from the anaerobic dissimilation of glucose (or some other compound). Energy (ATP) is generated through the dehydrogenation reactions that occur as glucose is broken down enzymatically. The simple organic end products formed from this incomplete biologic oxidation process also serve as final electron and hydrogen acceptors. On reduction, these organic end products are secreted into the medium as waste metabolites (usually alcohol or acid). The organic substrate compounds are incompletely oxidized by bacteria, yet yield sufficient energy for microbial growth. Glucose is the most common hexose used to study fermentation reactions.

For most microbial fermentations, glucose dissimilation occurs through the glycolytic pathway. The simple organic compound most commonly generated is pyruvate, or a compound derived enzymatically from pyruvate, such as acetaldehyde, a-acetolactate, acetyl ~ SCoA, or lactyl ~ SCoA. Acetaldehyde can then be reduced by NADH + H+ to ethanol, which is excreted by the cell. The end product of lactic acid fermentation, which occurs in streptococci (e.g., Streptococcus lactis) and many lactobacilli (e.g., Lactobacillus casei, L pentosus), is a single organic acid, lactic acid. Organisms that ferment glucose to multiple end products, such as acetic acid, ethanol, formic acid, and CO₂, are referred to as heterofermenters. Examples of heterofermentative bacteria include Lactobacillus, Leuconostoc, and Microbacterium species. Heterofermentative fermentations are more common among bacteria, as in the mixed-acid fermentations carried out by bacteria of the family Enterobacteriaceae (e.g., Escherichia coli, Salmonella, Shigella, and Proteus species). Many of these glucose fermenters usually produce CO₂ and H₂ with different combinations of acid end products (formate, acetate, lactate, and succinate). Many obligately anaerobic clostridia (e.g., Clostridium saccharobutyricum, C. thermosaccha-rolyticum) and Butyribacterium species ferment glucose with the production of butyrate, acetate, CO₂, and H₂, whereas other Clostridum species (C. acetobutylicum and C. butyricum) also form these fermentation end products plus others (butanol, acetone, isopropanol, formate, and ethanol).

Electron Transport and Oxidative Phosphorylation

The final stage of respiration occurs through a series of oxidation-reduction electron transfer reactions that yield the energy to drive oxidative phosphorylation; this in turn produces ATP. The enzymes involved in electron transport and oxidative phosphorylation reside on the bacterial inner (cytoplasmic) membrane. This membrane is invaginated to form structures called respiratory vesicles, lamellar vesicles, or mesosomes, which function as the bacterial equivalent of the eukaryotic mitochondrial membrane.

Respiratory electron transport chains vary greatly among bacteria, and in some organisms are absent. The respiratory electron transport chain of eukaryotic mitochondria oxidizes NADH + H+, NADPH + H+, and succinate (as well as the coacylated fatty acids such as acetyl~SCoA). The bacterial electron transport chain also oxidizes these compounds, but it can also directly oxidize, via non-pyridine nucleotide-dependent pathways, a larger variety of reduced substrates such as lactate, malate, formate, a-glycerophosphate, H₂, and glutamate. The respiratory electron carriers in bacterial electron transport systems are more varied than in eukaryotes, and the chain is usually branched at the site(s) reacting with molecular O₂. Some electron carriers, such as nonheme iron centers and ubiquinone (coenzyme Q), are common to both the bacterial and mammalian respiratory electron transport chains. In some bacteria, the naphthoquinones or vitamin K may be found with ubiquinone. In still other bacteria, vitamin K serves in the absence of ubiquinone. In mitochondrial respiration, only one cytochrome oxidase component is found (cytochrome a + a3 oxidase). In bacteria there are multiple cytochrome oxidases, including cytochromes a, d, o, and occasionally a + a3 .

In bacteria cytochrome oxidases usually occur as combinations of a1: d: o and a + a3: o. Bacteria also possess mixed-function oxidases such as cytochromes P-450 and P-420 and cytochromes c' and c'c', which also react with carbon monoxide. These diverse types of oxygen-reactive cytochromes undoubtedly have evolutionary significance. Bacteria were present before O₂ was formed; when O₂ became available as a metabolite, bacteria evolved to use it in different ways; this probably accounts for the diversity in bacterial oxygen-reactive hemoproteins.

Cytochrome oxidases in many pathogenic bacteria are studied by the bacterial oxidase reaction, which subdivides Gram-negative organisms into two major groups, oxidase positive and oxidase negative. This oxidase reaction is assayed for by using N,N,N', N'-tetramethyl-p-phenylenediamine oxidation (to Wurster's blue) or by using indophenol blue synthesis (with dimethyl-p-phenylenediamine and a-naphthol). Oxidase-positive bacteria contain integrated (cytochrome c type:oxidase) complexes, the oxidase component most frequently encountered is cytochrome o, and occasionally a + a3. The cytochrome oxidase responsible for the indophenol oxidase reaction complex was isolated from membranes of Azotobacter vinelandii, a bacterium with the highest respiratory rate of any known cell. The cytochrome oxidase was found to be an integrated cytochrome c4:o complex, which was shown to be present in Bacillus species. These Bacillus strains are also highly oxidase positive, and most are found in morphologic group II.

Autotrophy

Bacteria that grow solely at the expense of inorganic compounds (mineral ions), without using sunlight as an energy source, are called autotrophs, chemotrophs, chemoautotrophs, or chemolithotrophs. Like photosynthetic organisms, all autotrophs use CO₂ as a carbon source for growth; their nitrogen comes from inorganic compounds such as NH₃, NO₃^-, or N₂ (Table 4-1). Interestingly, the energy source for such organisms is the oxidation of specific inorganic compounds. Which inorganic compound is oxidized depends on the bacteria in question. Many autotrophs will not grow on media that contain organic matter, even agar.

Also found among the autotrophic microorganisms are the sulfur-oxidizing or sulfur-compound-oxidizing bacteria, which seldom exhibit a strictly autotrophic mode of metabolism like the obligate nitrifying bacteria (see discussion of nitrogen cycle below). The representative sulfur compounds oxidized by such bacteria are H₂S, S₂, and S₂O₃. Among the sulfur bacteria are two very interesting organisms; Thiobacillus ferrooxidans, which gets its energy for autotrophic growth by oxidizing elemental sulfur or ferrous iron, and T denitrificans, which gets its energy by oxidizing S₂O₃ anaerobically, using NO₃^- as the sole terminal electron acceptor. T denitrificans reduces NO₃ to molecular N₂, which is liberated as a gas; this biologic process is called denitrification.

All autotrophic bacteria must assimilate CO₂, which is reduced to glucose from which organic cellular matter is synthesized. The energy for this biosynthetic process is derived from the oxidation of inorganic compounds discussed in the previous paragraph. Note that all autotrophic and phototrophic bacteria possess essentially the same organic cellular constituents found in heterotrophic bacteria; from a nutritional viewpoint, however, the autotrophic mode of metabolism is unique, occurring only in bacteria.

Anerobic Respiration

Some bacteria exhibit a unique mode of respiration called anaerobic respiration. These heterotrophic bacteria that will not grow anaerobically unless a specific chemical component, which serves as a terminal electron acceptor, is added to the medium. Among these electron acceptors are NO₃, SO₄², the organic compound fumarate, and CO₂. Bacteria requiring one of these compounds for anaerobic growth are said to be anaerobic respirers.

A large group of anaerobic respirers are the nitrate reducers. The nitrate reducers are predominantly heterotrophic bacteria that possess a complex electron transport system(s) allowing the NO3 ion to serve anaerobically as a terminal acceptor of electrons (NO₃ 2e- NO₂; NO₃ 5e- N₂; or NO₃ 8e- NH₃). The nitrate reductase activity is common in bacteria and is routinely used in the simple nitrate reductase test to identify bacteria (see Bergey's Manual of Deterininative Bacteriology, 8th ed.).

The methanogens are among the most anaerobic bacteria known, being very sensitive to small concentrations of molecular O₂. They are also archaebacteria, which typically live in unusual and deleterious environments.

All of the above anaerobic respirers obtain chemical energy for growth by using these anaerobic energy-yielding oxidation reactions.

The Nitrogen Cycle

Nowhere can the total metabolic potential of bacteria and their diverse chemical-transforming capabilities be more fully appreciated than in the geochemical cycling of the element nitrogen. All the basic chemical elements (S, O, P, C, and H) required to sustain living organisms have geochemical cycles similar to the nitrogen cycle.

The nitrogen cycle is an ideal demonstration of the ecologic interdependence of bacteria, plants, and animals. Nitrogen is recycled when organisms use one form of nitrogen for growth and excrete another nitrogenous compound as a waste product. This waste product is in turn utilized by another type of organism as a growth or energy substrate.

The other important biologic processes in the nitrogen cycle include nitrification (the conversion of NH₃ to NO₃ by autotrophes in the soil; denitrification (the anaerobic conversion of NO₃ to N₂ gas) carried out by many heterotrophs); and nitrogen fixation (N₂ to NH₃, and cell protein). The latter is a very specialized prokaryotic process called diazotrophy, carried out by both free-living bacteria (such as Azotobacter, Derxia, Beijeringeia, and Azomona species) and symbionts (such as Rhizobium species) in conjunction with legume plants (such as soybeans, peas, clover, and bluebonnets). All plant life relies heavily on NO3- as a nitrogen source, and most animal life relies on plant life for nutrients.

Nutrition and Growth of Bacteria

Every organism must find in its environment all of the substances required for energy generation and cellular biosynthesis. The chemicals and elements of this environment that are utilized for bacterial growth are referred to as nutrients or nutritional requirements. In the laboratory, bacteria are grown in culture media which are designed to provide all the essential nutrients in solution for bacterial growth.

At an elementary level, the nutritional requirements of a bacterium such as E. coli are revealed by the cell's elemental composition, which consists of C, H, O, N, S. P, K, Mg, Fe, Ca, Mn, and traces of Zn, Co, Cu, and Mo. These elements are found in the form of water, inorganic ions, small molecules, and macromolecules which serve either a structural or functional role in the cells. The general physiological functions of the elements are outlined in the Table 2.

Table 2. Major elements, their sources and functions in bacterial cells.

Element	% of dry weight	Source	Function
Carbon	50	Organic compounds or CO2	Main constituent of cellular material
Oxygen	20	H2O, organic compounds, CO2, and O2	Constituent of cell material and cell water; O2 is electron acceptor in aerobic respiration
Nitrogen	14	NH3, NO3, organic compounds, N2	Constituent of amino acids, nucleic acids nucleotides, and coenzymes
Hydrogen	8	H2O, organic compounds, H2	Main constituent of organic compounds and cell water
Phosphorus	3	inorganic phosphates (PO4)	Constituent of nucleic acids, nucleotides, phospholipids, LPS, teichoic acids
Sulfur	1	SO4, H2S, SO, organic sulfur compounds	Constituent of cysteine, methionine, glutathione, several coenzymes
Potassium	1	Potassium salts	Main cellular inorganic cation and cofactor for certain enzymes
Magnesium	0.5	Magnesium salts	Inorganic cellular cation, cofactor for certain enzymatic reactions
Calcium	0.5	Calcium salts	Inorganic cellular cation, cofactor for certain enzymes and a component of endospores
Iron	0.2	Iron salts	Component of cytochromes and certain nonheme iron-proteins and a cofactor for some enzymatic reactions

The above table ignores the occurrence of trace elements in bacterial nutrition. Trace elements are metal ions required by certain cells in such small amounts that it is difficult to detect (measure) them, and it is not necessary to add them to culture media as nutrients. Trace elements are required in such small amounts that they are present as "contaminants" of the water or other media components. As metal ions, the trace elements usually act as cofactors for essential enzymatic reactions in the cell. One organism's trace element may be another's required element and vice-versa, but the usual cations that qualify as trace elements in bacterial nutrition are Mn, Co, Zn, Cu, and Mo.

In order to grow in nature or in the laboratory, a bacterium must have an energy source, a source of carbon and other required nutrients, and a permissive range of physical conditions such as O₂ concentration, temperature, and pH. Sometimes bacteria are referred to as individuals or groups based on their patterns of growth under various chemical (nutritional) or physical conditions. For example, phototrophs are organisms that use light as an energy source; anaerobes are organisms that grow without oxygen; thermophiles are organisms that grow at high temperatures.

Carbon and Energy Sources for Bacterial Growth

All living organisms require a source of energy. Organisms that use radiant energy (light) are called phototrophs. Organisms that use (oxidize) an organic form of carbon are called heterotrophs or chemo(hetero)trophs. Organisms that oxidize inorganic compounds are called lithotrophs.

The carbon requirements of organisms must be met by organic carbon (a chemical compound with a carbon-hydrogen bond) or by CO₂. Organisms that use organic carbon are heterotrophs and organisms that use CO₂ as a sole source of carbon for growth are called autotrophs.

Thus, on the basis of carbon and energy sources for growth four major nutritional types of procaryotes may be defined (Table 3).

Table 3. Major nutritional types of procaryotes

Nutritional Type	Energy Source	Carbon Source	Examples
Photoautotrophs	Light	CO2	Cyanobacteria, some Purple and Green Bacteria
Photoheterotrophs	Light	Organic compounds	Some Purple and Green Bacteria
Chemoautotrophs or Lithotrophs (Lithoautotrophs)	Inorganic compounds, e.g. H2, NH3, NO 2, H 2S	CO2	A few Bacteria and many Archaea
Chemoheterotrophs or Heterotrophs	Organic compounds	Organic compounds	Most Bacteria, some Archaea

Almost all eukaryotes are either photoautotrophic (e.g. plants and algae) or heterotrophic (e.g. animals, protozoa, fungi). Lithotrophy is unique to procaryotes and photoheterotrophy, common in the purple and green Bacteria, occurs only in a very few eukaryotic algae. Phototrophy has not been found in the Archaea.

This simplified scheme for use of carbon, either organic carbon or CO2, ignores the possibility that an organism, whether it is an autotroph or a heterotroph, may require small amounts of certain organic compounds for growth because they are essential substances that the organism is unable to synthesize from available nutrients. Such compounds are called growth factors.

Growth factors are required in small amounts by cells because they fulfill specific roles in biosynthesis. The need for a growth factor results from either a blocked or missing metabolic pathway in the cells. Growth factors are organized into three categories:

1. Purines and pyrimidines: required for synthesis of nucleic acids (DNA and RNA);

2. Amino acids: required for the synthesis of proteins;

3. Vitamins: needed as coenzymes and functional groups of certain enzymes.

Some bacteria (e.g E. coli) do not require any growth factors: they can synthesize all essential purines, pyrimidines, amino acids and vitamins, starting with their carbon source, as part of their own intermediary metabolism. Certain other bacteria (e.g. Lactobacillus) require purines, pyrimidines, vitamins and several amino acids in order to grow. These compounds must be added in advance to culture media that are used to grow these bacteria. The growth factors are not metabolized directly as sources of carbon or energy, rather they are assimilated by cells to fulfill their specific role in metabolism. Mutant strains of bacteria that require some growth factor not needed by the wild type (parent) strain are referred to as auxotrophs. Thus, a strain of E. coli that requires the amino acid tryptophan in order to grow would be called a tryptophan auxotroph and would be designated E. coli trp-.

Some vitamins that are frequently required by certain bacteria as growth factors are listed in Table 4. The function(s) of these vitamins in essential enzymatic reactions gives a clue why, if the cell cannot make the vitamin, it must be provided exogenously in order for growth to occur.

Table 4. Common vitamins required in the nutrition of certain procaryotes

Vitamin	Coenzyme form	Function
p-Aminobenzoic acid (PABA)		Precursor for the biosynthesis of folic acid
Folic acid	Tetrahydrofolate	Transfer of one-carbon units and required for synthesis of thymine, purine bases, serine, methionine and pantothenate
Biotin	Biotin	Biosynthetic reactions that require CO2 fixation
Lipoic acid	Lipoamide	Transfer of acyl groups in oxidation of keto acids
Mercaptoethane-sulfonic acid	Coenzyme M	CH4 production by methanogens
Nicotinic acid	NAD (nicotinamide adenine dinucleotide) and NADP	Electron carrier in dehydrogenation reactions
Pantothenic acid	Coenzyme A and the Acyl Carrier Protein (ACP)	Oxidation of keto acids and acyl group carriers in metabolism
Pyridoxine (B6)	Pyridoxal phosphate	Transamination, deamination, decarboxylation and racemation of amino acids
Riboflavin (B2)	FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide)	Oxidoreduction reactions
Thiamine (B1)	Thiamine pyrophosphate (TPP)	Decarboxylation of keto acids and transaminase reactions
Vitamin B12	Cobalamine coupled to adenine nucleoside	Transfer of methyl groups
Vitamin K	Quinones and napthoquinones	Electron transport processes

Culture Media for the Growth of Bacteria

For any bacterium to be propagated for any purpose it is necessary to provide the appropriate biochemical and biophysical environment. The biochemical (nutritional) environment is made available as a culture medium, and depending upon the special needs of particular bacteria (as well as particular investigators) a large variety and types of culture media have been developed with different purposes and uses. Culture media are employed in the isolation and maintenance of pure cultures of bacteria and are also used for identification of bacteria according to their biochemical and physiological properties. Nutrient media should be easily assimilable, and they should contain a known amount of nitrogen and carbohydrate substances, vitamins, a required salt concentration. In addition they should be isotonic, and sterile, and they should have buffer properties, an optimal viscosity, and a certain oxidation-reduction potential.

The manner in which bacteria are cultivated, and the purpose of culture media, vary widely. Liquid media are used for growth of pure batch cultures while solidified media are used widely for the isolation of pure cultures, for estimating viable bacterial populations, and a variety of other purposes. The usual gelling agent for solid or semisolid medium is agar, a hydrocolloid derived from red algae. Agar is used because of its unique physical properties (it melts at 100 degrees and remains liquid until cooled to 40 degrees, the temperature at which it gels) and because it cannot be metabolized by most bacteria. Hence as a medium component it is relatively inert; it simply holds (gels) nutrients that are in aquaeous solution.

Culture media may be classified into several categories depending on their composition or use. A chemically-defined (synthetic) medium is one in which the exact chemical composition is known.

Defined media are usually composed of pure biochemicals off the shelf; complex media usually contain complex materials of biological origin such as blood or milk or yeast extract or beef extract, the exact chemical composition of which is obviously undetermined. A defined medium is a minimal medium if it provides only the exact nutrients (including any growth factors) needed by the organism for growth. The use of defined minimal media requires the investigator to know the exact nutritional requirements of the organisms in question. Chemically-defined media are of value in studying the minimal nutritional requirements of microorganisms, for enrichment cultures, and for a wide variety of physiological studies. Complex media usually provide the full range of growth factors that may be required by an organism so they may be more handily used to cultivate unknown bacteria or bacteria whose nutritional requirement are complex (i.e., organisms that require a lot of growth factors).

Most pathogenic bacteria of animals, which have adapted themselves to growth in animal tissues, require complex media for their growth. Blood, serum and tissue extracts are frequently added to culture media for the cultivation of pathogens. Even so, for a few fastidious pathogens such as Treponema pallidum, the agent of syphilis, and Mycobacterium leprae, the cause of leprosy, artificial culture media and conditions have not been established. This fact thwarts the the ability to do basic research on these pathogens and the diseases that they cause.

Other concepts employed in the construction of culture media are the principles of selection and enrichment. A selective medium is one which has a component(s) added to it which will inhibit or prevent the growth of certain types or species of bacteria and/or promote the growth of desired species. One can also adjust the physical conditions of a culture medium, such as pH and temperature, to render it selective for organisms that are able to grow under these certain conditions.

A culture medium may also be a differential medium if allows the investigator to distinguish between different types of bacteria based on some observable trait in their pattern of growth on the medium. Thus a selective, differential medium for the isolation of Staphylococcus aureus, the most common bacterial pathogen of humans, contains a very high concentration of salt (which the staph will tolerate) that inhibits most other bacteria, mannitol as a source of fermentable sugar, and a pH indicator dye. From clinical specimens, only staphylococcus will grow. S. aureus is differentiated from S. epidermidis (a nonpathogenic component of the normal flora) on the basis of its ability to ferment mannitol. Mannitol-fermenting colonies (S. aureus) produce acid which reacts with the indicator dye forming a colored halo around the colonies; mannitol non-fermenters (S. epidermidis) use other non-fermentative substrates in the medium for growth and do not form a halo around their colonies.

An enrichment medium employs a slightly different twist. An enrichment medium contains some component that permits the growth of specific types or species of bacteria, usually because they alone can utilize the component from their environment. However, an enrichment medium may have selective features. An enrichment medium for nonsymbiotic nitrogen-fixing bacteria omits a source of added nitrogen to the medium. The medium is inoculated with a potential source of these bacteria (e.g. a soil sample) and incubated in the atmosphere wherein the only source of nitrogen available is N₂. A selective enrichment medium for growth of the extreme halophile (Halococcus) contains nearly 25 percent salt [NaCl], which is required by the extreme halophile and which inhibits the growth of all other procaryotes.

Thus, nutrient media can be subdivided into three main groups:

I. Ordinary (simple) media which include meat-peptone broth, meat-peptone agar, etc.

II. Special media (serum agar, serum broth, coagulated serum, potatoes, blood agar, blood broth, etc.).

Quite often elective media are employed in laboratory practice in which only certain species of bacteria grow well, and other species either grow poorly or do not grow at all. Enriched media are also employed in which the species of interest to the scientist grows more intensively and more rapidly than the accompanying bacteria. Thus, for example, on Endo's medium (elective) the growth of the Gram-positive microbes is inhibited while alkaline peptone water and alkaline meat-peptone agar serve as enriched media for the cholera vibrio. Nutrient media containing certain concentrations of penicillin are elective for penicillin-resistant strains of bacteria, but unfavourable for penicillin-sensitive strains.

III. Differential diagnostic media: (1) media for the determination of the proteolytic action of microbes (meat-peptone gelatine); (2) media for the determination of the fermentation of carbohydrates (Hiss media); media for the differentiation of bacteria which do and do not ferment lactose (Ploskirev, Drigalsky, Endo. etc.); (3) media for the determination of haemolytic activity (blood agar); (4) media for the determination of the reductive activity of micro-organisms; (5) media containing substances assimilated only by certain microbes.

Besides, in laboratory practice conservation media are used. They are used for primary seeding and transportation of the material under test. They prevent the death of pathogenic microbes and enhance the inhibition of saprophytes. This group of media includes a glycerin mixture composed of two parts 0.85 per cent salt solution, 1 part glycerin, and I part 15-20 per cent acid sodium phosphate, and also a glycerin preservative with lithium salts, a hypertonic salt solution, etc.

At present many nutrient media are prepared commercially as dry powders. They are convenient to work with, are stable, and quite effective.

Non-protein media are widely used for the cultivation of bacteria, on which many heterotrophic microbes including pathogenic species grow well. The composition of these media is complex and includes a large number of components.

When cultivating in synthetic media, the use of the method of radioactive tracers has permitted a more detailed differentiation of microbes according to the character of their biosynthesis.

Selective media are widely used for differentiating prototrophic and ULixotrophic bacteria. Prototrophs grow on a minimum medium which contains only salts and carbohydrates since they themselves are capable til' synthesizing the metaholites necessary for their development. Auxo-Irophs. in distinction, require definite media containing amino acids, vitamins, and other substances.

In consistency nutrient media may be solid (meat-peptone agar, meat-peptone gelatine, coagulated serum, potato, coagulated white of Ihc egg), semisolid (0.5 per cent meat-peptone agar), and liquid (peptone water, meat-peptone broth, sugar broth, etc.).

Physical and Environmental Requirements for Microbial Growth

The procaryotes exist in nature under an enormous range of physical conditions such as O₂ concentration, Hydrogen ion concentration (pH) and temperature. The exclusion limits of life on the planet, with regard to environmental parameters, are always set by some microorganism, most often a procaryote, and frequently an Archaeon. Applied to all microorganisms is a vocabulary of terms used to describe their growth (ability to grow) within a range of physical conditions. A thermophile grows at high temperatures, an acidophile grows at low pH, an osmophile grows at high solute concentration, and so on. This nomenclature will be employed in this section to describe the response of the procaryotes to a variety of physical conditions.

The Effect of Oxygen. Oxygen is a universal component of cells and is always provided in large amounts by H₂O. However, procaryotes display a wide range of responses to molecular oxygen O₂ (Table 6).

Table 6. Terms used to describe O₂ Relations of Microorganisms

Group	Environment		O2 Effect
	Aerobic	Anaerobic
Obligate Aerobe	Growth	No growth	Required (utilized for aerobic respiration)
Microaerophile	Growth if level not too high	No growth	Required but at levels below 0.2 atm
Obligate Anaerobe	No growth	Growth Toxic
Facultative Anaerobe (Facultative Aerobe)	Growth	Growth	Not required for growth but utilized when available
Aerotolerant Anaerobe	Growth	Growth	Not required and not utilized

Obligate aerobes require O₂ for growth; they use O₂ as a final electron acceptor in aerobic respiration.

Obligate anaerobes (occasionally called aerophobes) do not need or use O₂ as a nutrient. In fact, O₂ is a toxic substance, which either kills or inhibits their growth. Obligate anaerobic procaryotes may live by fermentation, anaerobic respiration, bacterial photosynthesis, or the novel process of methanogenesis.

Facultative anaerobes (or facultative aerobes) are organisms that can switch between aerobic and anaerobic types of metabolism. Under anaerobic conditions (no O₂) they grow by fermentation or anaerobic respiration, but in the presence of O₂ they switch to aerobic respiration.

Aerotolerant anaerobes are bacteria with an exclusively anaerobic (fermentative) type of metabolism but they are insensitive to the presence of O₂. They live by fermentation alone whether or not O₂ is present in their environment.

The response of an organism to O₂ in its environment depends upon the occurrence and distribution of various enzymes which react with O₂ and various oxygen radicals that are invariably generated by cells in the presence of O2. All cells contain enzymes capable of reacting with O₂. For example, oxidations of flavoproteins by O₂ invariably result in the formation of H₂0₂ (peroxide) as one major product and small quantities of an even more toxic free radical, superoxide or O₂. Also, chlorophyll and other pigments in cells can react with O₂ in the presence of light and generate singlet oxygen, another radical form of oxygen which is a potent oxidizing agent in biological systems.

In aerobes and aerotolerant anaerobes the potential for lethal accumulation of superoxide is prevented by the enzyme superoxide dismutase (Table 7).

Table 7. Distribution of superoxide dismutase, catalase and peroxidase in procaryotes

with different O₂ tolerances

Group	Superoxide dismutase	Catalase	Peroxidase
Obligate aerobes and most facultative anaerobes (e.g. Enterics)	+	+	-
Most aerotolerant anaerobes (e.g. Streptococci)	+	-	+
Obligate anaerobes (e.g. Clostridia, Methanogens, Bacteroides)	-	-	-

All organisms which can live in the presence of O₂ (whether or not they utilize it in their metabolism) contain superoxide dismutase. Nearly all organisms contain the enzyme catalase, which decomposes H₂O₂. Even though certain aerotolerant bacteria such as the lactic acid bacteria lack catalase, they decompose H₂O₂ by means of peroxidase enzymes which derive electrons from NADH₂ to reduce peroxide to H₂O. Obligate anaerobes lack superoxide dismutase and catalase and/or peroxidase, and therefore undergo lethal oxidations by various oxygen radicals when they are exposed to O₂.

All photosynthetic (and some nonphotosynthetic) organisms are protected from lethal oxidations of singlet oxygen by their possession of carotenoid pigments which physically react with the singlet oxygen radical and lower it to its nontoxic "ground" (triplet) state. Carotenoids are said to "quench" singlet oxygen radicals.

The Effect of pH on Growth. The pH, or hydrogen ion concentration, [H+], of natural environments varies from about 0.5 in the most acidic soils to about 10.5 in the most alkaline lakes (Table 8).

Table 8. Minimum, maximum and optimum pH for growth of certain procaryotes

Organism	Minimum pH	Optimum pH	Maximum pH
Lactobacillus acidophilus	4.0-4.6	5.8-6.6	6.8
Staphylococcus aureus	4.2	7.0-7.5	9.3
Escherichia coli	4.4	6.0-7.0	9.0
Clostridium sporogenes	5.0-5.8	6.0-7.6	8.5-9.0
Erwinia caratovora	5.6	7.1	9.3
Pseudomonas aeruginosa	5.6	6.6-7.0	8.0
Streptococcus pneumoniae	6.5	7.8	8.3
Nitrobacter spp	6.6	7.6-8.6	10.0

Appreciating that pH is measured on a logarithmic scale, the [H+] of natural environments varies over a billion-fold and some microorganisms are living at the extremes, as well as every point between the extremes! Most free-living procaryotes can grow over a range of 3 pH units, about a thousand fold change in [H+]. The range of pH over which an organism grows is defined by three cardinal points: the minimum pH, below which the organism cannot grow, the maximum pH, above which the organism cannot grow, and the optimum pH, at which the organism grows best. For most bacteria there is an orderly increase in growth rate between the minimum and the optimum and a corresponding orderly decrease in growth rate between the optimum and the maximum pH, reflecting the general effect of changing [H+] on the rates of enzymatic reaction.

Microorganisms which grow at an optimum pH well below neutrality (7.0) are called acidophiles. Those which grow best at neutral pH are called neutrophiles and those that grow best under alkaline conditions are called alkaliphiles. Obligate acidophiles, such as some Thiobacillus species, actually require a low pH for growth since their membranes dissolve and the cells lyse at neutrality. Several genera of Archaea, including Sulfolobus and Thermoplasma, are obligate acidophiles. Among eukaryotes, many fungi are acidophiles, and the champion of growth at low pH is the eukaryotic alga Cyanidium which can grow at a pH of 0.

In the construction and use of culture media, one must always consider the optimum pH for growth of a desired organism and incorporate buffers in order to maintain the pH of the medium in the changing milieu of bacterial waste products that accumulate during growth. Many pathogenic bacteria exhibit a relatively narrow range of pH over which they will grow. Most diagnostic media for the growth and identification of human pathogens have a pH near 7.

Enzymes and Their Role in Metabolism

Enzymes, organic catalysts of a highly molecular structure, are produced by the living cell. They are of a protein nature, are strictly specific in action, and play an important part in the metabolism of microorganisms. Their specificity is associated with active centres formed by a group of amino acids.

Enzymes of microbial origin have various effects and are highly active. They have found a wide application in industry, agriculture and medicine, and are gradually replacing preparations produced by higher plants and animals.

With the help of amylase produced by mould fungi starch is saccharified and this is employed in beer making, industrial alcohol production and bread making. Proteinases produced by microbes are used for removing the hair from hides, tanning hides, liquefying the gelatinous layer from films during regeneration, and for dry cleaning. Fibrinolysin produced by streptococci dissolves the thrombi in human blood vessels. Enzymes which hydrolyse cellulose aid in an easier assimilation of rough fodder.

Due to the application of microbial enzymes, the medical industry has been able to obtain alkaloids, polysaccharides, and steroids (hydrocortisone, prednisone, prednisolone. etc.).

Bacteria play an important role in the treatment of caoutchouc, collon. silk. coffee, cocoa, and tobacco: significant processes lake place under their effect which change these substances essentially in the needed direction. In specific weight the synthetic capacity of microorganisms is very high. The total weight of bacterial cytoplasm on earth is much higher than that of animal cytoplasm. The biochemical activity of microbes is of no less general biological importance than that of photosynthesis. The cessation of the existence of microorganisms would lead inevitably to the death of plants and animals.

Enzymes permit some species of micro-organisms to assimilate methane. butane, and other hydrocarbons, and to synthesize complex organic compounds from them. Thus, for example, with the help of the enzymatic ability of yeasts in special-type industrial installations protein-vitamin concentrates (PVC) can be obtained from waste products of petroleum (paraffins), which are employed in animal husbandry as a valuable nutrient substance supplementing rough fodder. Some soil micro-organisms destroy by means of enzymes chemical substances (carcinogens) which are detrimental to the human body because they induce malignant tumours.

Some enzymes are excreted by the cell into the environment (exoenzymes) for breaking down complex colloid nutrient materials while other enzymes are contained inside the cell (endoenzymes).

Depending on the conditions of origin of enzymes there are constitutive enzymes which are constantly found in the cell irrespective of the presence of a catalysing substrate. These include the main enzymes of cellular metabolism (lipase. carbohydrase. proteinase, oxydase, etc.). Adaptive enzymes occur only in the presence of the corresponding substrate (penicillinase, amino acid decarboxylase, alkaline phosphatase, B-galactosidase, etc.). The synthesis of induced enzymes in microbes occurs due to the presence in the cells of free amino acids and with the participation of ready proteins found in the bacteria.

According to chemical properties enzymes can be subdivided into three groups:

1 – enzymes composed only of proteins:

2 – enzymes containing in addition, to protein metallic ions essential for their activity, and assisting m the combination of the enzyme with the substrate, and taking part in the cyclic enzymatic transformations:

3 – enzymes which contain distinct organic molecules (coenzymes. prosthetic groups) essential for their activity. Some enzymes contain vitamins.

Bacterial enzymes are subdivided into some groups:

1. Hydrolases which catalyse the breakdown of the link between the carbon and nitrogen atoms, between the oxygen and sulphur atoms, binding one molecule of water (esterases. glucosidases, proteases. amidases, nucleases, etc.).

2. Transferases perform catalysis by transferring certain radicals from one molecule to another (transglucosidases, transacylases. transaminases).

3. Oxidative enzymes (oxyreductases) which catalyse the oxidation reduction processes (oxidases, dehydrogenases, peroxidases, catalases).

4. Isomerases and racemases play an important part in carbohydrate metabolism. They are found in most species of bacteria. Phosphohexoisomerase, galactovaldenase, phosphoglucomutase, hosphoglyceromutase pertain to the isomerases.

The absorption of food material by the cell is a rather complex process. Unicellular protozoa are characterized by a holozoic type of nutrition in which hard food particles are swallowed, digested and converted to soluble compounds. Bacteria, algae, fungi, and plants possess a holophytik type of nutrition. They absorb nutrients in a dissolved state. This difference, however, is not essential because the cells of protozoa, just like the cells of plant organisms, utilize nutrient substrates which are soluble in water or in the cell sap, while many bacteria and fungi can assimilate hard nutrients first splitting them by external digestion by means of exoenzymes. During diffusion the dissolved substance is transferred from the region of higher concentration outside the cell into the bacterial cell until the concentration becomes the same. The passage of a solvent through the cytoplasmic membrane of bacteria from a region where it is less concentrated to one where it is more concentrated is performed by osmosis. The concentration gradient and osmotic power on both sides of the cytoplasmic membrane are quite different, and depend on the difference in concentration of many substances contained in the cell and nutrient medium. The transfer of dissolved substances from the nutrient medium to the cell can take place by suction together with the solvent if the membrane is sufficiently porous.

It has been established that the cellular membranes are made up of lipid and protein molecules arranged in a certain sequence. The charged groups of molecules have their ends directed towards the surface of the membrane. On these charged ends the protein layers are adsorbed, composed of protein chains forming a meshwork on the external and internal surfaces of the membrane. The high selectivity which allows the cells to distinguish certain substances from others depends on the presence of enzymatic systems localized on the surface of bacterial cells. Due to the action of these enzymes, the insoluble substances in the membrane become soluble.

The cell membranes play an important role in metabolism. They are capable of changing rapidly their permeability to various substances and regulating in this way the entry of substances into the cell and their distribution in it, and the development of reactions in which these substances participate.

Some bacteria (Salmonella typhimurium} possess rudiments of memory. They recognize whether the medium is favourable or unfavourable to them. They 'run away' from an unfavourable one by means of flagella: when close to a favourable medium (glucose) Salmonella organisms swim to the 'bait'. This ability to recognize the needed direction is probably accomplished by the trial-and-error method.

In the process of bacterial nutrition great importance is attached to exchange adsorption. The active transport of ions takes place due to (he difference in charges on the surface of membranes in the cell wall and the surrounding medium of the micro-organisms. Besides, the role of transporters, as has been suggested, is performed by liposoluble substances X and Y. Compounds are formed with ions of potassium and sodium (KX and NaY) which are capable of diffusing through the cell wall, while the membrane remains unpenetrable for free transporters. Proteins concerned with the transport of amino acids have been isolated from the membranes of some micro-organisms, and protein systems responsible for the transfer of certain sugars in general and glucose in particular have been revealed.

Practical Use of the Fermentative Properties of Microbes

The widespread and theoretically founded application of microbiological processes in the technology of industries involving fermentation, treatment of flax, hides, farming, and canning of many food products became possible only in the second half of the 19th century. From the vital requirements of a vigorously developing industry, especially of the agricultural produce processing industry, there arose a need for a profound study of biochemical processes. The investigations by Pasteur in this field were prepared to a great extent by the development of industry, organic chemistry, and other sciences.

Microorganisms take part in the cycle of nitrogen (putrefaction), carbon (fermentation), sulphur, phosphorus, iron, and other elements which are important in the vital activity of organisms. Therapeutic muds and brine were produced as the result of the fermentative activity of definite microbial species. Micro-organisms are used as indicators for determining hydrolytic processes in seas and oceans, the soil requirements of fertilizers, and the exact amount of vitamins, amino acids and other substances which cannot be determined by chemical analytical methods. Certain species of microorganisms synthesize antibiotics, enzymes, hormones, vitamins, and amino acids which are industrially prepared and used in medicine, veterinary practice, and agriculture. The synthesis of proteins by means of special species of yeasts has been mastered.

Some soil bacteria are capable of rendering harmless (destroying) certain pesticides used in agriculture as well as chemical carcinogens. Hydrogenous bacteria may be used to produce fodder protein by cultivation on urea or ammonium sulphate. Some bacterial species are used for the control of methane in mines. Methanol, a monocarbon alcohol, is produced from methane by means of microbes.

Of great importance in medical microbiology is the utilization of the specific fermentative capacity of pathogenic bacteria for the determination of their species properties. Many bacteria ferment carbohydrates producing acid or acid and gas, while proteins are fermented with the production of indole, ammonia, hydrogen sulphide, etc.

Fermentative properties of microbes are used in the laboratory diagnosis of infectious diseases, and in studying microbes of the soil, water, and air.

Influence of Environmental Factors on Microbes Effect of Physical Factors

The effect of temperature. Microbes can withstand low temperatures fairly well. The cholera vibrio does not lose its viability at a temperature of -32°C. Some species of bacteria remain viable at a temperature of liquid air ( 190°C) and of liquid hydrogen (- 253°C). Diphtheria bacilli are able to withstand freezing for three months and enteric fever bacteria are able to live long in ice. Bacillus spores withstand a temperature of –253°C for 3 days. Many microorganisms remain viable at low temperatures, and viruses are especially resistant to low temperatures. Thus, for example, the virus of Japanese encephalitis in a 10 per cent brain suspension does not lose its pathogenicity at -70°C over a period of one year, the causative agents of influenza and trachoma at -70 C for 6 months and Coxsackie virus at —WC for 1.5 years. Low temperatures halt putrefying and fermentative processes. In sanitary-hygienic practice ice, cellars, and refrigerators for the storage of food products arc used according to this principle.

Only certain species of pathogenic bacteria are very sensitive to low temperatures (e. g. meningococcus, gonococcus, etc.). During short periods of cooling these species perish quite rapidly. This is taken into account in laboratory diagnosis, and materials under test for the presence of meningitis or gonorrhoea are conveyed to the laboratory protected from cold.

At low temperatures the processes of metabolism are inhibited, the bacteria die off as a result of ageing and starvation, and the cells are destroyed under the effect of the formation of ice crystals during freezing. Alternate high and low temperatures are lethal to microbes. It has been established, for instance, that sudden freezing as well as sudden healing causes a decrease in the life activities of pathogenic microbes.

Most asporogenic bacteria perish at a temperature of 58-60 ⁰C within 30-60 minutes. Bacillus spores are more resistant than vegetative cells. They withstand boiling from a few minutes to 3 hours, but perish under the effect of dry heat at 160-170°C in 1.0-1.5 hours. Heating at 120.6°C at 2 aim steam pressure kills them within 20-30 minutes, Individual and specific variations in the resistance of microbes to high temperatures have different limits and a rather large temperature range.

The inhibition of the activity of catalase. oxydase, dehydrogenase, protein denaturation, and an interruption of the osmotic barrier are the principles of the bacterial action of high temperatures. High temperatures cause a rather rapid destruction of viruses, but some of them (viruses of infectious hepatitis. poliomyelitis, etc.) are resistant to environmental factors. They remain viable long in water, in the faeces of sick people or carriers, and are resistant to heat at 60°C and to small concentrations of chlorine in water.

The Effect of Temperature on Growth. Microorganisms have been found growing in virtually all environments where there is liquid water, regardless of its temperature. In 1966, Professor Thomas D. Brock at Indiana University, made the amazing discovery in boiling hot springs of Yellowstone National Park that bacteria were not just surviving there, they were growing and flourishing. Boiling temperature could not inactivate any essential enzyme. Subsequently, procaryotes have been detected growing around black smokers and hydrothermal vents in the deep sea at temperatures at least as high as 115 degrees. Microorganisms have been found growing at very low temperatures as well. In supercooled solutions of H2O as low as -20 degrees, certain organisms can extract water for growth, and many forms of life flourish in the icy waters of the Antarctic, as well as household refrigerators, near 0 degrees.

A particular microorganism will exhibit a range of temperature over which it can grow, defined by three cardinal points in the same manner as pH. Considering the total span of temperature where liquid water exists, the procaryotes may be subdivided into several subclasses on the basis of one or another of their cardinal points for growth. For example, organisms with an optimum temperature near 37 degrees (the body temperature of warm-blooded animals) are called mesophiles (Table 9).

Table 9. Terms used to describe microorganisms in relation to temperature requirements for growth

Group	Minimum	Optimum	Maximum	Comments
Psychrophile	Below 0	10-15	Below 20	Grow best at relatively low T
Psychrotroph	0	15-30	Above 25	Able to grow at low T but prefer moderate T
Mesophile	10-15	30-40	Below 45	Most bacteria esp. those living in association with warm-blooded animals
Thermophile	45	50-85	Above 100 (boiling)	Among all thermophiles is wide variation in optimum and maximum T

Organisms with an optimum T between about 45 degrees and 70 degrees are thermophiles. Some Archaea with an optimum T of 80 degrees or higher and a maximum T as high as 115 degrees, are now referred to as extreme thermophiles or hyperthermophiles. The cold-loving organisms are psychrophiles defined by their ability to grow at 0 degrees. A variant of a psychrophile (which usually has an optimum T of 10-15 degrees) is a psychrotroph, which grows at 0 degrees but displays an optimum T in the mesophile range, nearer room temperature. Psychrotrophs are the scourge of food storage in refrigerators since they are invariably brought in from their mesophilic habitats and continue to grow in the refrigerated environment where they spoil the food. Of course, they grow slower at 2 degrees than at 25 degrees. Think how fast milk spoils on the counter top versus in the refrigerator.

Psychrophilic bacteria are adapted to their cool environment by having largely unsaturated fatty acids in their plasma membranes. Some psychrophiles, particularly those from the Antarctic have been found to contain polyunsaturated fatty acids, which generally do not occur in procaryotes. The degree of unsaturation of a fatty acid correlates with its solidification T or thermal transition stage (i.e., the temperature at which the lipid melts or solidifies); unsaturated fatty acids remain liquid at low T but are also denatured at moderate T; saturated fatty acids, as in the membranes of thermophilic bacteria, are stable at high temperatures, but they also solidify at relatively high T. Thus, saturated fatty acids (like butter) are solid at room temperature while unsaturated fatty acids (like canola oil) remain liquid in the refrigerator. Whether fatty acids in a membrane are in a liquid or a solid phase affects the fluidity of the membrane, which directly affects its ability to function. Psychrophiles also have enzymes that continue to function, albeit at a reduced rate, at temperatures at or near 0 degrees. Usually, psychrophile proteins and/or membranes, which adapt them to low temperatures, do not function at the body temperatures of warm-blooded animals (37 degrees) so that they are unable to grow at even moderate temperatures.

Thermophiles are adapted to temperatures above 60 degrees in a variety of ways. Often thermophiles have a high G + C content in their DNA such that the melting point of the DNA (the temperature at which the strands of the double helix separate) is at least as high as the organism's maximum T for growth. But this is not always the case, and the correlation is far from perfect, so thermophile DNA must be stabilized in these cells by other means. The membrane fatty acids of thermophilic bacteria are highly saturated allowing their membranes to remain stable and functional at high temperatures. The membranes of hyperthermophiles, virtually all of which are Archaea, are not composed of fatty acids but of repeating subunits of the C5 compound, phytane, a branched, saturated, "isoprenoid" substance, which contributes heavily to the ability of these bacteria to live in superheated environments. The structural proteins (e.g. ribosomal proteins, transport proteins (permeases) and enzymes of thermophiles and hyperthermophiles are very heat stable compared with their mesophilic counterparts. The proteins are modified in a number of ways including dehydration and through slight changes in their primary structure, which accounts for their thermal stability.

The effect of desiccation

Micro-organisms have a different resistance to desiccation to which gonococci, meningococci. treponemas, leptospiras. haemoglobinophilic bacteria, and phages are sensitive. On exposure to desiccation the cholera vibrio persists for 2 days. dysentery bacteria — for 7, plague — for 8. diphtheria — for 30, enteric fever — for 70, staphylococci and tubercle bacilli — for 90 days. The dry sputum of tuberculosis patients remains infectious for 10 months, the spores of anthrax bacillus remain viable for 10 years, and those of moulds for 20 years.

Desiccation is accompanied with dehydration of the cytoplasm and denaturation of bacterial proteins. Sublimation is one of the methods used for the preservation of food. It comprises dehydration at low temperature and high vacuum, which is attended with evaporation of water and rapid cooling and freezing. The ice formed in the food is easily sublimated, by-passing the liquid phase. The food may be stored for more than two years. In drying by sublimation all the sugars, vitamins, enzymes, and other components are preserved. Desiccation in a vacuum at a low temperature does not kill bacteria, rickettsiae. or viruses. This method of preserving cultures is employed in the manufacture of stable long-storage, live vaccines against tuberculosis, plague, tularaemia. brucellosis, smallpox, influenza, and other diseases.

Quick freezing of bacterial and viral suspensions at very low temperatures provokes conditions at which crystals do not form, and subsequent disruption of the micro-organisms does not occur.

The effect of light. Some bacteria (purple) withstand the effect of light fairly well. while others are injured. Direct sunlight has the greatest bactericidal action.

Investigations have established that different kinds of light have a bactericidal or sterilizing effect. These include ultraviolet rays (electromagnetic waves with a wave length of 200-300 nm). X-rays (electromagnetic rays with a wave length of 0.005-2.0 nm), gamma-rays (short wave X-rays), beta- particles or cathode rays (high speed electrons). alpha-particles (high speed helium nuclei) and neutrons.

The experiments in which short waves were used for the disinfection of wards, infectious material, for the conservation of products, the preparation of vaccines, for treating operating rooms and maternity wards. etc., have demonstrated that they have a rather high bactericidal effect. Viruses are very quickly inactivated under the effect of ultraviolet rays with a wave length of 260-300 nm. These waves are absorbed by the nucleic acid of viruses. Longer waves are weaker and do not render vi- ruses harmless.

Viruses in comparison to bacteria are less resistant to X-rays, and gamma-rays. Beta-rays are more markedly viricidal. Alpha-, beta-, and gamma-rays in small doses enhance multiplication but in large doses they are lethal to microbes. Viruses which are pathogenic to animals are inactivated by 44000-280000 roentgens. Thiobacteria which live in uranium ore deposits are highly resistant to radioactive rays. Bacteria were found in the water of atomic reactors at ionizing radiation concentration of 2-3 million rads.

Ionizing radiation can be used for practical purposes in sterilizing food products, and this method of cold sterilization has a number of advantages. The quality of the product is not changed as during heat sterilization which causes denaturation of its component parts (proteins, polysaccharides, vitamins). Radiation sterilization can be applied in the practice of treating biological preparations (vaccines, sera. phages, etc.).

Of interest is the phenomenon of photoreactivation described in 1949 by A. Kelner. If a suspension of bacteria is preliminarily exposed to visible light radiation, it becomes more resistant to ultraviolet radiation. If after exposure to strong ultraviolet light a suspension of colibacilli is irradiated with visible light, marked growth of the bacteria is observed when they are seeded on nutrient media.

The effect of high pressure and mechanical injury on microbes. Bacteria withstand easily atmospheric pressure. They do not noticeably alter at pressure from 100 to 900 aim at marine and oceanic depths of 1000-10000 m. Yeasts retain their viability at a pressure of 500 aim. Some bacteria, yeasts, and moulds withstand a pressure of 3000 aim and phytopathogenic viruses withstand 5000 aim.

The movement of liquid media has a harmful effect on microbes. The movement of water in rivers and streams, undulations in stagnant waters are factors important in self-purification of reservoirs from microbes.

Ultrasonic oscillation (waves with a frequency of about 20000 hertz per second) has bactericidal properties. At present this is used for the sterilization of food products, for the preparation of vaccines, and the disinfection of various objects.

The mechanism of the bactericidal action of ultrasonic oscillation is that in the cytoplasm of bacteria found in an aquatic medium a cavity is formed which is Filled with liquid vapours. A pressure of 10000 atmospheres occurs in the bubble, which leads to disintegration of the cytoplasmic structures. It is possible that highly reactive hydroxyl radicals originate in the cavities formed in the sonified water medium.

Of certain significance in rendering the air harmless is aeroionization. The negatively charged ions have a more lethal effect on the microbes.

Effect of Chemical Factors

Depending on the physicochemical composition of the medium, concentration, the length of contact and temperature chemical substances have a different effect on microbes. In small doses they act as stimulants, in bactericidal concentrations they paralyse the dehydrogenase activity of bacteria.

According to their effect on bacteria, bactericidal chemical substances can be subdivided into surface-active substances, dyes, phenols and their derivatives, salts of heavy metals, oxidizing agents, and the formaldehyde group.

Surface-active substances change the energy ratio. Bacterial cells lose their negative charge and acquire a positive charge which impairs the normal function of the cytoplasmic membrane.

Bactericidal substances with surface-active action include fatty acids and soaps which harm only the cell wall and do not penetrate into the cell.

Phenol, cresol, and related derivatives first of all injure the cell wall and then the cell proteins. Some substances of this group inhibit the function of the coenzyme (diphosphopiridine nucleotide) which participates in the dehydrogenation of glucose and lactic acid. Dyes are able to inhibit the growth of bacteria. The basis of this action is the marked affinity for the phosphoric acid groups of nucleoproteins. Dyes with bactericidal properties include brilliant green. rivanol, tripaflavine, acriflavine, etc.

Salts of heavy metals (lead, copper, fine, silver, mercury) cause coagulation of the cell proteins. When the salts of the heavy metal interact with the protein a metallic albuminate and a free acid are produced.

A whole series of metals (silver, gold, copper, zinc, tin. lead, etc.) have an oligodynamic action (bactericidal capacity). Thus, for example. silverware, silver-plated objects, silver-plated sand in contact with water render the metal bactericidal to many species of bacteria. The mechanism of the oligodynamic action is that the positively charged metallic ions arc adsorbed on the negatively charged bacterial surface. and alter the permeability of the cytoplasmic membrane. It is possible that during this process the nutrition and reproduction of bacteria are disturbed. Viruses also are quite sensitive to the salts of heavy metals under the influence of which they become irreversibly inactivated.

Oxidizing agents act on the sulphohydryl groups of active proteins. More powerful oxidizing agents are harmful also to other groups (phenol, thioelhyl, indole. amine).

Oxidizing agents include chlorine which impairs dehydrogenases, hydrolases. amylases and proteinases of bacteria and which is widely used in decontaminating water, and chloride of lime and chloramine used as disinfectants. In medicine iodine is used successfully as an anti-microbial substance in the form of iodine tincture which not only oxidizes the active groups of the proteins of bacterial cytoplasm, but brings about their denaturation. Potassium permanganate, hydrogen peroxide, and other substances also have oxidizing properties.

Many species of viruses are resistant to the action of ether, chloroform, ethyl and methyl alcohol, and volatile oils. Almost all viruses survive for long periods in the presence of whole or 50 per cent glycerin solution, in Ringer's and Tyrode's solutions. Viruses are destroyed by sodium hydroxide, potassium hydroxide, chloramine, chloride of lime, chlorine, and other oxidizing agents.

Formaldehyde is used as a 40 per cent solution known as formalin. Its antimicrobial action can be explained, as presumed, by its being united to the amino groups of proteins which causes their denaturation. Formaldehyde kills both the vegetative forms as well as the spores. It is applied for decontaminating diphtheria and tetanus toxins as a result of which they are transformed into antitoxins Some viruses (phages, tobacco mosaic virus) inactivated by formalin can sometimes renew their infectivity.

Fabrics possessing an antimicrobial effect have been produced, in which the molecules of the antibacterial substance are bound to the molecules of the material. The fabrics retain the bactericidal properties for a long period of time even after being washed repeatedly. They may be used for making clothes for sick persons, medical personnel, pharmaceutists, for the personnel of establishments of the food industry and for making filters for sterilizing water and air.

Effect of Biological Factors

In nature microorganisms constitute a component of the biocoenosis (a community of plants and animals living in a part of the habitat with more or less homogenous conditions of life).

Microbes are found in nature in associations among which there is a constant struggle for existence. Certain species which adapted themselves to a given medium have more marked antagonistic properties in relation to other species which have fallen into a new habitat. Thus, for example, lactic acid bacteria are antagonistic in relation to the causative agents of dysentery, plague, etc. Blue-pus bacteria inhibit the growth of dysentery, enteric fever microbes, anthrax bacilli, cholera vibrio, causative agents of plague, glanders, and staphylococci. meningococci. etc. The normal inhabitants of the human body (e. g. Colibacilli. enterococci, lactobacilli, microflora of the skin and nasopharynx, etc.) have especially potent antagonistic properties.

For many years a controversy raged on the possibility of intra-species antagonism among microbes. At the present time many scientists have established the antagonistic relationships not only between virulent and non-virulent strains of the same species. These properties are found in certain strains of colibacilli. Streptococcus pneumoniae, enteric fever, and dysentery bacteria, staphylococci. etc.

In certain conditions antagonistic properties appear in microbes due to a lack of nutrients, as a result of which some microbes are forced to feed at the expense of others. This phenomenon was named forced antagonism by I. Schiller. Antagonistic relations have been established by viruses when one virus protects the organism from penetration by another virus. In virology this has been called viral interference.

Among various groups of microbes there are several types of relationships: symbiosis, metabiosis, satellism, synergism, and antagonism. Symbiosis represents an intimate mutually beneficial relation of organisms of different species. They develop together better than separately. Sometimes the adaptation of two organisms becomes so profound that they lose their ability to exist separately (symbiosis of the fungus and blue-green algae, nitrogen-fixing bacteria and cellulose-decomposing bacteria, symbiosis of nodule bacteria with legume plants, various fungi with the roots of plants, yeast-like fungi and lamblias).

Metabiosis is that type of relationship in which one organism continues the process caused by another organism, liberating it from the products of life activities, and thus creating conditions for its further development (nitrifying and ammonizing bacteria).

During satellism one of the symbionts known as the favourable microbe incites the growth of the other (some yeasts and sarcinae producing amino acids, vitamins, etc., enhance the growth of microbes more strict in relation to nutrient media).

Synergism is characterized by the increase in the physiological functions of the microbial association (yeasts, lactobacilli, fusobacteria, and Borrelia organisms).

One of the forms of symbiosis is a virus-carrying form ~ a communal existence of some bacteria and protozoa with viruses (lysogenic bacteria retain the corresponding phages for long periods in their cells, during chronic tonsillitis besides the α-haemolytic streptococci, the adenoviruses take part in the infectious process, etc.).

During antagonistic relationships there is a struggle for oxygen, nutrients, and a habitat. Modern understanding of the problems of microbiology unfolds complex relationships among organisms and the essence of biological laws.

Biological factors have received widespread application in treating many infectious diseases with the products of the life activities of bacteria, fungi, higher plants, and animal tissues known as antibiotics. These effective drug preparations include penicillin, streptomycin. chloramphenicol. tetracycline, and many others.

In decontaminating the environment from pathogenic microorganisms by antagonism an important role is played by phages widespread in the soil and water and by phytoncides, volatile substances of many plants.

The influence of the environment is taken into account by the physician in combating harmful micro-organisms (sterilization, disinfection), vectors of causative agents of infectious disease (disinsection) and rodents — reservoirs of pathogenic microorganisms (deratization).

Forms of Symbiosis. According to the character of interrelationship with the plant and animal world, microbes can be subdivided into two groups: saprophytes and parasites. Saprophytes include micro-organisms unable to cause disease.

Parasites are microbes which live at the expense of plant and animal bodies. All kinds of associations of the macro-organisms and microorganisms constitute symbiosis in its broadest sense. Symbiosis has different forms: commensalism, mutualism and parasitism.

Commensalisin is a kind of symbiosis (association) of organisms in which one of them lives at the expense of the other without causing it any harm. The overwhelming majority of representatives of the normal microflora of the human body belong to microbe-commensals.

Mutualism is that kind of symbiosis in which both organisms concerned receive mutual benefit from their association. For example, the symbiosis of nodule bacteria with legume plants is characterized by typical mutualism. Nodule bacteria live in plant roots, while the legumes for their nutrition utilize nitrogenous compounds produced by the bacteria from atmospheric nitrogen A commonly encountered mutualism are the numerous lichens (the Arctic reindeer lichen and many others) which are formed of green or blue-green algae and Ascomycetes or Basidiomycetes fungus By means of photosynthesis, the algae provide themselves and the fungus with nutrition, while the fungus protects the algae, supplies them with water and mineral salts Some species of bacteria from the group of intestinal microflora live in symbiosis with animal organisms which they inhabit These microbe- mutualists feed on food remains which enter the lower part of the intestine, while the vitamins which they produce are used by the animals for biocatalytic reactions.

Parasitism is that state of symbiosis in which one organism (parasite) lives at the expense of another (host) and is harmful to it Many microbe-parasites are capable of causing infectious diseases in plants and animals.

Disease-producing species of micro-organisms are known as pathogenic organisms They have adapted themselves in the process of evolutionary development to a parasitic type of nutrition in tissues and fluids of the animal body The susceptible infected organism responds to the entry of the pathogenic microbe by non-specific and specific biological reactions. These are expressed in atypical or typical manifestations of the disease, and also in a variety of defense adaptations.

At one time J. Henle and then R. Koch (1878. 1882) formulated three conditions in the presence of which the given microbe can be recognized as a causative agent of a disease. Henle-Koch's triad consists in the following: (1) the microbe-causative agent should be discovered in all cases during a given disease, and is found neither in healthy persons nor in patients with other diseases; (2) the microbe-causative agent should be isolated from the patient's body in a pure culture: (3) the pure culture of the isolated microbe should cause the same disease in susceptible animals. At present this triad has lost its significance to a considerable degree.

For the origination and development of the infectious process three conditions are necessary. (I) the presence of d pathogenic microbe, (2) its penetration into a susceptible macro-organism, and (3) certain environrnental conditions in which the interaction between the micro-organism and macro-organism takes place.

The interrelationship of the pathogenic micro-organism and the susceptible macro-organism takes place under the complex conditions of the parasite coenosis, that is. in various relationships with other microbes and protozoa

The results of the penetration of pathogenic microbes into the human body depend not only on the reactivity of the macro-organism, but on the normal microflora of the human body), which can express itself antagonistically as well as synergistically

Besides pathogenic organisms, there is a comparatively large group of micro-organisms known as conditionally pathogenic micro-organisms living on the skin, in the intestine, in the respiratory tract and urogenital organs.

Pasteur discovered many of the basic principles of microbiology and, along with R. Koch, laid the foundation for the science of microbiology. In 1857 Napoleon III was having trouble with his sailors mutinying because their wine was spoiling after only a few weeks at sea. Naturally Napoleon was distraught because his hopes for world conquest were being scuttled (pardon the pun) over a little spoiled wine, so he begged Pasteur for help. Pasteur, armed with his trusty microscope, accepted the challenge and soon recognized that by looking at the spoiled wines he could distinguish between the contaminants that caused the spoilage and even predict the taste of the wine solely from his microscopic observations. He then reasoned that if one were to heat the wine to a point where its flavor was unaffected, but the harmful microbes were killed it wouldn't spoil. As we are aware this process, today known as pasteurization, worked exactly the way he predicted and is the foundation of the modern treatment of bottled liquids to prevent their spoilage. It is important to realize that pasteurization is NOT the same as sterilization. Pasteurization only kills organisms that may spoil the product, but it allows many microbes to survive, whereas sterilization kills all the living organisms in the treated material.

Pasteur also realized that the yeast that was present in all the wine produced the alcohol in wine. When he announced this, a number of famous scientists were enraged, because the current theory of wine production was that wine formation was the result of spontaneous chemical changes that occurred in the grape juice. Pasteur was attacked furiously at scientific meetings, to the point where certain scientists did humorous skits about Pasteur and his tiny little yeast "stills" turning out alcohol. Pasteur had the last laugh however as people all over the world soon realized that if he was right they could control the quality of wine by controlling the yeast that made it. In a short period many others verified his observations and the opposition sank without a sound.

Sterilization of instruments, needles, syringes, etc., is carried out by boiling. Dressings, glassware, and salt solutions are treated in autoclaves.

Pasteurization is widely employed to render milk harmless by heating it at 63°C for 30 minutes or at 71.6-80°C for 15-30 seconds and then cooling it. Pasteurization is also used to prevent the development of harmful microbes which turn wine, beer, and fruit juices sour; it does not destroy vitamins and does not deprive the beverages of their flavour.

DRY HEAT

Incineration. This is an excellent procedure for disposing of materials such as soiled dressings, used paper mouth wipes, sputum cups, and garbage. One must remember that if such articles are infectious, they should be thoroughly wrapped in newspaper with additional paper or sawdust to absorb the excess moisture. Disposable plastic liners for waste containers are inexpensive and may be easily closed on top to prevent scattering of refuse. The wrapping protects persons who must empty the trash cans, and it assures that the objects do not escape the fire, but it may also protect the microorganisms if incineration is not complete.

Adequate instructions should be given to workers responsable for burning disposable materials to insure complete burning. For example, a sputum cup containing secretions from a patient who has active tuberculosis is filled with paper or sawdust to absorb excess moisture. The cup is then placed in a plastic bag with shredded absorbent paper to prevent spilling If it is burned only on the outside, a soggy mass of dangerous infective material is left on the inside. Other possibilities will occur to the imaginative student

Ovens. Ovens are often used for sterilizing dry materials such as glassware, syringes and needles, powders, and gauze dressings. Petrolatum and other oily substances must also be sterilized with dry heat in an oven because moist heat (steam) will not penetrate materials insoluble in water.

In order to insure sterility the materials in the oven must reach a temperature of 165 to 170 C (329 to 338 t), and this temperature must be maintained for 120 or 90 minutes, respectively. This destroys all microorganisms, including spores. However, the oven must be maintained at that temperature for the entire time. This means that the oven door must remain closed during the sterilizing time opening the door will cool the articles below effective temperatures so that sterilization cannot be assured. Also, hot glassware will shatter immediately) in contact with cool air It is usual practice in a microbiology laboratory to let an oven cool completely before it is opened

It is practical to load the oven with glassware, pipettes, and so forth in the afternoon or evening and turn it on. In the morning, the oven is turned off and by lunchtime it is unloaded. This routine assures sterile glassware, once the setting of the temperature is regulated. A home oven, set at 330 t (model ate temperature), can be used as well as an oven built for laboratory or hospital equipment. It is wise to check the temperature in the oven with an oven thermometer (available at household supply stores)

Items may be secured in brown wrapping paper with a sting, but never with a lubber band. Some types of plastics, like the one used in connecting hoses, are heat-stable in an oven, but most plastics cannot be sterilized in this way.

MOIST HEAT

Boiling Water. Boiling water can never be trusted for absolute sterilization procedures because its maximum temperature is 100 C (at sea level). As indicated previously, spores can resist this temperature Boiling water can generally be used for contaminated dishes, bedding, and bedpans: for these articles neither sterility nor the destruction of spores is necessary) except under very unusual circumstances. All that is desired is disinfection or sanitization. Exposure to boiling water kills all pathogenic microorganisms in 10 minutes 01 less, but not bacterial spores or hepatitis viruses. At altitudes over 5,000 feet the boiling time should be increased by 50 per cent or moiré because water there boils at temperatures of only about 95 C or below.

Live Steam. Live steam (free flowing) is used in the laboratory in the preparation of culture media or in the home for processing canned foods. It must be remembered that steam does not exceed the temperature of 100 C unless it is under pressure.

To use free flowing steam effectively for sterilization, the fractional method must be used fractional sterilization, or tyndalization, is a process of exposure of substances (usually liquids) to live steam for 30 minutes on each of three successive days, with incubation during the intervals. During the incubations, spores germinate into vulnerable vegetative forms that are killed during the heating periods. This is a time-consuming process and is not used in modern laboratories. The use of membrane (Millipore) filters or similar rapid methods makes the preparation of heat-sensitive sterile solutions much easier.

Compressed Steam. In older to sterilize with steam certainly and quickly, steam under pressure in the autoclave is used (fig. 1). An autoclave is essentially a metal chamber with a door that can be closed very tightly. The inner chamber allows all air to be replaced by steam until the contents reach a temperature far above that of boiling water or live steam. The temperature depends on the pressure, commonly expressed in pounds per square inch, often written as psi. Steam under pressure hydrates rapidly and therefore coagulates very efficiently. Also, it brings about chemical changes somewhat like digestion, called hydrolysis. These characteristics give it special advantages in sterilization.

. An autoclave

By first allowing all the an in the chamber of the autoclave to escape and be replaced by the incoming steam, the spaces in the interior of masses of material may be brought quickly into contact with the steam. The escape of air is absolute!) essential since sterilization depends on the water vapor. Whenever an is trapped in the autoclave, sterilization is inefficient. One must be sure that:

1 All the air is allowed to escape and is replaced by steam

2 The pressure of the steam reaches at least 1 5 pounds to the square inch (psi) and remains there (In most automatic autoclaves it is now 18 pounds, permitting sterilization to be accomplished in less time )

3. The thermometer reaches at least 121 C without downward fluctuation for 15 minutes (Less time is required when 18 pounds of pressure is used).

If these conditions are met and if the masses 01 bundles are well separated and not too large, the autoclaved material will be sterile

The actual amount of water present as steam in the pressure chamber is usually small, consequently, the articles sterilized are not wet with much condensed steam when they are removed from the autoclave. All modern autoclaves are arranged so that all the steam is removed by vacuum after the sterilization period, to prevent dampening the articles inside.

The automatic autoclave is used in many laboratories, has the following settings:

1 Manual—used when the electrical power is off I he operator must then set and time all cycles

2 Slow exhaust—used for a wet load, for media or water (for dilutions)

3 Fast exhaust—used for killing microorganisms quickly on and in glassware that is to be washed

4 Fast exhaust and dry—used for pipettes, Petri plates, or dressings, a so-called dry loud.

After closing the door tightly, the operator sets the autoclave control to the desired setting, to the time interval that is necessary for the maximum preset temperature and pressure, and to ON. Lights go on as the autoclaving moves from pretimed cycle to cycle finally a bell rings, and the operator turns the setting to OFF and opens the door carefully Asbestos gloves protect the hands, when hot sterile materials are unloaded, but watch the right elbow—the inside of the open door is very hot.

The exhaust trap inside the autoclave must always be cleaned before starting a load, since dirt in the trap may delay the time needed for the various cycles. It is best to do this when the autoclave is still cold.

Since the effectiveness of an autoclave is dependent upon the penetration of steam into all articles and substances, the preparation of packs of dressings is very important, and the correct placement of articles in the autoclave is essential to adequate sterilization

Substitution of an autoclave for an oven by admitting steam only to the jacket and keeping the chamber dry is not advisable when sterilization is necessary because the temperature thus achieved (100 C) does not kill spores. The dryness of such an atmosphere may actually preserve some pathogens that would be quickly killed in a moist atmosphere

Cleaning Instruments. When sterilizing solutions, the pressure must be allowed to fall gradually so that the solutions will not boil. If the pressure falls rapidly, violent boiling occurs. Advantage is taken of this fact in autoclaving used surgical instruments. They are immersed in water in a perforated tray. After autoclaving the pressure is reduced suddenly. The water boils violently and washes the instruments clean.

Cleaning by Ultrasonic Energy. Machines are now available for cleaning surgical instruments, syringes, and so on by extremely rapid (ultrasonic) vibrations. These can clean and dry hundreds of instruments (perfectly) every five to ten minutes. They do not sterilize.

Indicators. Many institutions always include some sort of indicator inside bundles being sterilized, such as dyes that change color when the necessary temperature has been maintained for the required time. On glassware and bundles, labels are placed that read Not sterile before autoclaving or after insufficient autoclaving but read STERILE if sterilization has been fully effective. Another device, similar in principle, is cellulose tape having on it a chemical indicator that changes color when properly heated in the autoclave. One car use wax pellets that melt only at the necessary temperature but may not indicate lapse of time. Strips of paper containing bacterial spores can be dropped into broth in culture tubes after the sterilizing procedure. If the sterilizer has been properly operated, these broth cultures should remain sterile, even after seven days of incubation, since all spores have been lulled 1 hrs method does not give immediate indication of faulty operation, but it does constitute an absolute and permanent record.

Most modern autoclaves have a self-recording thermometer that plots the temperature the instrument has reached and the time of sterilization required for each "load". A permanent record provided in this way often proves to be verve valuable.

STERILIZATION WITHOUT HEAT

For many years heat was the only dependable and practicable means of destroying bacterial endospores. Now, at least three other means of killing microorganisms are available. These are use of the gas ethylene oxide, the vapors of beta propiolactone (BFL), and certain electromagnetic radiations (especially electron beams or cathode rays). The method of ultrasonic vibrations, although quite effective in destroying certain microorganisms, is not a practical means of large scale sterilization. Besides, it produces a heating effect. At present, we can only dream of an ultrasonic "dishwasher" that sanitizes duty dishes, preferably without any water.

Ultraviolet Light. This is satisfactory for the sterilization of smooth sin faces and of air in operating looms, unfortunately, UV radiation has virtually no power of penetration. Mercury-vapor lamps emitting 90 per cent UV radiation at 254 nm are used to decrease airborne infection. Ultraviolet lamps are also used to suppress surface-growing molds and other organisms in meat packing houses, bakeries, storage warehouses, and laboratories Sunlight is a good, inexpensive source of ultraviolet rays, which can induce genetic mutations in microorganisms. In excess, it can cause burns and even cancer

X-Rays. X-rays penetrate well but require very high energy and are costly and inefficient for sterilizing. Their use is therefore mostly for medical and experimental work and the production of mutants of microorganisms for genetic studies

Neutrons. Neutrons are very effective in killing microorganisms but are expensive and hard to control, and they involve dangerous radioactivity

Alpha Rays (Particles). Alpha rays are effective bactericides but have almost no power of penetration

Beta Rays (Particles). Beta rays have a slightly greater power of penetration than alpha lays but are still not practical for use in sterilization

Gamma Rays. These rays are high-energy radiations now mostly emitted from radioactive isotopes such as cobalt-60 or cesium-137, which are readily available by-products of atomic fission Gamma rays resemble x-rays in many respects. The U S Army Quartermaster Corps has used gamma rays and other radiations to sterilize food for military use X rays or gamma rays must be applied in 2 mrad (one mrad is 1/1 000 of a rad; a rad is 100 ergs of absorbed energy per gram of absorbing material) to 4 mrad doses to become a reliable sterilizing treatment of food. Foods exposed to effective radiation sterilization, however, undergo changes in color, chemical composition, taste, and sometimes even odor These problems are only gradually being overcome by temperature control and oxygen removal

Cathode Rays (Electrons). These are used mainly to kill microorganisms on sin faces of foods, fomites, and industrial articles. Since electrons have limited powers of penetration, they are at present not very useful for surgical sterilization. However, as a result of research on proper dosage and packaging, cathode lays are being developed for genial purposes such as food processing. This may completely revolutionize the food canning and frozen food industries as well as surgical sterilization techniques.

Pharmaceutical and medical products are adequately sterilized by treatment with a radiation dose of 2,5 mrad . The Association of the British Pharmaceutical Industry has reported that benzylpenicillin, streptomycin sulfate, and other antibiotics are satisfactorily sterilized by this method In addition, package radiation at dose levels of 2,5 mrad has become common procedure for the sterilization of disposable Petri plates, pipettes, syringes, needles, rubber gloves, tubing, and so on

Sterilization with Chemicals

Ethylene Oxide. This is a gas with the formula CH₂CH₂O. It is applied in special autoclaves under carefully controlled conditions of temperature and humidity Since pure ethylene oxide is explosive and irritating, it is generally mixed with carbon dioxide or another diluent in various proportions 10 per cent ethylene oxide to 90 per cent carbon dioxide (sold as Carboxide), 20 per cent ethylene oxide to 80 per cent carbon dioxide (sold as Oxyfume), or 11 per cent ethylene oxide to 89 per cent halogenated hydrocarbons (sold as Cryoxcide and Benvicide). Each preparation is effective when properly used. Oxyfume is very rapid in action but is more inflammable and moiré toxic than Carboxide, however, Carboxide requires high pressure Cryoxcide is more toxic and more expensive, but it is more convenient and requires less pressure. Other mixtures of ethylene oxide (e.g. , with Freon) are also commercially available All are mote costly and time-consuming than autoclaving with steam

Ethylene oxide is generally measured in terms of milligrams of the pure gas per liter of space. For sterilization, concentrations of 450 to 1,000 mg of gas/liter are necessary. Concentrations of 500 mg of gas/liter are generally effective in about four hours at approximately 1% F (58 C) and a relative humidity of about 40 per cent. Variations in any one of these factors require adjustments of the others For example, if the concentration of gas is increased to 1,000 mg/liter, the time may be reduced to two hours. Increases in temperature, up to a limit, also decrease the time required. At a relative humidity of 30 per cent, the action of ethylene oxide is about 10 times as rapid as at 95 per cent. The use of ethylene oxide, although as simple as autoclaving, generally requires special instructions (provided by the manufacturers) for each particular situation. At present ethylene oxide is used largely by commercial companies that dispense sterile packages of a variety of products

In general, seven steps are invoked after loading and closing the sterilizing chamber:

1 Draw out nearly all air with a vacuum pump

2 Admit a measured amount of water vapor

3 Admit the requires amount of ethylene oxide gas mixture

4 Raise the temperature to the required degree

5 Hold for the required time, turn off the heat

6 Draw out the gas with the vacuum pump

7 Admit filtered and sterilized air to the chamber

A fully automatic ethylene oxide autoclave requiring only proper supervision is available.

Beta-propiolactone (BPL). At about 20 C this substance is a colorless liquid It has a sweet but very irritating odor It is unstable at room temperatures but may be refrigerated at 4 CG for months without deterioration. Aqueous solutions effectively inactivate some viruses, including those of poliomyelitis and rabies, and also kill bacteria and bacterial spores. The vapors, in concentrations of about 1,5 mg of lactone per liter of air with a high relative humidity (75 to 80 per cent), at about 25 C, kill spores in a few minutes. A decrease in temperature, humidity, or concentration of the lactone vapors increases the time required to kill spores

Beta-propiolactone is not inflammable under ordinary conditions of use. It is, however, very irritating and may cause blisters if allowed in contact with skin for more than a few minutes. It is not injurious to most materials. BPL appeal s to act by forming chemical compounds with cell proteins. The necessity for high humidities during its use and also its cost are disadvantages Its activity at loom temperatures is a distinct advantage BPL does not penetrate as well as ethylene oxide and is therefore more suitable for disinfecting surfaces (e g , looms, buildings, and furniture) by fumigation

Aqueous solutions of BPL can be used to sterilize biological materials such as virus vaccines, tissues for grafting, and plasma.

Sterilization by Filtration

Many fluids may be sterilized without the use of heat, chemicals, or radiations. This is accomplished mechanically by passing the fluids to be sterilized through very fine filters Only fluids of low viscosity that do not contain numerous fine particles in suspension (e.g., silt, erythrocytes), which would clog the filter pores, can be satisfactorily sterilized in this way The method is applicable to fluids that are destroyed by heat and cannot be sterilized in any other way, such as fluids and medications for hypodermic or intravenous use, as well as culture media, especially tissue culture media and their liquid components, e g , serum.

Several types of filters are in common use. The Seitz filter, consisting of a mounted asbestos pad, is one of the older filters used. Others consist of diatomaceous earth (the Berkefeld filter), unglazed porcelain (the Chamber-land-Pasteur filters), or sintered glass of several varieties. The Sterifil aseptic filtration system consists of a tubelike arrangement that sucks up fluid around all sides of the tube into a Teflon hose-connected receiving flask. The advantage of this is that the filter is very inexpensive and can be thrown away when it clogs up

A widely used and practical filter is the membrane, molecular, or Millipore filter. It is available in a great variety of pore sizes, ranging from 0,45 mcm for virus studies to 0,01 mcm. These filters consist of paper-thin, porous membranes of material resembling cellulose acetate (plastic) One common form of these special filters is shown in Figure 9—11 In general, the porcelain, clay, paper, or plastic filtering element is held in some supporting structure, and the fluid to be filtered is forced through the filter into a receptacle by a vacuum or by pressure The filter, support, and receptacle are assembled and autoclaved before use Further details concerning these procedures need not be given here, since sterilization by filtration is rarely used without adequate information pertaining to the specific filtration problem, which would describe the advantages of one type of filter over another.

Summary. The most complete way to dispose of infectious materials is incineration, although precautions must be taken to prevent spilling and to assure that everything is fully burned. Things that cannot be incinerated are sterilized to free them not only of pathogens but of all living organisms. For dry materials, glassware, syringes, dressings, filters, and pipettes, this may be done in a sterilizer oven at 165 °C (329 F) for 2 hours This treatment destroys fungi, bacteria, spores, and viruses

Boiling water cannot be expected to kill bacterial spores, unless applied according to the tyndalization method (fractional sterilization) In order to sterilize with steam certainly and quickly, steam under pressure in an autoclave may be used at 15 psi for 15 minutes The temperature reached should be at least 121 °C, sufficient to destroy all bacteria, their spores, and all other microorganisms

The modern automatic autoclave is really only a glorified pressure cooker. It can be used manually, for a "wet load" of liquid materials, for a "dry load" containing glassware, or with fast exhaust—to kill microorganisms quickly so that contaminated dishes, plates, flasks, and pipettes can be safely washed

Ethylene oxide (in a mixture with carbon dioxide called Carboxide) and beta-propiolactone (BPL) are used routinely in hospitals for gas sterilization of all types of surgical and other materials

Ultraviolet light, employed routinely for sterilization of the air in operating rooms, cannot penetrate like x-rays into materials, but it is readily available in sunlight, which therefore has great bactericidal powers. Commercially, the use of UV in restaurants and so forth is impressive, but, like x-rays, they are inefficient for effective sterilization

Although they are highly bactericidal, neutrons are expensive to produce and difficult to control. Alpha and beta rays are not practical for use, but gamma rays are used widely to sterilize foods and pharmaceuticals Cathode rays (electrons) are applied to food canning, frozen food, and surgical sterilization, and are also used to sterilize disposable Petri plates, pipettes, tubing, and most packaged materials

Many fluids may be sterilized without the use of heat, chemicals, or radiation by means of filters. Several types of filters are in common use, such as the Seitz, the Berkefeld, the Chamberland-Pasteur, sintered glass of many varieties, and membrane filters such as the Millipore The great advantage of modern bacterial filters is that they are disposable.

Antiseptics is of great significance in medical practice. The people of Africa in ancient times knew the methods of treating wounds with the aid of ant bites which healed the edges of the wound no worse than if it had been stitched by modem medical techniques. Sunlight took the place of antiseptic substances. Yet in 1865. N. Pirogov pointed out the necessity of destroying the source of intrahospital infection and tried chlorine water, silver nitrate, iodine and other antiseptic substances in combating wound suppurations. In 1867-J. Lister used phenol extensively as an antiseptic.

The science of antiseptics played a large role in the development of surgery. The practical application of microbiology in surgery brought a decrease in the number of postoperative complications, including gangrene, and considerably diminished the death rate in surgical wards. J. Lister highly assessed the importance of antiseptics and the merits of L. Pasteur in this field.

This trend received further development after E. Bergman and others who introduced aseptics into surgical practice representing a whole system of measures directed at preventing the access of microbes into wounds. Aseptics is attained by disinfection of the air and equipment of the operating room, by sterilization of surgical instruments and material, and by disinfecting the hands of the surgeon and the skin on the operative field. Film and plastic isolators are used in the clinic for protection against the penetration of micro-organisms. Soft surgical Him isolators attached to the operative field fully prevent bacteria from entering the surgical wound from the environment, particularly from the upper respiratory passages of the personnel of the operating room. A widespread use of aseptics has permitted the maintenance of the health and lives of many millions of people.

Modem methods of aseptics have been perfected to a considerable extent. Consequently almost all operations are accompanied with primary healing of wounds without suppuration, while the incidence of postoperative septicaemia has been completely eliminated.

Controls of sterilization. We use chemical and biological controls of sterilzation products with the purpose of checking of effectivity its. The matter of these procedures consist of some steps (actions). There are three knids of media which we use for control of sterilization, whereas sugar broth of Hotinger, thiglicol media, Saburo broth. We put into these media (sugar broth of Hotinger, thiglicol media, Saburo broth) some products have been sterilized before and put into thermostat till 14 days. But there are one exception, all innoculated media we keep in thermostat 14 days at 37 ⁰C (centigrate) and just innoculated with checking products Saburo media at 22 ⁰C (centigrate). After this period of 14 days we examine growth on these media. For example, when growth on media are absent we might make conclusion about effective sterilization process.

Now, some words about chemical controls of sterilization. There are some chemical substances, which have certain point of smelt. Fe., powder of serum (point of smelt is 119 ⁰C (centigrate)), benzoic acid (point of smelt is 120-122 ⁰C (centigrate)), beta naphthol (point of smelt is 123 ⁰C (centigrate)), mannose (point of smelt is 132 ⁰C (centigrate)). We sterilized products with steam and pressure into autoclave we put into its the closed test-tubes with these chemical substances and some quantity of aniline dyes. The even colored contents of these test-tubes show that the temperature get according level and process of sterilization was effective.

The treating (processing) of arms before operation. The treating of arms of medical personnel which take part in the operation are necessary, The different chemical substances are used for surgical treating of arms, f.e., mixture of 1.71 ml per litre of hydrogen peroxide (H₂O₂) 30-33% and 0.81 ml per litre of formic acid 85%, which call “C-4”, chlorhexidini solution.

Before treating arms we are washing them with soap (without brush) during 1minutes and drying out. After that we are treating arms with “C-4”, and drying with napkin, and put gloves on arms. With regards to method of arms treating with chlorhexidini: we are performing the same washing procedure underlighted upon and processing arms for first by sterile cotton napkin for second by wads with 0.5% spirituosae solution of chlorhexidini during 2-3 minutes.

Respiration in Bacteria

Respiration in bacteria is a complex process which is accompanied with the liberation of energy required by the micro-organism for the synthesis of different organic compounds. Many microbes similar to vertebrates and plants utilize the molecular oxygen in the air for respiration.

The concept of respiration as a process of oxidation of organic substances with the production of energy has undergone considerable changes due to the discovery of anaerobic microbes unable to exist in the presence of oxygen. Pasteur established that the energy necessary for the life activity of some species of microbes is obtained in the process of fermentation (liberation of energy without the participation of oxygen).

All microbes according to type of respiration can be subdivided into obligate aerobes, facultative anaerobes and obligate anaerobes.

1. Obligate aerobes which develop well in an atmosphere containing 21 per cent of oxygen. They grow on the surfaces of liquid and solid nutrient media (brucellae, micrococci, tubercle bacilli, etc.).

2. Facultative anaerobes which can reproduce even in the absence of molecular oxygen (the majority of pathogenic and saprophytic microbes).

3. Obligate anaerobes for which the presence of molecular oxygen is a harmful growth-inhibiting factor (causative agents of tetanus, botulism, anaerobic infections, etc.).

Aerobic bacteria in the process of respiration oxidize different organic substances (carbohydrates, proteins, lipids, alcohols, organic acids, and other compounds). During complete oxidation of one gram-molecule of glucose a definite number of calories is liberated which corresponds to the potential energy store accumulated in the carbohydrate molecule during its photosynthesis in green plants from carbon dioxide and water

During incomplete (partial) aerobic oxidation, less energy is released corresponding to the degree of oxidation

A typical representative of the facultative aerobes is the colibacillus which in a carbohydrate medium begins to develop first as an anaerobe breaking down the carbohydrates by fermentation. Then it begins to utilize oxygen and grows like an aerobe, oxidizing the products of fermentation (lactic acid) farther to carbon dioxide and water. Facultative aerobes have a considerable advantage, as they can live in aerobic and anaerobic conditions.

Respiration in anaerobes takes place by fermentation of the substrate with the production of a small amount of energy. In the fermentation of one gram-molecule of glucose considerably less energy is produced than during aerobic respiration.

The mechanism of anaerobic respiration takes place in the following way. If carbohydrates make up the oxidizing substrate, then preliminarily they are broken down with the help of auxiliary enzymes. Thus, for example, glucose is phosphorylated employing ATP and ADP. As a result, hexose diphosphale is produced which under the influence of the enzyme aldolase breaks down into two components: phosphogly-ceraldehyde and dioxyacetone phosphate. The latter under the effect of oxyisomerase is transformed into phosphoglyceraldehyde and later on after a sequence of reactions produces pyruvic acid. This stage is the last in the anaerobic phase of transformation of carbon. The later stages are specific and are completed with the production of end products.

Anaerobic processes include alcohol fermentation by yeasts, lactic acid fermentation by lactobacilli, and butyric acid fermentation by butyric acid clostridia.

Anaerobes ferment mostly nitrogen-free compounds causing fermentation. However, there is no sharp boundary between the erobic and anaerobic types of respiration. Thus. for example, yeasts can change the anaerobic type of respiration to aerobic respiration. First of all, they break down sugar into alcohol and carbon dioxide, and during increased aeration glucose is broken down into water and carbon dioxide.

The presence of obligate anaerobes explains the rather great adaptability of living things and the completeness of the cycle ofsubstances in nature.

It has been established by investigations that the respiration in bac- teria takes place under the influence of enzymes of the oxidase and dehydrogenase types, which have a marked specificity and a multilateral activity. The oxidase and dehydrogenase processes of respiration are closely interconnected, supplementing each other, but at the same time differing in biological role and in enzymes.

The intensity of the processes of aerobic respiration depends on the age of the culture, temperature, and nutrient substrates. Actively growing cultures use 2500-5000 cu mm of oxygen per 1 mg of dry matter of bacleria per hour while starved cultures or cultures completely deprived of nitrogen nutrients require only 10-150 cu mm. A young culture produces considerably more heat energy than it uses for its synthetic and other life processes. A certain part of this energy is released into the environment. For instance, the colibacillus in the process of assimilation uses 31 per cent of the energy released, blue us bacteria – 28 per cent, Proteus vulgaris — 20 per cent, and salmonellae of enteric fever – 12 percent. The production by some microbes of an excess of heat energy in manure, turf and garbage can ause spon- taneous heating and spontaneous combustion.

In manure and garbage dumps due to the effect of the high temperature produced by thermophilic microbes, the eggs laid by flies and also the eggs of worms are unable to develop.

Increased respiration and an increased metabolism depend on the rate of cell reproduction, on the increase of the protein synthesis in the cell, which causes an increase in the reduction properties of the medium in which the microbes develop.

Biological oxidation comprises the removal of a negatively-charged electron, reduction - the addition of a negatively-charged electron.

Between the hydrogen acceptor (yellow enzyme) and oxygen there are intermediate hydrogen carriers w hich are participants of the long chain of the catalyst of biological oxidation.

The electrons are carried by cytochromes a, /?, and f which are pro- tein molecules bound with a chemical group of the haem. The haem contains an iron atom capable of undergoing oxidation and reduction alternately. Besides cytochromes, a new substance has been discovered, a carrier of electrons, called ubiquinon or cocnzyme.

Thus the processes of respiration in bacteria are very complex and represent a long chain of a sequence of oxidation-reduction reactions with the participation of many enzyme systems transporting the electrons from the system of the most negative potential to the system of the most positive potential. During gradual and fractional liberation of energy in respiration and during intermediate transport of hydrogen, the activity of cellular reactions increases. The biochemical mechanisms of respiration are described in detail in biochemistry textbooks.

The habitat of micro-organisms greatly influences the character of respiration. Thus, for example, upon cultivating the cholera-like vibrio in a medium containing glucose, its aerobic respiration can be decreased as a result of which it acquires the properties of a facultative anaerobe. Yeasts are also capable of changing their type of respiration depending on the presence or absence of oxygen.

G. McLeod explained that the toxic effect of oxygen on anaerobes is due to the production of hydrogen peroxide in the presence of oxygen. Anaerobes are unable to produce catalase. Only H,0, but not oxygen itself is toxic. However, this cannot be a complete explanation. Anaerobes can grow if there is oxygen in the medium, which does nt kill microbes, but only inhibits their life activities. Upon the addition of reducing agents to the medium, the microbes begin to grow. as reducing agents lower the oxidation-reduction potential. Glucose and other reducing substances act in the same way.

V. Engelhardt considers that in the presence of a high oxidation-reduction potential, the inactivation of vitally important enzymes takes place. Anaerobes then lose their ability to feed normally, and to carry out constructive processes. Hence they perish from starvation, and not from intoxication by oxygen or H₂0₂. The oxidation-reduction potential (rH,) is one of the factors on which the oxidation-reduction reactions in the nutrient medium depend. The oxidation-reduction potential expresses the quantitative character of the degree of aerobiosis. It becomes minimal upon saturating the medium with hydrogen, and maximal upon saturating the medium with oxygen. M. dark proposed to designate the unit of the oxidation-reduction potential as rH,-the negative logarithm of the partial pressure of gaseous hydrogen." The range of rHs from 0 to 42,6 characterizes all degrees of saturation of an aqueous solution with hydrogen and oxygen- Aerobes exist within the limits of rH, from 14 to 20 and more, facultative aerobes from 0 to 20 and more, and anaerobes from 0 to 12.

Aerobes are adapted to existence at a higher oxidation-reduction potential, anaerobes — at a lower rH,. Anaerobes are not passive micro-organisms, and they themselves cause the low rH, in the medium.

Seeded cultures of anaerobes prior to reproduction lower the rH, from 20-22 to 1-5. Thus anaerobes are characterized by a rather marked capability to adapt the medium to their requirements. Aerobes also have these properties, and they guard themselves from an excess of oxygen by a reduction barrier.

Upon controlling the oxidation-reduction potential of the nutrient medium, conditions can be obtained for the growth of anaerobes in the presence of oxygen by lowering the rH,, and also by cultivating the aerobes in anaerobic conditions by increasing the rH, of the medium.

The oxidation-reduction potential drops sharply when the bacterial culture dies, when it is lysed by a phage and when it is affected by lysozyme.

When preparing nutrient media the composition of the nutrient energy-yielding material, the reaction of the medium (pH), and its oxidation-reduction potential (rH;) are all taken into consideration.

Bacterial growth

Colony is bacterial cells of the same species which have grown from one bacterial cell on solid medium as isolated accumulation. Bacteriological investigation is based on isolating a pure culture of the causal organism and its identification. The term pure culture refers to a population of microorganism of the same species isolated on a nutrient medium.

The Medium. The technique used and the type of medium selected depend upon the nature of the investigation. In general, 3 situations may be encountered; (1) one may need to raise a crop of cells of a particular species that is on hand; (2) one may need to determine the numbers and types of organisms present in a given material; or (3) one may wish to isolate a particular type of microorganism from a natural source.

A. Growing Cells of a Given Species: Microorganisms observed microscopically to be growing in a natural environment may prove exceedingly difficult to grow in pure culture in an artificial medium. Certain parasitic forms, for example, have never been cultivated outside the host. In general, however, a suitable medium can be devised by carefully reproducing the conditions found in the organism's natural environment. The pH, temperature, and aeration are simple to duplicate; the nutrients present the major problem. The contribution made by the living environment is important and difficult to analyze; a parasite may require an extract of the host tissue, and a free-living form may require a substance excreted by a microorganism with which it is associated in nature.

B. Microbiologic Examination of Natural Materials: A given natural material may contain many different microenvironments, each providing a niche for a different species. Plating a sample of the materials under one set of conditions will allow a selected group of forms to produce colonies but will cause many other types to be overlooked. For this reason it is customary to plate out samples of the material using as many different media and conditions of incubation as is practicable, Six to 8 different culture conditions are not an unreasonable number if most of the forms present are to be discovered.

Since every type of organism present must have a chance to grow, solid media are used and crowding of colonies is avoided. Otherwise, competition will prevent some types from forming colonies.

C. Isolation of a Particular Type of Microorganism: A small sample of soil, if handled properly, will yield a different type of organism for every microenvironment present. For fertile soil (moist, aerated, rich in minerals and organic matter) this means that hundreds or even thousands of types can be isolated, This is done by selecting for the desired type. One gram of soil, for example, is inoculated into a flask of liquid medium that has been made up for the purpose of favoring one type of organism, eg, aerobic nitrogen fixers (Azotobacter). In this case the medium contains no combined nitrogen and is incubated aerobically. If cells of Azotobacter are present in the soil, they will grow well in this medium forms unable to fix nitrogen will grow only to the extent that the soil has introduced contaminating fixed nitrogen into the medium. When the culture is fully grown, therefore, the percentage of Azotobacter in the total population will have increased greatly; the method is thus called 'enrichment culture. Transfer of a sample of this culture to fresh medium will result in further enrichment of Azotobacter, after several serial transfers, the culture can be plated out on a solidified enrichment medium and colonies of Azotobacter isolated.

Liquid medium is used to permit competition and hence optimal selection, even when the desired type is represented in the soil as only a few cells in a population of millions. Advantage can be taken of "natural enrichment." For example, in looking for kerosene oxidizers, oil-laden soil is chosen, since such soil is already an enrichment environment for such forms.

Enrichment culture, then, is a procedure whereby the medium is prepared so as to duplicate the natural environment (“niche”) of the desired microorganism, thereby selecting for it. An important principle involved in such selection is the following: The organism selected for will be the type whose nutritional requirements are barely satisfied. Azotobacter, for example, grows best in a medium containing organic nitrogen, but its minimum requirement is the presence of N;;

hence it is selected for in a medium containing N; as the sole nitrogen source. If organic nitrogen is added to the medium, the conditions no longer select for Azotobacter but rather for a form for which organic nitrogen is the minimum requirement.

When searching for a particular type of organism in a natural material, it is advantageous to plate the organisms obtained on a differential medium if available. A differential medium is one that will cause the colonies of a particular type of organism to have a distinctive appearance. For example, colonies of Escherichia coli have a characteristic iridescent sheen on agar containing the dyes eosin and methylene blue (EMB agar). EMB agar containing a high concentration of one sugar will also cause organisms which ferment that sugar to form reddish colonies. Differential media are used for such purposes as recognizing the presence of enteric bacteria in water or milk and the presence of certain pathogens in clinical specimens from patients.

For selection the pure culture of microorganisms, it follows to separate numerous bacteria which are in tested material, one from other. It is possible to attain by methods which are based on two principles – mechanical and biological separation of bacteria.

Mechanical principle	Biological principle
METHODS 1. Factional dilutions (L. Pasteur’s technique) 2. Pour plate technique (Dilution in solid nutrient media by R. Koch’s technique) 3. Spread plate technique (Superficial dispersions by Drigalsky’s technique) 4. Streak plate technique	METHODS Take into account: – Respiration type (Fortner’s method); – bacterial motility (Shukevich’s method – resistance to acids (acid fast bacteria); – sporulation; – temperature optimum; – selective sensitiveness of laboratory animals to the bacteria and so on.

Methods based on mechanical principle

Method of factional dilutions (L. Pasteur’s technique) is based on mechanical disconnection of microorganisms by serial dilution in liquid nutrient media. The main lack of this technique: we can not make control the amount of microbal is tested tubes.

Pour plate technique (Dilution in solid nutrient media by R. Koch’s technique) is based on dilution of microbes and pouring the tested material with gelatin. After cooling the gelatin isolated colonies of microorganisms are formed and they easily can be transferred on a fresh nutrient medium by a platinum loop for obtaining a microbial pure culture.

Spread plate technique (Superficial dispersions by Drigalsky’s technique) is more perfect method which is widely widespread in everyday microbiological practice. There is quantitative technique that allows the determination of the number of bacteria in a sample.

Stages:

· Pipette the required amount of bacteria (from your dilution) on the surface of the Petri plate.

· Spread the inoculum over the surface of the agar medium using a hockey stick (spatula).

· Repeat this action on 3-4 Petri plates without sterilization of the hockey stick.

· Incubate the plate inverted at 37 ^oC.

There must be different number of microbial colony on the Petri plates.

Streak plate technique.

ADVANTAGES:

· Spread millions of cells over the surface;

· Individual cells deposited at a distance from all others;

· Divide forming distinct colonies;

· Distinct colonies do not touch any other colonies;

· Clone of a single bacteria à pure culture

You streak the plate on 3 different portion of the Petri plate, so you can draw the section that you will streak on the bottom of your plate.

Stages:

· Using a sterile loop take a loopful of your bacteria from the broth

· Streak a vertical line

· Then streak gently across section 1

· Zig-zag pattern until a 1/3 of the plate is covered

· Do not dig into the agar

· Sterilize the loop à let it cool

· Rotate the plate about 90 degrees and spread the bacteria from the first streak into a second area

· Do only one streak (or very few) in the first area and once you are in the second area do not go back to the first

· Do a zig-zag pattern until the 2nd area is covered

· Sterilize again à do the same for 3rd area

· Make sure that your red hot loop is cool enough prior to touch the bacteria

· After you waited a few seconds

· Stab it into the agar in a position away from bacteria à will cool it

· If you stab where bacteria are à production of aerosol

· Incubate the plate inverted at 37 ^oC.

In a day it is necessary to examine the colonies for future investigation.

Or:

Thus, substantial advantage of Pour plate technique (dilution in solid nutrient media by R. Koch’s technique), spread plate technique (superficial dispersions by Drigalsky’s technique), and streak plate technique consists in that they create the ability to obtain isolated (distinct) colonies of microorganisms which can be transferred on slant agar for pure culture obtaining.

Methods based on biological principle

Biological principle of disconnection of bacteria foresees the purposeful search of methods which take into account the numerous features of microbal species. Among the most widespread methods it is possible to select the followings:

1. Respiration type. All of microorganisms according to the type of respiration are divided into two basic groups: aerobic (Corynebacterium diphtheriae, Vibrio сholerae and others) and anaerobic (Clostridium tetani, Clostridium botulinum, Clostridium perfringens and so on). If tested material from which it follows to select anaerobic bacteria to warm up preliminary, and then cultivate in anaerobic terms, these bacteria will grow exactly.

2. Sporulation. It is known that some microbes (bacilli and clostridia) form endospores. There are Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Bacillus subtilis, Bacillus cereus among them. Spores are resistant against different external environment factors. That’s why, if tested material would be heated previously and then inoculated in nutrient medium spore-forming bacteria would be grown.

3. Resistance of microbes against acids and alkali. Some microbes (Mycobacterium tuberculosis, Mycobacterium bovis) as a result of their chemical structure features are resistant agains acids. That’s why tested material with this bacteria previously is treated with 10 % sulfuric acid and later inoculated on proper nutrient medium An extraneous flora perishes, and mycobacteria as a result of their resistance to acids grow.

Vibrio сholerae is a halophylic bacterium, and for its growth it is inoculated in 1 % alkaline peptone water. Already in 4-6 hrs it growth like a tender bluish pellicle on the surface of medium.

4. Bacteria motility. Some microbes (Proteus vulgaris) have a tendency to creeping growth and is able to spread quickly on the surface of moist nutrient medium because they have flagella. So such bacteria are inoculated in the drop of condensation liquid which appears after the cooling the slant agar. In 16-18 hrs they spread on all surface of nutrient medium. If material from the upper part of agar would be taken we will have a pure culture of microbe.

5. A susceptibility of microbes to different chemicals, antibiotics etc. As a result of features of metabolism some bacteria have a different susceptibility to some chemical factors. For example, staphylococci, aerobic bacilli can grow in nutrient media which have 7,5–10 % to the sodium chloride. That is why for the selection of these bacteria this substance is added into yalk-salt afar and mannitol-salt agar for their selection. Other bacteria under the influence of such concentration of sodium chloride do not grow practically.

Some antibiotics (nistatin) is used for inhibition for pathogenic fungi growth if it is necessary to obtain only bacteria. Adding the Penicillin in nutrient medium inhibit the growth only gram-positive bacteria. Presence of Furazolidon makes favorite condition for Corynebacteria and Micriococci.

6. Ability of microorganisms to penetrate through unharmed skin. Some pathogenic bacteria (Yersinia pestis) as a result of presence a lot of aggression enzymes are able to penetrate through an intact skin. For this purpose body wool of laboratory animal is shaven and tested material with different bacteria a rubbed in this skin area. Later some microbes may be obtain from the blood or internal organs.

7. A sensitiveness of laboratory animals is to the exciters of infectious diseases. Some laboratory animals show a high susceptibility to the different microorganisms.

For example, after any method of Streptococcus pneumoniae introduction into a mouse generalized pneumococcal infection are developed. An analogical picture is observed after injection of Mycobacterium tuberculosis into Guinean pig or Mycobacterium bovis into the rabbit.

• 8. Temperature optimum. The cardinal temperatures:

- Minimum

- Optimum

- Maximum

Microorganisms can be grouped by the temperature ranges they require

• Psychrophiles, low temperature optima (4°C) – Polaromonas vacuolata

• Mesophils midrange (39°C) – Escherichia coli

• Thermophiles high (60°C) – Bacillus stearothermophilus

• Hyperthermophiles very high (>80°C) – Thermococcus celer

In everyday practice bacteriologists use such concepts as a species, a strain and pure culture of microorganisms.

Species – a collection of bacterial cells which share an overall similar pattern of traits in contrast to other bacteria whose pattern differs significantly

A strain is a subset of a bacterial species differing from other bacteria of the same species by some minor but identifiable difference. A strain is "a population of organisms that descends from a single organism or pure culture isolate. Strains within a species may differ slightly from one another in many ways."

Culture: population of microorganisms grown under well defined conditions.

Pure culture – one that contains one type of microorganism.

Isolation and identification of a pure culture

First day

1. Microscopic examination of the tested material.

2. Streaking of the material tested onto nutrient media (solid, liquid).

Second day

1. Investigation of the cultural properties.

2. Sub-inoculation of colonies onto solid media to enrich for a pure culture.

Third day

1. Checking of the purity of the isolated culture.

2. Investigation of biochemical properties: (a) sugarlytic, (b) proteolytic.

3. Determination of antigenic properties.

4. Study of phagosensitivity, phagotyping, colicinogensitivity, colicinogenotyping, sensitivity to antibiotics, and other properties.

Main Principles of the Cultivation of Microorganisms

Bacterial cultivation. In laboratory conditions microorganisms can be grown in nutrient media in incubation chambers maintained at a constant temperature. According to the type of heating, incubation chambers can be subdivided into electric, gas and kerosene. Each incubation chamber has a thermoregulator which maintains a constant temperature. Temperature conditions are of great importance for the growth and reproduction of bacteria. In relation to conditions of temperature all micro-organisms can be subdivided into three groups: psychrophilic (Gk. psychros cold, philein love), mesophilic (Gk. mesos intermediate), thermophilic (Gk. thermos warm). Microorganisms may reproduce within a wide temperature regimen range of –10 to +80 °C.

Of great importance in the life activities of bacteria is the concentration of hydrogen ions in the nutrient medium, i. e. pH, which is expressed by the negative logarithm of the concentration of hydrogeons. The pH characterizes the degree of acidity or alkalinity, from extremely acid (pH 0) to extremely alkaline (pH 14) conditions.

During evolution each microbial species adapted itself to existence within certain limits of hydrogen ion concentration beyond the range of which its life processes are unable to take place; It has been suggested that pH influences the activity of enzymes. Depending on the pH, weak acids in an acid medium occur as molecules, and in an alkaline medium as ions. Saprophytes can live in conditions within a wide range of a pH from 0.6 to 11.0, while pathogenic species of microbes grow within certain limits of hydrogen ion concentration Nutrient media should be easily assimilable, and they should contain a known amount of nitrogen and carbohydrate substances, vitamins, a required salt concentration. In addition they should be isotonic, and sterile, and they should have buffer properties, an optimal viscosity, and a certain oxidation reduction potential.

During the whole history of microbiology nutrient media have gradually been perfected. Before Pasteur only infusions and decoctions were used as media for growing microbes. Pasteur and Nageli introduced non-protein media for the cultivation of microbes. Koch and Loeffler employed meat broth, peptone, and sodium chloride for growing microbes. This medium is a meat-peptone broth from which meat-peptone agar is prepared by adding 1-2 per cent industrial agar.

Agar (in Malayan - jelly) is compact fibrous material produced from some seaweed, forms in water solutions a solid gel. Agar contains 70-75% polysaccharides, 2-3% proteins and other nitrogen-containing substances, 2-4% ashes. Main components of agar high molecular weight substances — agarose and agaropectin. Agar dissolves in water while heating and solidifies at room temperature. It is manufactured as colourless plates or powder.

Because of the ability of agar upon cooling to give the nutrient medium a solid gel consistency, and due to its high resistance towards the microbial enzymes, it has received wide application in bacteriological techniques for preparing semisolid, solid, and dry nutrient media.

For the preparation of nutrient media M. Hottinger suggested the use of products of the tryptic breakdown of proteins which do not contain peptones, but contain the low molecular polypeptides and free amino acids. L. Martin employed papain as an enzyme for the break-down of proteins. In recent years all the essential amino acids and vitamins used for the cultivation of bacteria have been obtained in a pure state.

Isolation and Identification of Pure Culture of Aerobic Bacteria

First day. Prepare smears of the tested material and study them under the microscope. Then, using a spatula or a bacteriological loop, streak the material onto a solid medium in a Petri dish. This ensures mechanical separation of microorganisms on the surface of the nutrient medium, which allows for their growth in isolated colonies. In individual cases the material to be studied is streaked onto the liquid enrichment medium and then transferred to Petri dishes with a solid nutrient medium. Place these dishes in a 37 ⁰C incubator for 18-24 hrs.

Incubator

Second day. Following a 24-hour incubation, the cultural properties of bacteria (nature of their growth on solid and liquid nutrient media) are studied.

Macroscopic examination of colonies in transmitted and reflected light. Turn the dish with its bottom to the eyes and examine the colonies in transmitted light. In the presence of various types of colonies count them and describe each of them. The following properties are paid attention to; (a) size of colonies (largo, 4-5 mm in diameter or more; medium, 2-4 mm; small, 1-2 mm; minute, less than 1 mm); (b) configuration of colonies {regularly or irregularly rounded, rosette-shaped, rhizoid, etc.); (c) degree of transparency (non-transparent, semitransparent, transparent).

In a reflected light, examine the colonies from the top without opening the lid. The following data are registered in the protocol: (a) colour of the colonies (colourless, pigmented, the colour of the pigment); (b) nature of the surface (smooth, glassy, moist, wrinkled, lustreless, dry, etc.); (c) position of the colonies on the nutrient medium (protruding above the medium, submerged into the medium; flat, at the level of the medium; flattened, slightly above the medium).

Microscopic examination of colonies. Mount the dish, bottom upward, on the stage of the microscope, lower the condenser, and, using an 8 x objective, study the colonies, registering in the protocol their structure (homogeneous or amorphous, granular, fibriliar, etc.) and the nature of their edges (smooth, wavy, jagged, fringy, etc.).

Use some portion of the colonies to prepare Gram-stained smears for microscopic examination. In the presence of uniform bacteria, transfer the remainder of colonies to an agar slant for obtaining a sufficient amount of pure culture. Place the test tubes with the inoculated medium into a 37 °C incubator for 18-24 hrs.

Third day. Using the culture which has grown on the agar slant prepare smears and stain them by the Gram method. Such characteristics as homogeneity of the growth, form, size, and staining of microorganisms permit definite judgement as to purity of the culture. To identify the isolated pure culture, supplement the study of morphological, tinctorial, and cultural features with determination of their enzymatic and antigenic attributes, phago- and bacterio-cinosensitivity, toxigenicity, and other properties characterizing their species specificity.

To demonstrate carbohydrate-splitting enzymes, Hiss' media are utilized. When bacteria ferment carbohydrates with acid formation, the colour of the medium changes due to the indicator present in it. Depending on the kind and species of bacteria studied, select media with respective mono- and disaccharides (glucose, lactose, maltose, sucrose), polysaccharides (starch, glycogen, inulin), higher alcohols (glycerol, mannitol). In the process of fermentation of the above substances aldehydes, acids, and gaseous products (CO₂, H₂, etc.) are formed.

To demonstrate proteolytic enzymes in bacteria, transfer the latter to a gelatin column. Allow the inoculated culture to stand at room temperature (20-22 °C) for several days, recording not only the development of liquefaction per se but its character as well (laminar, in the form of a nail or a fir-tree, etc.)

Proteolytic action of enzymes of microorganisms can also be observed following their streaking onto coagulated serum, with depressions forming around colonies (liquefaction). A casein clot is split in milk to form peptone, which is manifested by the fact that milk turns yellowish (milk peptonization).

More profound splitting of protein is evidenced by the formation of indol, ammonia, hydrogen sulphide, and other compounds. To detect the gaseous substances, inoculate microorganisms into a meat-peptone broth or in a 1 per cent peptone water. Leave the inoculated cultures in an incubator for 24-72 hrs.

To demonstrate indol by Morel's method, soak narrow strips of filter paper with hot saturated solution of oxalic acid (indicator paper) and let them dry. Place the indicator paper between the test tube wall and stopper so that it does not touch the streaked medium. When indol is released by the 2nd-3rd day, the lower part of the paper strip turns pink as a result of its interaction with oxalic acid.

The telltale sign of the presence of ammonia is a change in the colour of a pink litmus paper fastened between the tube wall and the stopper (it turns blue). Hydrogen sulphide is detected by means of a filter paper strip saturated with lead acetate solution, which is fastened between the tube wall and the stopper. Upon interaction between hydrogen sulphide and lead acetate the paper darkens as a result of lead sulphide formation.

To determine catalase, pour 1-2 ml of a 1 per cent hydrogen peroxide solution over the surface of a 24-hour culture of an agar slant. The appearance of gas bubbles is considered as a positive reaction. Use a culture known to contain catalase as a control.

The reduction ability of microorganisms is studied using methylene blue, thinning, litmus, indigo carmine, neutral red, etc. Add one of the above dyes to nutrient broth or agar. The medium decolorizes if the microorganism has a reduction ability. The most widely employed is Rothberger's medium (meat-peptone agar containing 1 per cent of glucose and several drops of a saturated solution of neutral red). If the reaction is positive, a red colour of the agar changes into yellow, yellow-green, and fluorescent, while glucose fermentation is characterized by cracks in the medium.

Antigen properties of the isolated culture are investigated by the agglutination test and other serological tests.

Species identification of aerobic bacteria is performed by comparing their morphological, cultural, biochemical, antigenic, and other properties.

On solid nutrient media microbes form colonies of different shapes and sizes which are ggregations of individuals connected by bands of cytoplasm providing for a certain structure of bacterial groupings. The colonies may be flat. convex, dome-shaped, or pitted; their surface – smooth (S-forms). rough (R-forms). ridged, or bumpy; their edges may be straight, serralcd. fibrous, or lasseled. The shape of the colonies also differs: e.g. round, rosette-shaped, star-shaped, tree-like. According lo their size the colonies may be divided into large (4-5 mm in diameter), intermediate (2-4 mm), small (1-2 mm), and dwarf (less than 1 mm).

The colonies differ in their consistency, density, and colour. They may be transparent and opaque, coloured and colourless, moist, dry, and slimy.

In liquid nutrient media microbes grow producing a duffuse suspen- sion. film. or precipitate visible to the naked eye.

The growth of bacteria in the laboratory is carried out in test tubes, Petri dishes, and flasks.

In institutes for production of vaccines the cultivation of aerobes is carried out by deep stab methods. This method permits a more rational use of the nutrient substrate, and a large microbial mass can be obtained. The cultures are grown in reactors. Aeration is produced by passing a stream of air through the medium. The method of aeration is used in laboratory investigations to promote rapid growth of bacteria and to study some processes of metabolism.

Reproduction in microbes takes place more intensively in a flowing nutrient medium which is constantly being renewed. For this purpose a spare tank with nutrient medium is installed, from which the mediumenters the cultivator and is carefully mixed with the culture.

Colonies of a different structure.

After this the excess of cultural fluid together with the suspended bacterial cells flows out. When the rate of flow of cells from the cultivator is equal to the rate of reproduction, the number of the microbial population remains constant.

Modem plant equipment is supplied with devices for automatic control over reproduction and other microbiological processes.

In usual laboratory conditions anaerobes develop in stationary or portable anaerostats containing rarefied air up to 1-8 mm or in vacuum desiccators.

Stationary anaerostat (jar)

Portable anaerostat

For successfully cultivating anaerobes it is necessary to seed a large amount of material into the nutrient medium. The nutrient medium should have a certain viscosity which is attained by adding 0.2 per cent agar. The air is removed by boiling prior to seeding, and to inhibit thesubsequent entry of air, the medium is covered with a layer of oil 0.5-1 cm thick. Anaerobiosis is obtained by the adsorption of oxygen on porous substances (pumice, cotton wool, coal) and by adding reducing substances (carbohydrates, peptone, cysteine. pieces of liver, spleen, kidneys, brain, etc.). After seeding, the test tubes are filled up with liquid vaseline. Growth of the anaerobes is usually carried out on a Kitt-Tarozzi

Isolation and identification of a pure culture

First day

1. Microscopic examination of the tested material.

2. Streaking of the material tested onto nutrient media (solid, liquid).

Second day

1. Investigation of the cultural properties.

2. Sub-inoculation of colonies onto solid media to enrich for a pure culture.

Third day

1. Checking of the purity of the isolated culture.

2. Investigation of biochemical properties: (a) sugarlytic, (b) proteolytic.

3. Determination of antigenic properties.

4. Study of phagosensitivity, phagotyping, colicinogensitivity, colicinogenotyping, sensitivity to antibiotics, and other properties.

With regards to obtaining microorganisms in pure culture, are based on mechanical divorced of bacteria tested material inoculate onto surface media in Petri dish by bacteriological loop or pipette and after that streak plating evenly. After that again that glass spatula (don’t burn through the flame) was used for streak plating onto the same second media in Petri dish.The seeding has been done by bacteriological loop too. With that purpose in upper part of Petri dish has been made dense streaking, set free bacteriological loop from superfluous material. After that are made paralel streaks at the last part of the agar. Somever are applied method of laminar dilution, the matter of this method is a stiring diferrent serial dillution tested materials with melting and colling agar in tubes.After that its are flooded into Petri dishes and put down into incubator.The tested materials are boiled of short duration or heat on 80 ⁰C for destroy bacteria without spores. The spores of microorganisms leave still alive and ater reinoculate this materials they are grown.

Fortner method. The agar media is divided into two parts. Onto the one part inoculate E.coli or Serratia marcescens (these microorganisms absorb intensively oxygen) and onto second part taested material. Closely stop up this Petri dish by parafin and put down into the thermostat. This method is used for obtainig anerobe culture.

Reproduction and Growth of Microorganisms

Reproduction in microbes constitutes the ability of self-multiplication, i.e. the increase in the number of individuals per unit volume The growth of micro-organisms represents the increase of the mass of bacterial cytoplasm as a result of the synthesis of cellular material.

Bacteria reproduce by simple transverse division, vegetative reproduction, which occurs in different planes and produces many kinds of cells (clusters, chains, pairs, packets, etc.). They also reproduce by budding, by means of the cleavage of segmented filaments, by reproducing cells similar to spores, by producing minute motile conidia. And by conjugation, which brings us closely to the concept of sexual reproduction in bacteria DNA replication is an important condition in the process of amitotic binary fission of bacteria, the hydrogen bonds are ruptured and two DNA strands are formed, each one is contained in the daughter cells The single-stranded DNA are eventually linked by means of hydrogen bonds and again form double-chain DNA responsible for genetic information DNA replication and cell fission occur at a definite rate characteristic of each species. Actinomycetes and many fungi (phycomycetes, ascomycetes, etc) reproduce predominantly by sporulation

The transverse division of bacteria is not only a process of cell division of one mother cell into two equal daughter cells, but represents d constant separation of daughter cells from the mother cell, the former in their turn become mother cells. After a certain number of generations, the mother cells age and perish. This explanation has annulled the metaphysical concept of 'bacterial immortality'.

The rate of cell division differs among bacteria It depends on the species of microbe, the age of the culture, on the nutrient medium, temperature, concentration of carbon dioxide, and on many other factors.

The length of the generation off coli, Clostridium perfringens, Streptococcus faecalis is 15 minutes, while for the cells of a mammalian tissue culture it is 24 hours Thus, bacteria reproduce almost 100 times faster than cells of tissue culture The increase in the number of cells can be expressed in the following way:

0— 1— 2— 3 — 4— 5— n number of generations

The total amount of bacteria (N) after n generations will be equal to 2ⁿ per cell of seeded material If we take the original amount of bacteria inoculated into the nutrient medium as a single individual, and the time for one division as 30 minutes, then theoretically the total amount of bacteria produced per 24 hours would be equal N=2⁴⁸. Upon division every 20 minutes, in 36 hours the microbial mass will be equal to 400 tons. Thermophilic microbes divide even more rapidly.

However, in natural as well as in artificial conditions, the reproduction of bacteria is of a considerably smaller scale. It is limited by the effect of a number of environmental factors. Reproduction in bacteria conforms to certain laws. Fig. 1 illustrates schematically the rate of reproduction of bacteria in arbitrary units, and the size of the bacterial population expressed as the logarithm of the numbers of live cells per millimeter of the medium.

There are eight principal phases of reproduction which are designated on the diagram by Roman numerals.

1. An initial stationary phase represents the time from the moment of seeding the bacteria on the nutrient medium. Reproduction does not occur in this phase. The length of the initial stationary phase after seeding is 1-2 hours.

2. The lag phase of reproduction during which bacterial reproduction is not intensive, while the growth rate is accelerated. The second phase may last almost two hours.

3. Phase of exponential (logarithmic) growth which is characterized by a maximal division rate and decrease in cell size. The length of this period ranges from 5 to 6 hours.

4. Phase of negative growth acceleration during which the rate of bacterial reproduction ceases to be maximal, and the number of dividing cells diminishes. This phase lasts almost two hours.

5. A maximal stationary phase when the number of newly produced bacteria is almost equal to the number of dead organisms. This phase continues for two hours.

6. Accelerated death phase during which the equilibrium between the stationary phase and the bacterial death rate is interrupted. This continues for 3 hours.

7. Logarithmic death phase when the cells die at a constant rate. This continues almost 5 hours.

8. Decelerated death-rate phase in which those cells which remain alive enter a dormant state.

Graph of the reproduction of bacteria

The length of these phases is arbitrary, as it can vary depending on the bacterial species and the conditions of cultivation. Thus, for example, the colibacilli divide every 15-17 minutes, salmonellae of enteric fever — every 23 minutes, pathogenic streptococci — every 30 minutes, diphtheria bacilli — every 34 minutes and tubercle bacilli — every 18 hours.

Methods based on biological principle

Biological principle of disconnection of bacteria foresees the purposeful search of methods which take into account the numerous features of microbal species. Among the most widespread methods it is possible to select the followings:

1. Respiration type. All of microorganisms according to the type of respiration are divided into two basic groups: aerobic (Corynebacterium diphtheriae, Vibrio сholerae and others) and anaerobic (Clostridium tetani, Clostridium botulinum, Clostridium perfringens and so on). If tested material from which it follows to select anaerobic bacteria to warm up preliminary, and then cultivate in anaerobic terms, these bacteria will grow exactly.

2. Sporulation. It is known that some microbes (bacilli and clostridia) form endospores. There are Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Bacillus subtilis, Bacillus cereus among them. Spores are resistant against different external environment factors. That’s why, if tested material would be heated previously and then inoculated in nutrient medium spore-forming bacteria would be grown.

3. Resistance of microbes against acids and alkali. Some microbes (Mycobacterium tuberculosis, Mycobacterium bovis) as a result of their chemical structure features are resistant agains acids. That’s why tested material with this bacteria previously is treated with 10 % sulfuric acid and later inoculated on proper nutrient medium An extraneous flora perishes, and mycobacteria as a result of their resistance to acids grow.

Vibrio сholerae is a halophylic bacterium, and for its growth it is inoculated in 1 % alkaline peptone water. Already in 4-6 hrs it growth like a tender bluish pellicle on the surface of medium.

4. Bacteria motility. Some microbes (Proteus vulgaris) have a tendency to creeping growth and is able to spread quickly on the surface of moist nutrient medium because they have flagella. So such bacteria are inoculated in the drop of condensation liquid which appears after the cooling the slant agar. In 16-18 hrs they spread on all surface of nutrient medium. If material from the upper part of agar would be taken we will have a pure culture of microbe.

5. A susceptibility of microbes to different chemicals, antibiotics etc. As a result of features of metabolism some bacteria have a different susceptibility to some chemical factors. For example, staphylococci, aerobic bacilli can grow in nutrient media which have 7,5–10 % to the sodium chloride. That is why for the selection of these bacteria this substance is added into yalk-salt afar and mannitol-salt agar for their selection. Other bacteria under the influence of such concentration of sodium chloride do not grow practically.

Some antibiotics (nistatin) is used for inhibition for pathogenic fungi growth if it is necessary to obtain only bacteria. Adding the Penicillin in nutrient medium inhibit the growth only gram-positive bacteria. Presence of Furazolidon makes favorite condition for Corynebacteria and Micriococci.

6. Ability of microorganisms to penetrate through unharmed skin. Some pathogenic bacteria (Yersinia pestis) as a result of presence a lot of aggression enzymes are able to penetrate through an intact skin. For this purpose body wool of laboratory animal is shaven and tested material with different bacteria a rubbed in this skin area. Later some microbes may be obtain from the blood or internal organs.

7. A sensitiveness of laboratory animals is to the exciters of infectious diseases. Some laboratory animals show a high susceptibility to the different microorganisms.

For example, after any method of Streptococcus pneumoniae introduction into a mouse generalized pneumococcal infection are developed. An analogical picture is observed after injection of Mycobacterium tuberculosis into Guinean pig or Mycobacterium bovis into the rabbit.

• 8. Temperature optimum. The cardinal temperatures:

- Minimum

- Optimum

- Maximum

Microorganisms can be grouped by the temperature ranges they require

• Psychrophiles, low temperature optima (4°C) – Polaromonas vacuolata

• Mesophils midrange (39°C) – Escherichia coli

• Thermophiles high (60°C) – Bacillus stearothermophilus

• Hyperthermophiles very high (>80°C) – Thermococcus celer

In everyday practice bacteriologists use such concepts as a species, a strain and pure culture of microorganisms.

Species – a collection of bacterial cells which share an overall similar pattern of traits in contrast to other bacteria whose pattern differs significantly

A strain is a subset of a bacterial species differing from other bacteria of the same species by some minor but identifiable difference. A strain is "a population of organisms that descends from a single organism or pure culture isolate. Strains within a species may differ slightly from one another in many ways."

Culture: population of microorganisms grown under well defined conditions.

Pure culture – one that contains one type of microorganism.

Isolation and identification of a pure culture

First day

1. Microscopic examination of the tested material.

2. Streaking of the material tested onto nutrient media (solid, liquid).

Second day

1. Investigation of the cultural properties.

2. Sub-inoculation of colonies onto solid media to enrich for a pure culture.

Third day

1. Checking of the purity of the isolated culture.

2. Investigation of biochemical properties: (a) sugarlytic, (b) proteolytic.

3. Determination of antigenic properties.

4. Study of phagosensitivity, phagotyping, colicinogensitivity, colicinogenotyping, sensitivity to antibiotics, and other properties.

Main Principles of the Cultivation of Microorganisms

Bacterial cultivation. In laboratory conditions microorganisms can be grown in nutrient media in incubation chambers maintained at a constant temperature. According to the type of heating, incubation chambers can be subdivided into electric, gas and kerosene. Each incubation chamber has a thermoregulator which maintains a constant temperature. Temperature conditions are of great importance for the growth and reproduction of bacteria. In relation to conditions of temperature all micro-organisms can be subdivided into three groups: psychrophilic (Gk. psychros cold, philein love), mesophilic (Gk. mesos intermediate), thermophilic (Gk. thermos warm). Microorganisms may reproduce within a wide temperature regimen range of –10 to +80 °C.

Of great importance in the life activities of bacteria is the concentration of hydrogen ions in the nutrient medium, i. e. pH, which is expressed by the negative logarithm of the concentration of hydrogeons. The pH characterizes the degree of acidity or alkalinity, from extremely acid (pH 0) to extremely alkaline (pH 14) conditions.

During evolution each microbial species adapted itself to existence within certain limits of hydrogen ion concentration beyond the range of which its life processes are unable to take place; It has been suggested that pH influences the activity of enzymes. Depending on the pH, weak acids in an acid medium occur as molecules, and in an alkaline medium as ions. Saprophytes can live in conditions within a wide range of a pH from 0.6 to 11.0, while pathogenic species of microbes grow within certain limits of hydrogen ion concentration Nutrient media should be easily assimilable, and they should contain a known amount of nitrogen and carbohydrate substances, vitamins, a required salt concentration. In addition they should be isotonic, and sterile, and they should have buffer properties, an optimal viscosity, and a certain oxidation reduction potential.

During the whole history of microbiology nutrient media have gradually been perfected. Before Pasteur only infusions and decoctions were used as media for growing microbes. Pasteur and Nageli introduced non-protein media for the cultivation of microbes. Koch and Loeffler employed meat broth, peptone, and sodium chloride for growing microbes. This medium is a meat-peptone broth from which meat-peptone agar is prepared by adding 1-2 per cent industrial agar.

Agar (in Malayan - jelly) is compact fibrous material produced from some seaweed, forms in water solutions a solid gel. Agar contains 70-75% polysaccharides, 2-3% proteins and other nitrogen-containing substances, 2-4% ashes. Main components of agar high molecular weight substances — agarose and agaropectin. Agar dissolves in water while heating and solidifies at room temperature. It is manufactured as colourless plates or powder.

Because of the ability of agar upon cooling to give the nutrient medium a solid gel consistency, and due to its high resistance towards the microbial enzymes, it has received wide application in bacteriological techniques for preparing semisolid, solid, and dry nutrient media.

For the preparation of nutrient media M. Hottinger suggested the use of products of the tryptic breakdown of proteins which do not contain peptones, but contain the low molecular polypeptides and free amino acids. L. Martin employed papain as an enzyme for the break-down of proteins. In recent years all the essential amino acids and vitamins used for the cultivation of bacteria have been obtained in a pure state.

Isolation and Identification of Pure Culture of Aerobic Bacteria

First day. Prepare smears of the tested material and study them under the microscope. Then, using a spatula or a bacteriological loop, streak the material onto a solid medium in a Petri dish. This ensures mechanical separation of microorganisms on the surface of the nutrient medium, which allows for their growth in isolated colonies. In individual cases the material to be studied is streaked onto the liquid enrichment medium and then transferred to Petri dishes with a solid nutrient medium. Place these dishes in a 37 ⁰C incubator for 18-24 hrs.

Incubator

Second day. Following a 24-hour incubation, the cultural properties of bacteria (nature of their growth on solid and liquid nutrient media) are studied.

Macroscopic examination of colonies in transmitted and reflected light. Turn the dish with its bottom to the eyes and examine the colonies in transmitted light. In the presence of various types of colonies count them and describe each of them. The following properties are paid attention to; (a) size of colonies (largo, 4-5 mm in diameter or more; medium, 2-4 mm; small, 1-2 mm; minute, less than 1 mm); (b) configuration of colonies {regularly or irregularly rounded, rosette-shaped, rhizoid, etc.); (c) degree of transparency (non-transparent, semitransparent, transparent).

In a reflected light, examine the colonies from the top without opening the lid. The following data are registered in the protocol: (a) colour of the colonies (colourless, pigmented, the colour of the pigment); (b) nature of the surface (smooth, glassy, moist, wrinkled, lustreless, dry, etc.); (c) position of the colonies on the nutrient medium (protruding above the medium, submerged into the medium; flat, at the level of the medium; flattened, slightly above the medium).

Microscopic examination of colonies. Mount the dish, bottom upward, on the stage of the microscope, lower the condenser, and, using an 8 x objective, study the colonies, registering in the protocol their structure (homogeneous or amorphous, granular, fibriliar, etc.) and the nature of their edges (smooth, wavy, jagged, fringy, etc.).

Use some portion of the colonies to prepare Gram-stained smears for microscopic examination. In the presence of uniform bacteria, transfer the remainder of colonies to an agar slant for obtaining a sufficient amount of pure culture. Place the test tubes with the inoculated medium into a 37 °C incubator for 18-24 hrs.

Third day. Using the culture which has grown on the agar slant prepare smears and stain them by the Gram method. Such characteristics as homogeneity of the growth, form, size, and staining of microorganisms permit definite judgement as to purity of the culture. To identify the isolated pure culture, supplement the study of morphological, tinctorial, and cultural features with determination of their enzymatic and antigenic attributes, phago- and bacterio-cinosensitivity, toxigenicity, and other properties characterizing their species specificity.

To demonstrate carbohydrate-splitting enzymes, Hiss' media are utilized. When bacteria ferment carbohydrates with acid formation, the colour of the medium changes due to the indicator present in it. Depending on the kind and species of bacteria studied, select media with respective mono- and disaccharides (glucose, lactose, maltose, sucrose), polysaccharides (starch, glycogen, inulin), higher alcohols (glycerol, mannitol). In the process of fermentation of the above substances aldehydes, acids, and gaseous products (CO₂, H₂, etc.) are formed.

To demonstrate proteolytic enzymes in bacteria, transfer the latter to a gelatin column. Allow the inoculated culture to stand at room temperature (20-22 °C) for several days, recording not only the development of liquefaction per se but its character as well (laminar, in the form of a nail or a fir-tree, etc.)

Proteolytic action of enzymes of microorganisms can also be observed following their streaking onto coagulated serum, with depressions forming around colonies (liquefaction). A casein clot is split in milk to form peptone, which is manifested by the fact that milk turns yellowish (milk peptonization).

More profound splitting of protein is evidenced by the formation of indol, ammonia, hydrogen sulphide, and other compounds. To detect the gaseous substances, inoculate microorganisms into a meat-peptone broth or in a 1 per cent peptone water. Leave the inoculated cultures in an incubator for 24-72 hrs.

To demonstrate indol by Morel's method, soak narrow strips of filter paper with hot saturated solution of oxalic acid (indicator paper) and let them dry. Place the indicator paper between the test tube wall and stopper so that it does not touch the streaked medium. When indol is released by the 2nd-3rd day, the lower part of the paper strip turns pink as a result of its interaction with oxalic acid.

The telltale sign of the presence of ammonia is a change in the colour of a pink litmus paper fastened between the tube wall and the stopper (it turns blue). Hydrogen sulphide is detected by means of a filter paper strip saturated with lead acetate solution, which is fastened between the tube wall and the stopper. Upon interaction between hydrogen sulphide and lead acetate the paper darkens as a result of lead sulphide formation.

To determine catalase, pour 1-2 ml of a 1 per cent hydrogen peroxide solution over the surface of a 24-hour culture of an agar slant. The appearance of gas bubbles is considered as a positive reaction. Use a culture known to contain catalase as a control.

The reduction ability of microorganisms is studied using methylene blue, thinning, litmus, indigo carmine, neutral red, etc. Add one of the above dyes to nutrient broth or agar. The medium decolorizes if the microorganism has a reduction ability. The most widely employed is Rothberger's medium (meat-peptone agar containing 1 per cent of glucose and several drops of a saturated solution of neutral red). If the reaction is positive, a red colour of the agar changes into yellow, yellow-green, and fluorescent, while glucose fermentation is characterized by cracks in the medium.

Antigen properties of the isolated culture are investigated by the agglutination test and other serological tests.

Species identification of aerobic bacteria is performed by comparing their morphological, cultural, biochemical, antigenic, and other properties.

On solid nutrient media microbes form colonies of different shapes and sizes which are ggregations of individuals connected by bands of cytoplasm providing for a certain structure of bacterial groupings. The colonies may be flat. convex, dome-shaped, or pitted; their surface – smooth (S-forms). rough (R-forms). ridged, or bumpy; their edges may be straight, serralcd. fibrous, or lasseled. The shape of the colonies also differs: e.g. round, rosette-shaped, star-shaped, tree-like. According lo their size the colonies may be divided into large (4-5 mm in diameter), intermediate (2-4 mm), small (1-2 mm), and dwarf (less than 1 mm).

The colonies differ in their consistency, density, and colour. They may be transparent and opaque, coloured and colourless, moist, dry, and slimy.

In liquid nutrient media microbes grow producing a duffuse suspen- sion. film. or precipitate visible to the naked eye.

The growth of bacteria in the laboratory is carried out in test tubes, Petri dishes, and flasks.

In institutes for production of vaccines the cultivation of aerobes is carried out by deep stab methods. This method permits a more rational use of the nutrient substrate, and a large microbial mass can be obtained. The cultures are grown in reactors. Aeration is produced by passing a stream of air through the medium. The method of aeration is used in laboratory investigations to promote rapid growth of bacteria and to study some processes of metabolism.

Reproduction in microbes takes place more intensively in a flowing nutrient medium which is constantly being renewed. For this purpose a spare tank with nutrient medium is installed, from which the mediumenters the cultivator and is carefully mixed with the culture.

Colonies of a different structure.

After this the excess of cultural fluid together with the suspended bacterial cells flows out. When the rate of flow of cells from the cultivator is equal to the rate of reproduction, the number of the microbial population remains constant.

Modem plant equipment is supplied with devices for automatic control over reproduction and other microbiological processes.

In usual laboratory conditions anaerobes develop in stationary or portable anaerostats containing rarefied air up to 1-8 mm or in vacuum desiccators.

Stationary anaerostat (jar)

Portable anaerostat

For successfully cultivating anaerobes it is necessary to seed a large amount of material into the nutrient medium. The nutrient medium should have a certain viscosity which is attained by adding 0.2 per cent agar. The air is removed by boiling prior to seeding, and to inhibit thesubsequent entry of air, the medium is covered with a layer of oil 0.5-1 cm thick. Anaerobiosis is obtained by the adsorption of oxygen on porous substances (pumice, cotton wool, coal) and by adding reducing substances (carbohydrates, peptone, cysteine. pieces of liver, spleen, kidneys, brain, etc.). After seeding, the test tubes are filled up with liquid vaseline. Growth of the anaerobes is usually carried out on a Kitt-Tarozzi

Isolation and identification of a pure culture

First day

1. Microscopic examination of the tested material.

2. Streaking of the material tested onto nutrient media (solid, liquid).

Second day

1. Investigation of the cultural properties.

2. Sub-inoculation of colonies onto solid media to enrich for a pure culture.

Third day

1. Checking of the purity of the isolated culture.

2. Investigation of biochemical properties: (a) sugarlytic, (b) proteolytic.

3. Determination of antigenic properties.

4. Study of phagosensitivity, phagotyping, colicinogensitivity, colicinogenotyping, sensitivity to antibiotics, and other properties.

With regards to obtaining microorganisms in pure culture, are based on mechanical divorced of bacteria tested material inoculate onto surface media in Petri dish by bacteriological loop or pipette and after that streak plating evenly. After that again that glass spatula (don’t burn through the flame) was used for streak plating onto the same second media in Petri dish.The seeding has been done by bacteriological loop too. With that purpose in upper part of Petri dish has been made dense streaking, set free bacteriological loop from superfluous material. After that are made paralel streaks at the last part of the agar. Somever are applied method of laminar dilution, the matter of this method is a stiring diferrent serial dillution tested materials with melting and colling agar in tubes.After that its are flooded into Petri dishes and put down into incubator.The tested materials are boiled of short duration or heat on 80 ⁰C for destroy bacteria without spores. The spores of microorganisms leave still alive and ater reinoculate this materials they are grown.

Fortner method. The agar media is divided into two parts. Onto the one part inoculate E.coli or Serratia marcescens (these microorganisms absorb intensively oxygen) and onto second part taested material. Closely stop up this Petri dish by parafin and put down into the thermostat. This method is used for obtainig anerobe culture.

Reproduction and Growth of Microorganisms

Reproduction in microbes constitutes the ability of self-multiplication, i.e. the increase in the number of individuals per unit volume The growth of micro-organisms represents the increase of the mass of bacterial cytoplasm as a result of the synthesis of cellular material.

Bacteria reproduce by simple transverse division, vegetative reproduction, which occurs in different planes and produces many kinds of cells (clusters, chains, pairs, packets, etc.). They also reproduce by budding, by means of the cleavage of segmented filaments, by reproducing cells similar to spores, by producing minute motile conidia. And by conjugation, which brings us closely to the concept of sexual reproduction in bacteria DNA replication is an important condition in the process of amitotic binary fission of bacteria, the hydrogen bonds are ruptured and two DNA strands are formed, each one is contained in the daughter cells The single-stranded DNA are eventually linked by means of hydrogen bonds and again form double-chain DNA responsible for genetic information DNA replication and cell fission occur at a definite rate characteristic of each species. Actinomycetes and many fungi (phycomycetes, ascomycetes, etc) reproduce predominantly by sporulation

The transverse division of bacteria is not only a process of cell division of one mother cell into two equal daughter cells, but represents d constant separation of daughter cells from the mother cell, the former in their turn become mother cells. After a certain number of generations, the mother cells age and perish. This explanation has annulled the metaphysical concept of 'bacterial immortality'.

The rate of cell division differs among bacteria It depends on the species of microbe, the age of the culture, on the nutrient medium, temperature, concentration of carbon dioxide, and on many other factors.

The length of the generation off coli, Clostridium perfringens, Streptococcus faecalis is 15 minutes, while for the cells of a mammalian tissue culture it is 24 hours Thus, bacteria reproduce almost 100 times faster than cells of tissue culture The increase in the number of cells can be expressed in the following way:

0— 1— 2— 3 — 4— 5— n number of generations

The total amount of bacteria (N) after n generations will be equal to 2ⁿ per cell of seeded material If we take the original amount of bacteria inoculated into the nutrient medium as a single individual, and the time for one division as 30 minutes, then theoretically the total amount of bacteria produced per 24 hours would be equal N=2⁴⁸. Upon division every 20 minutes, in 36 hours the microbial mass will be equal to 400 tons. Thermophilic microbes divide even more rapidly.

However, in natural as well as in artificial conditions, the reproduction of bacteria is of a considerably smaller scale. It is limited by the effect of a number of environmental factors. Reproduction in bacteria conforms to certain laws. Fig. 1 illustrates schematically the rate of reproduction of bacteria in arbitrary units, and the size of the bacterial population expressed as the logarithm of the numbers of live cells per millimeter of the medium.

There are eight principal phases of reproduction which are designated on the diagram by Roman numerals.

1. An initial stationary phase represents the time from the moment of seeding the bacteria on the nutrient medium. Reproduction does not occur in this phase. The length of the initial stationary phase after seeding is 1-2 hours.

2. The lag phase of reproduction during which bacterial reproduction is not intensive, while the growth rate is accelerated. The second phase may last almost two hours.

3. Phase of exponential (logarithmic) growth which is characterized by a maximal division rate and decrease in cell size. The length of this period ranges from 5 to 6 hours.

4. Phase of negative growth acceleration during which the rate of bacterial reproduction ceases to be maximal, and the number of dividing cells diminishes. This phase lasts almost two hours.

5. A maximal stationary phase when the number of newly produced bacteria is almost equal to the number of dead organisms. This phase continues for two hours.

6. Accelerated death phase during which the equilibrium between the stationary phase and the bacterial death rate is interrupted. This continues for 3 hours.

7. Logarithmic death phase when the cells die at a constant rate. This continues almost 5 hours.

8. Decelerated death-rate phase in which those cells which remain alive enter a dormant state.

Graph of the reproduction of bacteria

The length of these phases is arbitrary, as it can vary depending on the bacterial species and the conditions of cultivation. Thus, for example, the colibacilli divide every 15-17 minutes, salmonellae of enteric fever — every 23 minutes, pathogenic streptococci — every 30 minutes, diphtheria bacilli — every 34 minutes and tubercle bacilli — every 18 hours.

References

1. Review of Medical Microbiology /E. Jawetz, J. Melnick, E. A. Adelberg/ Lange Medical Publication, Los Altos, California, 2002. – P.46-87.

2. Medical Microbiology and Immunology: Examination and Board Rewiew /W. Levinson, E. Jawetz.– 2003.– P.14-16

3. Handbook on Microbiology. Laboratory diagnosis of Infectious Disease/ Ed by Yu.S. Krivoshein, 1989, P. 29-74.

4. Essentials of Medical Microbiology / W.A. Volk at al., – Lippincott-Raven, Philadelphia-New-York