General Microbiology - Schlegel, H. 1987

Microbial Growth
Inhibition of Growth and Destruction of Microorganisms by Various Agents

A number of chemical agents slow down or completely inhibit the growth of microorganisms. If a substance suppresses bacterial growth, but growth resumes once the substance is removed, this is referred to a bacteriostatic effect. Bactericidal substances, on the other hand, cause Cell death. However, either effect depends on the concentration of the active substance. Furthermore, some bacterial strains are resistant to general cellular and metabolic poisons (such as hydrogen sulfide, phenol, or carbon monoxide) and may even utilize them as Energy Sources. For many antimicrobial agents, the subcellular target and MECHANISM OF ACTION have been elucidated to varying degrees.

Damage to surface structures or cell layers. Ethanol at a sufficiently high concentration (70%) causes protein coagulation and exerts a bactericidal effect. Phenols, cresols, neutral soaps, and surface-active agents (detergents) affect the outer cell layers and disrupt the selective permeability of The Plasma Membrane. Cell membranes consist primarily of Lipids and Proteins. Detergents have a polar Structure, with their molecules containing both lipophilic groups (long hydrocarbon chains or aromatic rings) and hydrophilic ionized groups. By accumulating in lipoprotein membranes (which also have a polar structure), detergents disrupt their Functions. Because these substances possess a broad spectrum of antimicrobial activity, they are commonly used to disinfect various surfaces and clothing. Certain Polypeptide Antibiotics (polymyxin, colistin, bacitracin, subtilin) and plant-derived antimicrobial substances are similar in their mode of action to detergents.

Damage to Enzymes and metabolic disruption. Certain heavy metals (copper, silver, mercury, etc.) act as potent enzyme poisons even at low concentrations (the so-called "oligodynamic effect"). Whether in the form of salts (HgCl₂, CuCl, AgNO₃) or Organic compounds (such as p-hydroxymercuribenzoate), they bind to SH-groups, thereby profoundly altering the Tertiary and Quaternary structure of enzyme proteins. The functional sulfhydryl group of coenzyme A is also blocked. Cyanide acts as a respiratory poison by binding iron, thereby blocking the function of cytochrome c oxidase, the terminal respiratory enzyme. Carbon monoxide inhibits Respiration by competing with free oxygen for cytochrome c oxidase, acting via "competitive inhibition." Antimycin A disrupts Electron transport along the Respiratory Chain by inhibiting cytochrome c reductase. 2,4-Dinitrophenol uncouples oxidation and phosphorylation processes in Mitochondria. Arsenate inhibits substrate-level phosphorylation. Fluoroacetate blocks The Tricarboxylic Acid Cycle. Initially, like acetate, it is activated and used as a precursor for citrate (so-called Lethal synthesis), and the resulting fluorocitrate inhibits aconitase, thereby halting the further conversion of citrate.

Competitive inhibition. An example of competitive inhibition is the action of malonate, which suppresses The conversion of succinate to fumarate. This action is extremely specific and manifests even at low concentrations of malonic acid. While the inhibition caused by cyanide (at a given concentration) cannot be reversed by increasing the Substrate Concentration (i.e., the partial pressure of O₂), the inhibition caused by malonate can be partially or fully reversed by increasing the concentration of succinate. It is believed that the normal metabolite, succinate, competes with its structural analogue, or antimetabolite, malonate, for the catalytic site of the succinate dehydrogenase enzyme. Competitive inhibition is based on the structural similarity between inhibitors and normal cellular metabolites. An antimetabolite that has entered The Cell can affect BIOSYNTHETIC PROCESSES IN various ways. In the diagram below, three metabolites are shown in black, and three antimetabolites in red.

Class="center">

Disruption of cellular component synthesis. The best-known example of growth inhibition resulting from the incorporation of a structural analogue into a cellular component is the action of sulfanilic acid derivatives. The antibacterial effect of sulfonamides was discovered purely empirically (Domagk); only later was the key to understanding this mechanism found in the structural similarity between sulfonamides and p-aminobenzoic acid (see the structural formulas above). p-Aminobenzoic acid is a component of a coenzyme, specifically tetrahydrofolic acid. In most Bacteria, tetrahydrofolic acid is synthesized from simpler components. However, when p-aminobenzoic acid or sulfonamides are added to the growth medium, these substances readily penetrate the cell and are incorporated into Folic acid. When a sulfonamide is incorporated, it leads to the synthesis of a non-functional coenzyme and, ultimately, to the cessation of cell growth. The Effect of sulfonamides can be reversed by increasing the concentration of p-aminobenzoic acid, as the underlying inhibition is competitive in nature. In animal organisms, folic acid is neither formed de novo nor synthesized from simpler components; animals must obtain it preformed in their diet. Therefore, sulfonamides cannot be incorporated into this coenzyme in animal Cells and thus exert no harmful effect. The feasibility of using sulfonamides as therapeutic agents is due to the limited synthetic capabilities of the animal Organism.

The inhibition of succinate dehydrogenase by malonate and the suppression of bacterial growth by sulfanilic acid derivatives are Examples of antagonistic relationships between normal cellular metabolites and their structural analogues. Antagonism between metabolites and antimetabolites (structural analogues) can manifest at various levels. Structural analogues may prevent the incorporation of normal metabolites and thereby the synthesis of individual cellular components. They may also be incorporated into polymers, which can lead to reduced activity or even complete inactivation of an enzyme, or disrupt nucleic acid function.

Inhibition of Protein Synthesis by antibiotics. Protein synthesis in prokaryotes is specifically inhibited by a number of antibiotics. Their action is directed against 70S ribosome function. Streptomycin and neomycin inhibit the binding of Amino Acids to one another. Erythromycin disrupts the function of the 50S subunits. Tetracyclines prevent the attachment of aminoacyl-tRNA to Ribosomes. Chloramphenicol suppresses the Incorporation of Amino acids into proteins, apparently by hindering amino acid binding mediated by peptidyl transferase. Chloramphenicol (levomycetin) is used in medicine as a highly effective bacteriostatic agent, and in biochemical research as a selective protein synthesis inhibitor that does not affect other metabolic processes. These antibiotics naturally affect the ribosomes of Mitochondria and METABOLISM/14.html">Chloroplasts in Eukaryotic cells as well. However, because the outer membrane of mitochondria, for example, is poorly permeable to streptomycin, this antibiotic at the low concentrations used in therapy has virtually no effect on eukaryotic cells. The division of these Organelles in eukaryotes ceases only when concentrations 1,000 times higher are used. If eukaryotes (Yeasts, Euglena, meristematic zones of higher plants) are treated with high concentrations of streptomycin, the number of mitochondria and chloroplasts will decrease during growth, yielding Cells and Tissues with a severely depleted number of these organelles.

Inhibition of nucleic acid synthesis by antibiotics. Certain antibiotics inhibit nucleic acid synthesis. Mitomycin C selectively prevents DNA Synthesis without initially affecting RNA and Protein synthesis. Its action is believed to be based on The formation of cross-links in the DNA double helix and the breakage of its strands. Actinomycin D forms a complex with double-stranded DNA by binding to guanine residues; it disrupts the synthesis of all Three types of RNA, but does not affect DNA Replication. Rifampicin targets DNA-dependent RNA polymerase, thereby suppressing mRNA synthesis in bacteria.

Inhibition of Cell wall synthesis. The suppression of peptidoglycan synthesis in prokaryotes by penicillin, cephalosporin, and other cell wall-active agents was already discussed in Section 2.2.3.

Death and destruction of microorganisms. The death of microorganisms refers to the irreversible loss of The ability to grow and reproduce; under laboratory conditions, this usually means the loss of the capacity to form colonies. Many types of damage that typically lead to cell death can be reversible under certain conditions. The phenomenon of photoreactivation following ultraviolet irradiation or exposure to high temperatures is well known (Sec. 15.2.2). Quantitative data regarding the death of microorganisms (whether natural or agent-induced) can only be obtained for a population, not for individual cells. In some cases, The rate of decline in the number of living cells in a population at any given time is proportional to the Number of viable cells present; the cell death process then obeys first-order reaction kinetics $N = N_0 \cdot e^{-kt}$ (where $k$ is the rate constant of death). This applies, for example, to radiation sterilization.

6.6.1 Methods of sterilization

The destruction of microorganisms is an essential element of microbiological work and The basis of food preservation; therefore, it deserves a closer look. The process of freeing a material from living microorganisms or their dormant forms is called sterilization. Sterilization should be distinguished from partial reduction (pasteurization) as well as preservation. If a sterile medium or microbial culture becomes contaminated by accidentally introduced microorganisms, this is referred to as contamination. Concepts such as disinfection (the destruction of all pathogenic microorganisms), asepsis and antisepsis, and infection are used primarily in hygiene rather than in microbiology.

Microorganisms exhibit varying sensitivities to agents used for their destruction. There are species-specific differences in sensitivity, as well as variations depending on moisture content and the pH of the medium, the age of vegetative cells or spores, and so forth. The effectiveness of various agents used to destroy microorganisms is characterized by the $D_{10}$ value (the time required to cause the death of 90% of the cells in a given population under specified environmental conditions; see Table 6.5).

Complete or partial sterilization is accomplished using moist heat, dry heat, filtration, irradiation, or various chemical agents.

Moist heat. Vegetative cells of most bacteria and Fungi are killed within 5–10 min at temperatures around 60°C, Yeast and mold spores only at temperatures above 80°C, and bacterial spores above 120°C (15 min). The duration of moist heat exposure required to destroy the spores of certain extraordinarily heat-resistant bacterial species can be derived from the data in Table 6.5. It should be noted that The ultimate outcome of sterilization also depends on the degree of contamination of the treated material—that is, for instance, on the number of heat-resistant spores: the greater their number, the longer the heating must be.

Table 6.5. $D_{10}$ values (in seconds) for spore Suspensions of three aerobic bacterial species (after I. Miller, O. Kandler, Milchwiss., 22 [1967], 686, modified)

To achieve temperatures above the boiling point of Water, an autoclave is used. The Temperature of saturated steam depends on pressure (Fig. 6.12). In the presence of air, a given pressure corresponds to a significantly lower temperature. Since the destruction of microorganisms by moist heat depends on temperature rather than pressure, the autoclave must only be closed after all air has been displaced by steam. Air is removed along with the escaping steam or by evacuation. During autoclaving, temperature should be measured rather than pressure, although for reasons of simplicity and safety, pressure is still commonly measured. The duration of sterilization naturally depends on the volume (heat capacity) of the vessels in which it is performed (Table 6.6).

Fig. 6.12. Saturated water vapor pressure.

Table 6.6. Sterilization times for liquids in various vessels using an autoclave (121–123°C)

The same effect can often be achieved through fractional sterilization in flowing steam at 100°C (tyndallization). In this method, the liquid is sterilized at 100°C for 30 minutes on three consecutive days; between heating sessions, it is kept in an incubator to allow spores to germinate, so that the resulting vegetative cells can be destroyed during the subsequent heating.

For many purposes, partial sterilization—that is, the destruction of vegetative forms of microorganisms—is sufficient. This effect is usually achieved by pasteurization, which involves holding the material at 75 or 80°C for 5–10 minutes. Milk, in particular, is partially sterilized by pasteurization; however, to avoid spoiling its flavor, the exposure time is shortened in this case. Two methods of milk pasteurization are employed: high-temperature short-time (HTST) pasteurization (20 s at 71.5–74°C) and flash pasteurization (2–5 s at 85–87°C). Complete sterilization of milk is achieved through ultra-high-temperature (UHT) Processing. In this method, superheated steam is injected into the milk, raising the temperature of the mixture to 135–150°C. The milk is exposed to this temperature for 1–2 seconds. Then, by passing the milk through a nozzle, the pressure is reduced while the milk is simultaneously cooled; this process also removes the water introduced as steam.

Methods of preserving berries and stone fruits should also be considered as a form of partial sterilization. Conventional heating of canned goods for 20 min at 80°C destroys only vegetative cells and the spores of many fungi, whereas bacterial spores remain viable. Low pH levels, resulting from the presence of acids in fruit juice, prevent bacterial spores from germinating. Pasteurised strawberries are frequently affected by the so-called "strawberry fungus" Byssochlamys nivea. Its ascospores can withstand 86°C, at which temperature D₁₀ is 14 min.

Dry heat. Bacterial spores tolerate higher temperatures and for longer periods during dry-heat sterilization than during moist-heat sterilization. Consequently, heat-resistant glassware, powders, oils, and similar Materials are sterilized for 2 h at 160°C in a dry-heat sterilizer. When sterilizing materials with high heat capacity or thermal insulation properties, heating time must be taken into account. In all cases, it is advisable to monitor the temperature using indicators or to verify sterilization completeness (by placing a soil sample containing spores into the apparatus, which is subsequently cultured). Whenever the material being sterilized permits, a 30-minute heating cycle at 180°C is currently applied. Experience shows that this destroys all spores. Heat sterilization is based on the coagulation of cellular proteins.

Filtration. Solutions containing thermolabile substances are most conveniently sterilized by filtration. Unglazed porcelain cylinders (Chamberland candles) were already in use in Pasteur's laboratory. Berkefeld filters (made of compressed kieselguhr) are utilized in laboratories and for sterilizing drinking water. Asbestos pads (in Seitz filters), Glass filters, and membrane filters are also widely employed. Some of these are available with varying pore sizes, enabling the Separation of organisms of different Sizes and Shapes.

Irradiation. Ultraviolet, X-, and gamma rays are used for complete or partial sterilization. In laboratory settings, ultraviolet rays are of the greatest importance. THE SPECTRUM OF UV lamps is dominated by radiation around 260 nm, which is primarily absorbed by Nucleic Acids and, with sufficiently prolonged exposure, causes the death of all bacteria (see Section 15.2.2 and Fig. 15.5). UV irradiation is used for the partial sterilization of rooms; bacteria are killed very rapidly under these conditions, whereas fungal spores, which are far less sensitive to ultraviolet light, are destroyed significantly more slowly. Ionizing radiation is used to sterilize foodstuffs and other compact materials.

Chemical agents. In the sterilization of foods, Pharmaceuticals, and various types of equipment, as well as in laboratory practice, The Use of Ethylene oxide has proven highly effective. It kills both vegetative cells and spores, but is only active if the materials being sterilized contain a certain amount of water (5-15%). Ethylene oxide is applied as a gas mixture (with N₂ or CO₂) in which its proportion ranges from 2 to 50%.

To preserve thermolabile substances present in nutrient media, sterilization with β-propiolactone was introduced into practice. It is considerably more active than ethylene oxide, but appears to possess fairly strong carcinogenic properties and causes a number of other adverse physiological effects. It is added at a concentration of 0.2% to prepared nutrient media, which are then incubated for 2 h at 37°C. If left overnight, the propiolactone decomposes completely.

CARBOHYDRATES remain unaffected by this process. Beverages are also sterilized using diethyl pyrocarbonate (0.003-0.02%).

For the sterilization of seeds used in the cultivation of sterile plants, conventional antimicrobial agents are suitable, such as bromine water (1%), corrosive sublimate (HgCl₂; a 1% solution in alcohol), AgNO₃ (0.05%), calcium hypochlorite [1% Ca(ClO)₂], Uspulun, and others, applied for 5-30 min. Prior to this Treatment, the seeds should be washed with soap or another surfactant to ensure complete surface wetting.

6.6.2 Preservation Methods

Organic materials decompose As a result of microbial activity unless specific preventive measures are taken. A variety of methods can be employed to preserve organic substances. Methods ensuring the preservation of food products are of paramount importance. These issues fall within The Scope of food microbiology.

Food products become unfit for consumption not only through microbial decomposition (aerobic oxidation or anaerobic putrefaction), but also as a result of colonization by bacteria and fungi that produce toxins. The most significant toxin producers that contaminate food are Clostridium botulinum and various species of staphylococci. C. botulinum secretes an exotoxin that is extremely potent even in small quantities and attacks The Nervous system. Staphylococci produce an enterotoxin that causes so-called food poisoning. Certain fungi produce mycotoxins, of which the best known is aflatoxin (a metabolite of the fungus Aspergillus flavus).

The preservation methods used to protect food products from microorganisms are highly diverse, encompassing both Physical and Chemical techniques.

Physical methods. We have already discussed sterilization using high temperatures. Metal cans are generally heated in an autoclave. For preserving acidic fruit juices, pasteurization is sufficient; this kills only vegetative cells while spores remain viable, though bacterial endospores do not germinate in acidic environments.

Fruit juices, mineral waters, and pharmaceutical preparations are sterilized by passing them through fine-pored asbestos or Cellulose filters. In winemaking, centrifugation and filtration are employed to halt Fermentation at the desired stage (in order to retain "residual sugar").

The traditional and widespread method of food preservation by drying is based on the fact that microorganisms require a specific level of moisture for growth (typically more than 10% water). Rolled oats, dried fruits, hay, and grain in silos are preserved precisely due to their dry state; in humid air, once damp, they rapidly spoil under the action of fungi and bacteria.

The application of irradiation for preservation remains limited. Ultraviolet rays are primarily used to sterilize air in dairies, cold storage facilities, bakeries, and similar environments. Food preservation using ionizing radiation is theoretically viable due to the high penetrating power of these rays; however, this method has not yet come into widespread use. A reliable method that is even beginning to compete with pickling and Other forms of home preservation in domestic settings is low-temperature storage. In deep-freezing chambers, products are kept at temperatures below -20°C. At such temperatures, microbial viability does not noticeably decline and their toxins are not destroyed, but growth ceases entirely. Even psychrophilic bacteria are unable to grow at temperatures below -12°C.

Chemical methods. Preservation by acidification is based on the fact that at low pH levels and in the absence of air, very few microorganisms can grow. Simple pasteurization is sufficient to destroy them. Heat-resistant spores do not germinate at pH levels below 5.0. Natural acidification resulting from Lactic acid fermentation is utilized in The production of sauerkraut, silage, pickles, and raw-smoked sausages (salami, cervelat). Vinegar, lactic, tartaric, or citric acid are frequently added to foods for preservation purposes. When exposed to air, unpasteurized acidic products are decomposed by yeasts and other fungi.

Meat and fish products are preserved by smoking. Smoking involves exposure to the distillation products contained in wood smoke—phenols, cresols, aldehydes, acetic acid, and formic acid. All of these substances possess antiseptic properties, an effect that is enhanced by the removal of a portion of moisture from the treated product.

For salting, products are placed in a 14-25% sodium chloride solution. This reduces the water content of the product and suppresses the growth of spoilage microorganisms. Under these conditions, only a few halophilic bacteria retain the ability to multiply.

High concentrations of sugar (approximately 50% sucrose) also inhibit Microbial growth. Marmalade and various syrups are preserved primarily due to their high acid and sugar content.

The preservation of certain food products necessitates the use of chemical preservatives. Sulfurous acid was previously added to wine for this purpose; wine and fruit juices can (if necessary at all) be preserved by The addition of diethyl pyrocarbonate.

Sorbic, benzoic, or formic acid are also used to preserve food products. Citrus fruits are treated with diphenyl or o-phenylphenol. Attempts are currently being made to utilize antibiotics for preservation.