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Microbial Control

 

Introduction
Defination of Terms
Factors Influencing Antimicrobial Activity

Cell Injury

The Physical Environment
The Chemical Environment
Standardization of Disinfectants

 

Introduction

In the proceeding chapters, optimum physical and chemical conditions required for the growth of microorganisms have been described. A knowledge of the methods for killing, removing, and inhibiting (preventing growth of)microorganisms is an essential aspect of microbial control. There are four main reasons for killing, inhibiting or removing microorganisms. They are: (1) to prevent transmission of disease and infection in man, animals, and plants, (2) to prevent decomposition and spoilage of food and food products, (3) to prevent interference by contaminating microorganisms in industrial processes that depend on pure cultures and (4) to prevent contamination of materials used in pure culture work in laboratories, so that studies on one type of organism in a particular environmental condition will not be confused by the presence and growth of other types at the same time.

Microorganisms can be killed, removed or inhibited by various physical and chemical agents. It should be realized, however, that physical factors probably exert their influence upon the cell by influencing chemical changes in the environment or within the cell. A number of physical and chemical agents are available. They act in many different ways, and each has its own limited practical appli­cation. In the first place it is important to understand certain modern terms used to describe these agents and their results.

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Definition Of Terms

Several modern terms used to describe the physical processes and chemical agents employed in controlling microorganisms are being explained below.

Sterilization. Sterilization is a process of destroying all forms of life by physical or chemical agents. The term may also include the removal of organisms by means of centrifugation or filtration. It refers to the complete absence or destruction of microorganisms, and should not be used in a relative sense. An object or. substance is sterile or non-sterile; it can never be semisterile or almost sterile.

Disinfection. Disinfection means the killing or removal of organisms capable of causing infection. Disinfection is usually accom­plished by chemical agents called disinfectants. It is generally agreed that this means an agent that kills vegetative cells, but not heat-resistant spores. A disinfectant is normally applied to inanimate objects such as floors, utensils, buildings, equipment, laundry, etc.

Antiseptic. The term antiseptic is used to designate any substance which would prevent sepsis, either by killing microorganisms or by inhibiting their growth and activity. It is closely similar to a disinfectant. An antiseptic can be applied to body tissues without causing injury to the tissue. On the other hand, most disinfectants are too destructive of tissues.

Sanitizer.   A sanitizer is an agent that reduces the microbial population to safe levels,  as may be judged by public health require­ments. Sanitizers are commonly applied to inanimate objects.Germicide. A germicide is an agent that kills vegetative cells, but not necessarily the resistant spore forms of germs. The term bactericide is synonymous with germicide, but is more specifically applied to a substance (hat kills bacteria. Similarly, the terms fungicide, virucide and sporicide refer to agents that kill fungi, viruses, and spores, respectively.

Microbiostasis. Microbiostasis is a condition in which the growth of microorganisms is prevented. In the presence of a microbiostatic agent microbial metabolism is inhibited. The microorganisms die over a period of time without significant multiplication. However, removal of microbiostatic agents and the presence of favourable environmental conditions result in growth of microorganisms.

Antimicrobial agent. An antimicrobial agent interferes with the growth and activity of microbes. In common usage the term denotes inhibition of growth.

Preservative. A preservative prevents the growth of micro­organisms, it is used most frequently in connection with food preservation.

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Factors Influencing Antimicrobial Activity

Herbert Spencer once defined life as "a continuing series of chemical reactions among molecules of great complexity." While this definition has some limitations, it can serve as starting-point to emphasize that anything which can interfere with the continuing reactions or with the molecular complexity can kill. But this leaves still a further factor. Not all of the reactions of a cell occur in all parts of it. There are structures within it, and interference with these structures can also kill. Also it must be recognized that one cannot say simply that heat will kill. Obviously one must specify how much heat. Thus many factors must be considered in the application of any physical or chemical agent to inhibit or destroy microbial popula­tions. Broadly, they are classified into two groups (1) factors related to a killing agent, and (2) factors related to organisms to be killed. The factors are listed in the Table.

Table.  Factors influencing antimicrobial action .

Related to filling agent

Related to organisms to be killed

1.

2.

Intensity (if physical), concentration (if chemical) The time during which the agent acts.

1 .  The number of organisms to be

2.Kind of organisms.

 3.  The physiological state of the

killed.

3.

The   temperature   at   which the agent acts.

organisms.

4.  The environmental condition in the killing takes place.

which

 
The intensity or concentration. The term intensity is applied to physical factors. Temperature can be considered as an illustration of intensity. The very heat resistant spore of Clostridium botulinum may be boiled at 100C for hours, with little effect. At 113C it can be killed in 60 minutes, at 114C in 39 minutes and at 119C in 10 minutes. Since intensity and time are interrelated, the effectiveness of killing in terms of the time required to kill can be expressed. Obviously the higher the temperature, the more effective is the agent.
The term concentration is applied to chemical agents- As an illustration of concentration, phenol can be considered. For example 0.1.% phenol will not kill E. coli, but will only prevent its growth. At 10 times this amount, i.e., 1%, phenol will kill in 20-30 minutes. And 5% phenol will kill in 2-3 minutes. However, there is an optimal, concentration of a chemical agent, beyond which it accomplishes increasingly less, and is wasteful.
The time an agent can act. No agent, as ordinarily used, acts instantly. Sufficient time for contact and for whatever physical and chemical reactions that occur must be allowed. This is described as the dynamics of disinfection, which is as follows:
1.    The process is a gradual operation in which the   number of organisms killed in unit time is greater in the beginning, but becomes less and less as the exposure period is increased.
2.    The number of surviving organisms in unit volume plotted against time gives a smooth curve.

3.    The log of number of the surviving organisms in unit volume plotted against time gives a straight line curve.  The curve is similar to that obtained when one  plots the decay of a radioac­tive substance against time. It is a monomolecular reaction, or a reaction of the first order, provided the anti-microbial agent is present in large excess,

Fig. Dynamics of disinfection

Secondly, time is inversely proportional to intensity or the concen­tration factor, i.e. the longer the time, the smaller the intensity or con­centration factor. This is dearly evident in the examples given in the previous discussion. At certain times a longer time at lesser temperature is preferred  to prevent the deterioration of flavour, texture, etc., of biological material

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Cell Injury

Damage to the cell wall. The lowering of surface tension of the medium in which microorganisms are suspended often injures the cell wall. Substances which reduce surface tension are called surfactants. Scaps, bile salts, hexylresorcinol, and many cationic and anionic deter­gents are surfactants. The damage is caused, at least in part, by the dissolving or emulsifying effects of the surfactants on lipids. Several hydrolytic enzymes present in natural sources, or elaborated by some microorganisms, hydrolyze the microbial cell wall. The enzyme lysozyme found in tears, leucocytes, mucous secretions, etc., hydrolyzes the cell walls of many bacteria, especially gram positive bacteria. -gluconase, chitinase, cellulase and proteolytic enzymes produced by several micro­bial species are capable of hydrolyzing the cell wall of other species. Antibiotics such as penicillin, cycloserine, etc., inhibit the formation of cell wall material in a growing bacterial culture, with the resulting formation of protoplasts. Partial or complete removal of the cell wall leads to osmotic rupturing of the unsupported cell membrane and dissolves the cell (lysis).

Damage to the cytoplasmic membrane. Surfactants, which dissolve lipids, disrupt the cytoplasmic membrane. This destroys the selective permeability of nutrients and metabolites. This results in the disruption of normal metabolic processes, leading to the inhibition of growth and the death of the cell. The antimicrobial activity of phenolic-compounds, synthetic detergents, soaps, quaternary ammonium com­pounds, antibiotics (e. g. polymyxin), etc., is due to their effects on the cell membrane.

Denaturation and coagulation of protein. Native configura­tion of protein molecules is essential for normal metabolic activity of the cell. A condition or substance which alters this native state, i.e., denatures or coagulates the proteins, may damage or kill the cell. High temperatures, alcohol, formaldehyde, etc., are important denaturants or coagulants.

Inhibition  of  metabolic reactions.  Metabolic activity of the cell is determined by various enzyme-catalyzed reactions, An agent which inhibits any one of these reactions therefore alters the normal metabolic  pattern of a  cell.   Inhibition of the   energy-generating metabolic  pathways (catabolism) may be particularly detrimental. Glycolysis, the tricarboxylic acid cycle, biological oxidation and oxidative phosphorylation are important pathways of catabolism. Enzymes in these key pathways are inhibited by a number of agents. For example, fluoride inhibits glycolysis, and Trivalent arsenic compounds block the tricarboxylic acid cycle. Cyanide is a potent inhibitor of cytochrome oxidase, while dinitrophenol uncouples oxidative phosphorylation. Oxidizing agents stronger than molecular oxygen are often microbicidal. Such agents include halogens; hydrogen peroxide, sodium perborate, potassium permanganate, etc. A sulphydryl group(-SH) is an important constituent of many enzymes. An alteration of this group by au oxidizing agent inactivates the enzyme.

Inhibition of biosynthetic reactions by specific compounds more commonly leads to stoppage of growth and division (microbiostasis), Compounds which are structural analogues of the substrates or the coenzymes of enzymatic reactions compete for the enzymes and thereby prevent the synthesis of essential metabolites. Such compounds are known as antimetobolites. Sulphonamide drugs and antibiotics function as competitive inhibitors of certain essential reactions of biosynthetic pathways

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The Physical Environment

Microorganisms occur everywhere on the surface of the earth and are able to grow and survive under a wide range of environmental

conditions. However, there are limitations to the variations in the environment that a particular species can tolerate. Drastic changes in the physical environment results in 'the inhibition or destruction of microorganisms. A variety of physical factors that control microbial growth are briefly described.

1. Temperature. Temperature is a factor always present in the environment of any living organism. Each particular type of microorganism has an optimum, minimum and maximum temperature at which it will grow. Temperatures above a maximum exert a killing effect,, whereas temperatures below a minimum inhibit microbial growth. Heat is the most widely used method of sterilization, because it is one of the most effective, reliable, and economic sterilizing agents.

Vegetative cells of microorganisms are killed by exposures to temperatures between 60 to 70oC for 5 to 10 minutes. Higher tempera­tures are required to kill microbial spores. Endospores of bacteria are the most heat resistant of all living things. Temperatures above 100oC for extended periods of time are required to kill bacterial spores. Spores of yeasts, moulds, and actinomycetes are less heat resistant, . and are killed at temperatures between 80 to 90oC. The susceptibility of most viruses to heat is similar to that of the vegetative cells.

Certain terms are used as a common basis for comparative studies in heat resistance of microorganisms. One of these is thermal death point. This is denned as the lowest temperature required to kill a particular type of microorganism when heated in a standard nutrient broth for 10 minutes. Since all the cells in a given population do not die at one instant, some dying much more quickly than others, a thermal death rate is sometimes preferred. .However, this is more difficult to determine. As death occurs over a period of time, another term, thermal death time, is also used. This is defined at the shortest period of time required to kill a microbial suspension at a prescribed temperature and under specific conditions. These terms express a time-temperature relationship to killing, i.e. temperature can be varied when time is constant, or time can be varied when temperature is constant. In experimental determination of these values, several factors must be considered. Conditions producing variations are: (1) composition of the medium, (2) hydrogen ion concentration (pH) of the medium, (3) the number and age of the cells, (4) presence of spores in a culture of spore forming specie!, and (5) water content

Degrees centigrade

Time and temperature required to kill spores of Clostridium botulinum)

of the medium. The killing action of heat is, therefore, a time-temperature relationship affected by various environmental condi­tions.

Practical procedures by which heat is employed for sterilization are conveniently divided into two categories, moist heat and dry heat. The killing efficacy of moist heat and dry heat vary considerably. Heat kills orga­nisms by denaturation or coagu­lation of cellular proteins, and the presence of moisture facili­tates this procedure. This is illustrated by studying the coagulation temperature of a protein at different water contents.

Relationship  between moisture content and temperature of coagulation of egg albumin.

Temperature of

coagulation, C

Amount of water, percent

170

0

147

10

75

25

56

50

 
As the percentage of moisture is increased, the temperature of coagulation becomes progressively less. Similarly, the remarkable heat resistance of spores is attributed to their low water content illustrates the time and temperature required to kill the spores of Clostridium botulinum with moist heat and with dry heat. Moist heat kills the spores in 5-20 minutes at 120C, while dry heat requires 2 hours at this temperature.
 
Moist Heat : boiling in water The practice of exposing glassware, instruments, fruit jars, etc., to boiling water for 5 minutes will destroy all
vegetative cells. However, some bacterial spores will remain alive even after boiling for many hours. Materials cannot be sterilized with certainty, and boiling water is therefore not used as a method of sterilization in the laboratory.
 
Moist heat: fractional sterilization or tyndallization.  To accomplish fractional sterilization or tyndallization, free flowing steam at a temperature of 100C is used. John Tyndall devised a process of sterilization by steaming for 20 minutes at 100C for three consecutive days, with an incubation period of 24 hours at room temperature. During the incubation period spores germinate into vegetative cells which are killed at 100C on subsequent exposure to heat. If the spores are present and do not germinate during the incubation period, the method will fail to sterilize the material. Failure may be due to the following.: (1) The medium may be unsuitable for the germination of spores, for example water. (2) Spores of anaerobic bacteria will not germinate if the material is in contact with air, and spores of aerobic bacteria will not germinate if the material is not freely exposed to air. Another disadvantage is that it is lime-consuming. The method is principally used for the sterilization of materials which are hydrolyzed or decomposed at a temperature over 100C. An apparatus known as the Arnold sterilizer is used for this technique Relationship between steam pressure and temperature.

Steam pressure, 1 b /sq. inch.

Temperature, C

0

100.0

5

109.0

10

115.0

15

121.5

20

126.Q

25

130.5

 
Moist heat: steam under pressure. Saturated steam under pressure is the most practical and dependable agent for sterilization. Steam under pressure is hotter than boiling water or free flowing steam. Water boils at about 100C, depending upon the vapour pressure of the atmosphere. If the vapour pressure is increased, the temperature will be increased. The relationship between steam pressure and temperature is shown.
The laboratory apparatus designed to use steam under pressure is called an autoclave. It is a cylindrical metal vessel having double walls, except at the opening. It is built to withstand a steam pressure of at least 60 lb per sq. in. It is fitted with various gauges, pipes, valves, clocks and wheels. In principle, it is very much similar to a home pressure cooker, which is in fact is a simple form of autoclave.
Certain precautions are necessary to prevent sterilization failures. The most important single cause is incomplete evacuation of air from the chamber. It must be remembered that it is the compressed steam (moisture, hydration) that sterilizes and not compressed air (dry, and usually not as hot as steam). The temperature of a mixture of steam and air at given pressure is less than that of pure steam alone. This means that although the autoclave is kept at the desired pressure, the temperature required to give complete sterilization is not obtained. For example, pure steam at 15 pounds pressure has a temperature of 121.5C. If the steam is mixed with an equal amount of air, at the same pressure, the temperature is only 112C. Thus it is not the pressure that kills the organisms but the high temperature of the steam. Another important precaution to be observed is that the steam must have access to the materials  to be treated. Nonfluid materials should be loosely packed to allow space for the circulation of steam.
The autoclave is used to sterilize anything that is not injured by steam and high temperature. This includes most types of solid and liquid media, solutions, rubber tubing and stoppers, discarded cultures and contaminated media prior to washing, etc. Some materials, however, cannot be sterilized by autoclaving. Substances immiscible in water, such as fats, and oils, cannot be reached by the steam; hence organisms contained in them will survive. Furthermore, certain ingredients used in culture media, such as growth factors, some sugars, etc., are decom­posed by extensive heat treatment. These materials are therefore sterilized by other methods.
 
A variety of sterilizing indicators are now available which show whether the heat penetration has been adequate. Two general types of sterility checks are commonly employed. One consists of some thermo-labile dye applied on pieces of papers, guaze, or thread, which are inserted at various places in the autoclave. The dye gives a characteris­tic colour if a desired temperature is attained for a sufficient time. A second method employs the use of very heat-resistant spores in sealed ampoules, or on strips of filter paper inserted in the autoclave. If the spores grow in a suitable medium, then it shows that the sterili­zation process was inadequate.
Dry heat : hot-air sterilizer. As mentioned earlier, dry heat is much less effective as a killing agent than moist heat. Dry heat dehydrates the cells, and the resulting lack of water reduces the proba­bility of protein coagulation. Dry heat acts mostly by oxidizing or bur­ning the cell constituents. The apparatus employed for this type of sterilization is an electric or gas oven. It is operated at a temperature of 160C to 180Cfor 1 to 2 hours. Only dry articles not injured by baking (glassware, instruments, oils, powders, and similar substances) are thus sterilized. Solutions containing water, alcohol, or other volatile substances will boil to dryness.
Dry heat: incineration. Dry heat in its extreme incinerates, i.e. actually burns up the cell constituents. Incineration is used for the disposal of infected materials. This, of course, is what is done in the laboratory when inoculating needles, forceps, etc. are introduced into the flame of a bunsen burner. However, care should be taken to prevent spattering, because the droplets which fly off are likely to carry viable organisms.
Low temperatures. Living processes do not appear to go on below OC This is mainly due to the formation of ice crystals in the cells. These cut the membrane or coagulate the proteins. Microorganisms are generally resistant to freezing, and many survive extreme cold. The initial exposure of the microbial population to freezing temperatures kills some organisms, but those which survive are capable of remaining viable for years. These organisms are considered dormant, that is they do not perform any detectable metabolic activity. Low temperatures, therefore, cannot be depended upon for sterilization or disinfection. Generally, low temperatures are used for the preservation of foods. Frozen foods, therefore, are not sterile. Organisms are present, and when conditions change and the temperature rises, they are ready to grow. Thus high temperatures may be considered as microbicidal and low temperatures (freezing and lower) as microbiostatic.
2. Desiccation
Just as water and its availability is a condition of life, so is its lack a factor in death. Microorganisms are seldom harmed by an excess of water, except indirectly, as through restriction of other nutrients. Dehydration of microbial cells and their environment restricts the metabolic activities, followed by a decline in the total viable population. The time of survival of microorganisms after desiccation varies, some being more resistant than others. Spores may be dried and kept for years under laboratory conditions. Many vegetative cells are killed in a few hours by drying, for example gonococci and meningococci. Extensive generalization is not possible, since some pathogens such as haemolytic streptococci survive for weeks in floor dust, dry blankets, or when smeared on dry paper. Similarly, tubercle bacilli dried in sputum remain viable for even longer periods. As an agent of sterilization, drying is not adequate. Like low temperatures, desiccation may be considered microbiostatic.
These generalizations of the effects of dehydration apply to air drying. By using proper conditions, microorganisms can be preserved for long periods of time. This is achieved through the process of lyophilization. In this process microorganisms are subjected to extreme dehydration in the frozen state, and then sealed under a vacuum. 3. Osmotic pressure
Osmosis is diffusion in which solvent and small solute mole­cules pass through a semipermeable membrane. It tends to equalize the concentration of the substances on either side of the membrane. The term osmotic pressure refers to the unbalanced pressure that gives rise to the phenomenon of diffusion and osmosis in a solution in which there are differences in concentration. A microbial cell is a small system surrounded by a semipermeable membrane which will allow water and small molecules to pass quite rapidly. As an illustration, assume that microbial cells are suspended in a solution containing a high concentration of salt or sugar. Water will pass from the region of lower concentration of dissolved substance (interior of the cell) into the solution surrounding the cell. This will continue until an equili­brium is established between the osmotic pressure inside and outside the cell. The cell becomes dehydrated, and the cytoplasmic membrane is drawn away from the cell wall with the cytoplasmic contents,. The Cell is then said to be plasmolysed, and the process is called plasmolysis. The solution on the outside is hypertonic with respect to the solution inside, the cell. On the other hand if, microbial cells are suspended in a solution containing a very low concentration of salt or sugar or plain distilled water, then water will flow from the solution into the cell. Water continues to move inward" until a balancing osmotic pressure is attained. This pressure may cause swelling, and may even burst the cell releasing its constituents. The cell is then plasmoptyzed, and the process is called plasmoptysis. In this case, the solution on the outside is hypotonic with respect to the solution on the inside of the cell. Microorganisms usually do not exhibit striking changes because of their rigid cell walls, and are less sensitive to osmotic effects than plant and animal cells. A greater increase in the osmotic pressure of the surrounding solution is necessary before any destructive action in microorganisms is observed.
Microorganisms are generally inhibited by high concentrations of salt (10 to 15 percent) and sugar (50 to 70 percent). The use of high osmotic pressure finds practical applications in the preservation of some foods from microbial attack. Jams, jellies, and condensed milk are preserved by concentrated sugar solutions. Meat, corned beef, fish, etc., are preserved by salting. In .such cases the mechanism of microbial inhibition is plasmolysis. The cells are dehydrated, and hence are unable to metabolize and grow. Microorganisms are not necessarily killed. Osmotic pressure, therefore, is also microbiostatic.
Adaptability of some microbial species to varying osmotic pressure is quite amazing; for example in organisms indigenous to oceans, where salt concentration is quite high. Some microorganisms are found in commercial pickling brines, where the salt concentration is between 20 to 30 percent. Microorganisms which require large amounts of salt in media before they grow are called halophilic. Similarly some microorganisms are capable of growing in high concen­trations of sugar, and are termed saccharophilic. The concentration of these two ingredients as natural preservatives in the food industry may often prove inadequate to prevent spoilage.
4. Surface tension.

The interface, or boundary between a liquid and a gas, is characterized by unbalanced forces of attraction between the molecules in the surface of the liquid and in the interior. As can been seen from Fig., a molecule at the surface of the liquid-air interface is pulled strongly towards the interior by the molecules beneath it, whereas molecules in the interior of the liquid are attracted uniformly in all directions. The molecular forces at the liquid-air interface imparts a distinctive characteristic, known as surface tension, to the surface of a liquid.

Fig.. Forces acting upon molecules of liquid. (A) In the body of the fluid, forces are equally distributed. (B) At the liquid-air interface, forces are unevenly distributed.

Presence of nutrients as well as waste products in the media may either increase or decrease the surface tension. Substances such as soaps, synthetic detergents, ethanol, glycerol, etc., which lower the surface tension, are inhibitory for the growth of microorganisms. Toxicity of disinfectants is enhanced by lowered surface tension. Apparently, the lowered surface tension increases the accumulation of the toxic agent at the cell surface, and this in effect increases its concentration.

5. Radiations.

Energy transmitted through space or through a material medium is generally called radiation.  Major types of radiation are electro­magnetic, acoustic and particle radiation. Electromagnetic radiation is the most significant. Fig. shows the broad bands of electro­magnetic radiations distinguished by their wavelengths.

Ionizing radiations. Gamma rays and x-rays have energies of more than about 10 electron volts.

Fig. Spectrum charts. The nanometer (nm) used to express the wavelength of light and ultraviolet   radiations, is equal to I /1,000,000th of millimeter or 10A

They also have great penetrating power. They pull electrons away from molecules and ionize them, and are therefore called ionizing radiations. When they pass through the cell they form free hydrogen and hydroxyl radicals and some per­oxides, which in turn react with cellular constituents and bring about damage to the cell. Since the damage is produced in a wide variety of constituents, ionizing radiations are non-specific in their effects. There are several indications which link their damaging effects to oxygen. One main assumption is that free radicals oxidize vital materials in the cell. In spite of their great penetrating power and toxicity, they are impractical for purpose of sterilization because of the following reasons:

1.Their large scale production is expensive,

2.Their efficient   utilization is difficult, since   radiations are given off in all directions from their point of origin,

3.Their continuous use is hazardous to the operators. 

Ultraviolet light.  The ultraviolet portion of electromagnetic radiations includes all radiations from 150 to 3900A. It is less ener­getic and does not ionize. It is absorbed quite specifically by many cellular constituents, but primarily by the nucleic acids. It produces a variety of abnormal structures in DNA, such as thymine dimers. These structures interfere in the replication of DNA, and therefore produce Mutations.

Ultraviolet light around wavelength 2600A is a powerful steri­lizing agent. It is as toxic to spores as to the vegetative cells. Many lamps are available which emit a high concentration of ultraviolet rays in the most effective region around 2537 A. They are called germicidal lamps or sterilizing lamps. These lamps are used to reduce the microbial population in hospitals, school rooms and packaging rooms and in the food, dairy and pharmaceutical industries. The chief difficulty is that ultraviolet rays have very little ability to penetrate matter. For example, a thin layer of glass filters off a large percentage of the rays. It can, therefore, act on direct contact only.

Damage to DNA caused by ultraviolet rays can be repaired by visible light. This phenomenon is called photo reactivation. A microbial suspension is irradiated in ultraviolet light, until a small fraction is able to form colonies in a nutrient medium, and thus appear viable. A portion of the irradiated cells is then exposed to visible light for several minutes and plated. The fraction of survivors in this portion is much higher. That is, the organisms killed by ultraviolet light are restored to viability by visible light. This is due to a light-dependent enzyme that removes thymine dimers and restores the normal DNA structures. A second mechanism, dark reactivation, which is not light dependent, requires the presence of two enzymes. A hydrolytic enzyme excises the dimers and a polymerase replaces the excised regions by copying the undamaged DNA strand.

Ultrasonic vibrations. Organisms maybe subjected to another physical factor which certainly should be classed as radiation. This is acoustic radiation. It is a wave propagated in air, that is the sound wave. Microorganisms ate not susceptible to normal sound. They are, however, sensitive to ultrasonic waves of 20 kilocycles per second. Ultrasonic vibrations create gas bubble cavities in the liquid. When the cavities collapse, extremely high pressures are produced. The extreme fluctuations of pressures associated with cavitation break the cell wall structure, and intracellular constituents are liberated- Sonication is not very practical for sterilization but is very useful as a method of disrupting cells for the purpose of isolating sub-cellular structures, organelles, enzymes, endotoxins, antigens, etc.

Electricity. Electric currents at varying voltages, intensity, and periods of action have been investigated as means of destroying microorganisms. Equipment using electricity has been designed and used for pasteurization of milk and fruit juices and disinfection of water, sewage, etc. The killing effect may be due to :

(1)  Heat produced by resistance to the passage of the current and,

(2) Formation of such germicidal materials as ozone and chlorine by the action of the current upon the suspending fluid.

6. Filtration.

Filtration used extensively to sterilize a liquid or a gas. Human and animal serum  in culture media is easily coagulated  by heat.

Enzymes, toxins, and antibiotics in solution are also easily destroyed by heat. Such solutions are best sterilized by filtration. If filters remove bacteria naturally they will also remove larger organisms. A number of bacteriological and serological filters are available for this purpose (Fig. 18,7). Some of these are briefly described.

Porcelain filters. Filters of this type, called Chamber land filters are composed of hydrous aluminium silicate or kaolin, and are baked at a temperature as high as possible without sintering the clay. The filters are prepared in various degrees of porosity from 0.65 to 15. They are cleaned by heating to burn off the organic matter in the pores.

Diatomaceous filters. Diatomaceous earth is a fine, usually white, siliceous powder composed chiefly or wholly of the remains of diatoms, and is also called kieselguhr. It is prepared by mixing diatomaceous earth, asbestos, and organic matter such as plaster of Paris, in different proportions, depending upon the size of pores. The mixture is subjected to high pressure, and then baked in an oven at 1000 to 2000C to bind the materials together. They are called Berkefeld or Mandler filters. They are cleaned by washing. The filter is reversed and placed in a special metal holder connected to a faucet. The water passes from within outward, removing all foreign matter from pores.

Fritted-glass filters. These are formed by heating powdered glass in a disk form to a temperature just below its melting point. This causes the particles to adhere to one another without destroying the porosity of the disk. The disk is then carefully fused in a glass funnel and cleaned by treatment with sulphuric acid containing sodium nitrate. The strong acid quickly oxidizes and dissolves organic matter. The acid is then removed by thorough washing.

Asbestos filters. These are composed of matted asbestos fibres in disk form, and are called Seitz filters. The disk is clamped tightly between two smooth metal rims by screw clamps. Bacteria and other suspended particles are adsorbed by the filter disks. These disks are discarded after use.

Ultra filters. These are composed of cellulose esters and called polypore or milipore filters. The filters are discarded after use. All these filters are available in several grades, based on the average size of the pores. These filters do not act -merely as mechanical sieves, because porosity alone is not the only factor preventing the passage of organisms. Efficiency of filtration is also controlled by the electric charge on the filter, the electric charge carried by the organisms, and the nature of the material being filtered. Normally a negative pressure is applied to the filter flask by the use of a vacuum or water pump to force the fluid through the filter. Occasionally, a positive pressure is imposed above the fluid in the filter chamber to force the fluid through the filter.

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The Chemical Environment

 In natural conditions microorganisms are seldom exposed to injurious chemicals in concentrations high enough to cause their death. Man, however has been interested in searching for toxic chemicals which kill or inhibit the growth of microorganisms, mainly to prevent infection or destructive actions of microorganisms. The search for such chemicals goes on continually in an effort to find those that have the highest toxicity for microorganisms with the lowest possible poisonous effects on man, animals, and plants. The chemical substances used for this purpose are many, and are divided into three main groups.

1.      Antiseptics and disinfectants.  These substances show very little specificity. Acids and alkalies, soaps, halogens, phenols, heavy metals, etc., are able to kill or inhibit many types of organisms.

2.      Antimetabolites.    These are structural   analogues of the inter­mediates in the metabolism of the cell.   A false molecule is built up which is functionally inactive. Some of these, for example sulphonamides, have a great deal of specificity, while others have much less specificity.

3.                    Antibiotics.   These are substances  formed   by one organism which are toxic to another.   Highest specificity is found among these substances.

Antiseptics and disinfectants

1. Acids and alkalies. There is a limit to the degree of acidity or alkalinity that microorganisms can tolerate. The germicidal efficiency of acids is proportional to the hydrogen-ion (H+ ) concentrations of their solutions. Most mineral acids (HCl, H2SO4, HNO3, H3P04, etc.) act primarily as generators of hydrogen ions. However, many acids, which are only weakly ionized and do not increase hydrogen ion concentration to any appreciable extent, also produce toxic effects. This may be due to the additional effects of the anions, or to the undissociated molecules. For example, benzole, acetic, salicylic, and sulphu­rous acids, which are almost undissociated in strongly acid solutions, are a hundred times more germicidal than in a neutral solution, where they are completely dissociated.

Similarly, the germicidal action of alkalies is dependent on dis­sociation and release of hydroxyl ions (OFT) in solution. But there are some exceptions. Ba(OH)2 is less dissociated than KOH, but is considerably more toxic. This is due to the high toxicity of Ba++ ions. In general, H+ exerts more toxic effects than an equal number of OH- Strong acids and alkalies are not especially useful, because they are corrosive.

2. Halogens. Chlorine, iodine, bromine and fluorine in the free state as well as their compounds are actively germicidal. Out of these only chlorine and iodine are of practical use. Bromine is strongly irritant and difficult to handle: fluorine is even more so.

Chlorine is one of the most widely used disinfectants either in the form of a gas or in certain chemical combinations. Chlorine gas is used to disinfect filtered water of municipal water supplies and sewage disposal plants. It is usually handled in tanks or tubes, like oxygen, and therefore requires special equipment from which it can be dispensed. It is applied to drinking water in a final concentration of 0.1 to 1 parts per million.

Compounds of chlorine can be handled more conveniently for individual and household uses. Calcium kypochlorite (Ca(OCl)2)

and sodium hypo chlorite (NaClO) are very widely used for disin­fection of dairy equipment and eating utensils in restaurants. Many organic chlorine compounds which liberate their chlorine slowly are more effective. These are collectively called chloramines, wherein one Or more of the hydrogen atoms in an amino or imino group are replaced with chlorine.

The germicidal action of chlorine and its compounds is due to the formation of hypochlorous acid when free chlorine reacts with water:

Cl2 + H2O HCl + HClO (hypochlorous acid)

Similarly, hypo chlorites and chloramines undergo hydrolysis with the formation of hypochlorous acid :

OCl + H2o hclo + oh-

The hypochlorous acid formed is further decomposed :

2 HCIO -> 2 HC1 + O2

The oxygen released in this reaction (nascent oxygen) brings about microbial destruction by oxidation. Combination of chlorine with proteins of the cell membrane and enzymes is also responsible for the death of microorganisms.

Iodine is one of the oldest and most effective germicidal agents. As tincture of iodine (2% I2, 2% KI in 90% ethanol), this halogen has been a household disinfectant for wounds, cuts, and scratches. It is poisonous and irritating, but is a highly effective bactericidal agent.

Iodine is also combined with surface-active agents to give com­pounds called iodophores. These are non-irritating, non-staining, and virtually odourless. The iodophores release iodine slowly when diluted with water, and also lower the surface tension of the solution. Germi­cidal activity of iodine is due to its combination with enzymes and proteins, which in turn are inactivated.

3.Heavy metals. Most of the heavy metals, either alone or in certain chemical compounds, are germicidal. The most widely used compounds of heavy metals are those of mercury, silver and copper. These metals are so toxic that small quantities of their salts present on the surface of the metal is inhibitory to the growth of microorganisms. Intensity in low concentration is called the oligodynamic action (oligos=small and dynamis = power, in Greek). However, extremely low concentrations stimulate growth of microorganisms. This can be demonstrated in the laboratory by placing a clean piece of metal (coin) on a plate inoculated with micrococci. After incubation a zone of inhibition (no growth) surrounds the metal. However, an increased density of growth at the outer margin of the zone of inhibition is also seen. This may be due to: (I) stimulation by extremely low concen­tration of the metallic ions, or (2) less competition for food at the edge of the inhibition zone, or (3) both 

4.Phenol and its derivatives. Phenol or carbolic acid was used by Lister in 1867 for the development of antiseptic surgery, and is still

Fig. Phenol and some of its derivatives.

used as a standard in evaluating other disinfectants. Phenol is now no longer used as a disinfectant because it is expensive and because its derivatives are more effective and less costly. There have been two types of work on the phenol-like compound. One is to find more active inhibitors by substitution into the phenol structure and to obtain decreasing corrosiveness; the other is a careful study of factors which affect its action. Fig. shows phenol and some of its derivatives which are more effective than phenol.

Phenol is soluble in water, but most of its derivatives are only slightly soluble. However, they may be held in suspension by mixing with soap to obtain colloidal solutions. The emulsification of such disinfectants results in the formation of more potent germicidal prepara­tions. In the emulsified state the particles of germicide are adsorbed to the surface of the emulsifying agent (soap). This results in an increased concentration in the vicinity of the organisms.

The cresols are more germicidal than phenol when they arc emulsified in liquid soaps and alkalies. Lysol is a commercial preparation containing cresols. It is mostly used as a disinfectant for inanimate objects. Hexylresorcinols is marketed as a solution in glycerin and water, and these preparations are employed as general antiseptics in mouth washes, gargles, and cough-drops. Hexachlorophene is solubilized in acetone, alcohol, or dilute alkali. It is widely used in many soaps, oils and creams as an antiseptic to control the microbial flora of the skin.

The primary mode of action of phenolic compounds is to damage the cytoplasmic membrane. This is followed by leakage of constituents from the cell. These compounds also precipitate the cellular proteins, and therefore act as gross protoplasmic poisons.

5.Alcohols. A number of aliphatic alcohols are effective microbicides. Their germicidal activity increases with the molecular weight as far as the amyl derivative (5 carbon atoms). Since the alcohols decrease in solubility as the molecular weights increase, the higher members of the series are generally not employed as germicides. Ethyl alcohol and isopropyl alcohol are the most commonly used disinfectants as they are non-toxic on external application. Methyl alcohol is highly' poisonous, and is not generally employed as a germicide. Alcohols are protein coagulants and lipid solvents. They are also dehydrating agents, a property that interferes greatly with their coagulating power. A very high concentration (95 to 100 percent) of alcohol removes so much of water from the cell that the alcohol is unable to penetrate. The severe dehydration would result in a micro-biostatic condition. 70 percent alcohol is, therefore, more effective. Similarly, in spores where the water content is very low, alcohols have little effect. Alcohols therefore cannot be relied upon to produce a sterile condition. Alcohol is effective in reducing the microbial flora of skin, and is used prior to hypodermic injections and also for the disinfection of clinical oral thermometer. Viruses and cells with lipid envelopes are also damaged by alcohols.

6. Dyes.   A number of dyes have been found to inhibit bacterial growth.   Generally speaking, they are relatively more toxic to gram positive than to gram negative bacteria.   Brilliant green, malachite green, crystal violet and basic fuchsin are frequently added to selective culture media to inhibit the growth of gram   positive bacteria. This helps in the isolation of gram negative bacteria from materials contaminated with gram positive bacteria. Some dyes are used as antiseptics for the treatment of infections caused by gram positive bacteria and certain species of fungi. Theacridine dyes, e.g.  acriflavine, are highly bactericidal.   They are also strong mutagenic agents. Generally, basic dyes combine with the acidic material of the cell, especially nucleic acids to form salts.

 7.Detergents. Compounds which are primarily employed for clean­sing surfaces are called detergent for example common soaps. Soaps are prepared by boiling oils and fats with potassium or sodium hydroxide. Soaps are actively lethal for certain spirochetes such as Treponema pallidwn, for certain streptococci such as Streptococcus pneumoniae. and a few other organisms. With these few exceptions, they are relatively ineffective as general disinfectants.

Synthetic detergents consisting of anionic and cationic compo­unds have replaced soaps for many cleansing jobs. These compounds, because of their strong surface-tension reducing action, are frequently microbiostatic and may even be microbicidal. Chemically, detergents are of three types :

(1)      Anionic detergents- those which ionize, with the   detergent property resident in  the anion.

(2)     Cationic detergents - those which ionize with the detergent property resident in the cation e.g., (R-N(CH3)3) Cl- quaternary ammonium compounds.

(3)        Non-ionic detergents - those which do not ionize. Cationic detergents are more germicidal than anionic and non-ionic detergents. Most of the cationic detergents are quaternary ammonium compounds As a group they are called quater­naries, and are marketed under specific trade names. They are highly active against gram positive bacteria and are also quite active against gram negative bacteria. They are also active against fungi and certain pathogenic Protozoa. Viruses appear to be more resistant that bacteria and fungi. They are, however, effective against certain viruses which have lipid-rich components, e. g., envelopes, because of their emulsifying action on lipids. Their primary mode of action is disrup­tion of cell walls and membranes. They also inactivate enzymes and denature proteins.

 Quaternaries are widely used for sterilization of surgical instru­ments and as antiseptics for surgical and gynecological procedures, since they are painless and harmless to the tissues. They are also used as skin antiseptics and as sanitizing agents in public eating establish­ments, dairies and food-processing plants.

8. Gaseous sterilization. Materials and substances that are adversely affected by heat and liquid solutions are sterilized by means of gases. This has been practiced for years under the designation of fumigation. Commonly used gasses were sulphur dioxide, chlorine arid formaldehyde which were widely employed for the disinfection of rooms occupied by sick people. Sulphur dioxide and chlorine are poisonous and damage the materials. They are now replaced by formaldehyde, ethylene oxide, and -propiolactone.

 

Formaldehyde (H'CHO)'. This gas is generated by heating a concen­trated solution of formaldehyde. Formaldehyde in aqueous solution is known as formalin, and contains 37 to 40 percent formaldehyde. Vapo­rization, of formaldehyde, either from formalin or paraformaldehyde, is used to sterilize an enclosed area. For best results a relative humidity of about 70 percent and a temperature of about 22C are required. Since it penetrates only slightly, it acts only as a surface disinfectant. Also, the residual gas persists even after prolonged airing. It kills both vegetative cells and spores. It is a strong reducing agent and inac­tivates enzymes and other cell constituents.

Ethylene oxide. It is liquid at a temperature below 10.8 C, its boiling point. Above this temperature it vaporizes rapidly. In the pure state it is very toxic if inhaled and causes blisters if its vapours remain in contact with the skin. It is highly flammable. This is overcome by mixing it with carbon dioxide or fluorinated hydrocarbons (freons). A mixture of 10 percent ethylene oxide and 90 percent carbon dioxide is sold as carboxide. Carbon dioxide and freons do not alter the microbicidal activity of ethylene oxide. Ethylene oxide has proved better than formaldehyde because of following reasons: (1) it is relatively inexpensive, readily available, and released in pure state; (2) it dose not polymerize or condense on surfaces; (3) it exhibits deep penetration; (4) it is quickly removed by simple airing of treated materials.

Ethylene oxide is very effective against microorganisms, including spores. It acts by alkylation with organic compounds by replacing an active hydrogen atom. e. g., the hydrogen atom in a free carboxyl, amino, or sulfyhydryl group.

It is widely used as a sterilizing agent in many biological or non-biological material. It is also used in the liquid state in 1 percent concentration for sterilizing organic fluids. Ethylene oxide is mixed with the fluids at 10QC. After some time ethylene oxide is driven off at higher temperature. However, its most important disadvantage is its slow action upon microorganisms.

t-propiolactoiie (BPL). In pure form at 20C it is colourless, pungent liquid. Its boiling point is 162.3C and it is atomised into the air. Like formaldehyde it requires high relative humidity and a tempe­rature of 25C, and has low penetrating power. However, it is noncorrosive, does not condense on surfaces, is not explosive, and is easily removed. It kills most microorganisms, including spores. It probably acts by alkylation, as does ethylene oxide. However, it is slowly converted to inactive -hydroxypropionic acid by hydrolysis in aqueous solution :

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Standardization of disinfectants

To standardize antimicrobial agents, several kinds of properties are evaluated. These are :

(1) the ability to prevent growth of organisms, (2) the ability to kill organisms already grown, (3) the spectrum of activity or the types of organisms affected by an agent, and (4) the toxicity, or the ability to be used in or on living medium.

Phenol coefficient. A number of different methods have been developed to measure the effectiveness of disinfectants. However, the most widely used method to evaluate disinfectants is to rate them according to their phenol coefficient. The phenol coefficient is defined as the killing power of a disinfectant against a test organism, as compared to that of phenol under identical conditions. The conditions which must be specified are as follows : (1) organism used in the test (2) age of the culture, (3) temperature of the test, (4) time of action of disinfectant, (5) presence and amount of organic matter in the culture, (6) proportion of disinfectant to the culture, (7) composition and reaction of the transfer medium, (8) temperature and time of the incubation of the transfer medium.

The most commonly used method is the one recommended by Association of Official Agricultural Chemists (AOAC) and Food and Drug Administration (FDA), U. S. A., and is summarized as follows:

A series of dilutions of phenol is prepared in tubes of sterile distilled water. A similar series of dilutions of the disinfectant to be tested is prepared. The tubes are kept in a water bath at a tempe­rature of 20C. To each dilution tube 0.5 ml of 24-hr broth culture of the test organism (selected strains of Staphylococcus aureus, or Salmonella typhosa) is added. At intervals of 5, 10 and 15 minutes, a standard loopful is transferred from each dilution tube to a corres­ponding tube containing 10 ml of sterile nutrient broth. The subculture tubes are incubated at 37C for 24 to 48 hours, and presence or absence of growth is noted.

The phenol coefficient is then calculated as the ratio of the highest dilution of the disinfectant killing the organism in 10 minutes (no growth in subculture tube) but not in 5 minutes (growth in sub­culture tube) to the corresponding dilutions of phenol that will kill under like conditions. For example, in Table 18.5 the phenol coefficient would be 150/90=1.6 This means the disinfectant X is 1.6 times more effective than phenol.

The phenol coefficient of a disinfectant is of great value in determining the dilution at which it can be used effectively. The test gives no information as to its effects on living tissue, and therefore it is of doubtful value for clinical application.

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