Methods of sterilization and disinfection.

Methods of sterilization and disinfection.

Methods of examination of bacterial antibiotic susceptibility. The main principles of rational antibiotic therapy of diseases

 

[Strict or Obligate] Aerobe: An organism that is capable of using oxygen as a terminal electron acceptor [i.e., aerobic respiration], can tolerate a level of oxygen equivalent to or higher than that present in an air atmosphere (21% oxygen), and has a strictly respiratory type of metabolism. Some aerobes may also be capable of growing anaerobically with electron acceptors other than oxygen [i.e., anaerobic respiration].

 The following guidelines are intended to ensure that all biohazardous waste is processed in a safe and timely manner, as well as in accordance with all applicable regulations.

Each generator of biohazardous waste has an obligation to handle and dispose their material in a manner which affords protection from leakage and injury or exposure to anyone handling their waste material.

1.	Each individual working with biohazardous material or contaminated items is responsible for their decontamination, disinfection, and appropriate preparation prior to disposal or reuse.
2.	All laboratories, in which work with biohazardous materials is carried out, must have labeled, leak-proof, covered containers for temporary holding of infectious materials awaiting disinfection or disposal. (Keep the containers closed at all times, unless adding waste.)

 

 

DEFINITIONS

Decontamination is a term used to describe a process or treatment that renders a medical device, instrument, or environmental surface safe to handle. A decontamination procedure can range from sterilization to simple cleaning with soap and water. Sterilization, disinfection and antisepsis are all forms of decontamination.

Sterilization is the use of a physical or chemical procedure to destroy all microbial life, including highly resistant bacterial endospores.

Disinfection eliminates virtually all pathogenic non-spore-forming microorganisms but not necessarily all microbial forms on inanimate objects (work surfaces, equipment, etc.). Effectiveness is influenced by the kinds and numbers of organisms, the amount of organic matter, the object to be disinfected and chemical exposure time, temperature and concentration.

Disinfectants are substances that are applied to non-living objects to destroy microorganisms that are living on the objects.[1] Disinfection does not necessarily kill all microorganisms, especially resistant bacterial spores; it is less effective than sterilisation, which is an extreme physical and/or chemical process that kills all types of life.[1] Disinfectants are different from other antimicrobial agents such as antibiotics, which destroy microorganisms within the body, and antiseptics, which destroy microorganisms on living tissue. Disinfectants are also different from biocides — the latter are intended to destroy all forms of life, not just microorganisms. Disinfectants work by destroying the cell wall of microbes or interfering with the metabolism.

Sanitisers are substances that simultaneously clean and disinfect.[2]

Bacterial endospores are most resistant to disinfectants, but some viruses and bacteria also possess some tolerance.

Disinfectants are frequently used in hospitals, dental surgeries, kitchens, and bathrooms to kill infectious organisms.

A perfect disinfectant would also offer complete and full microbiological sterilisation, without harming humans and useful forms of life, be inexpensive, and non-corrosive. However, most disinfectants are also, by nature, potentially harmful (even toxic) to humans or animals. Most modern household disinfectants contain Bitrex, an exceptionally bitter substance added to discourage ingestion, as a safety measure. Those that are used indoors should never be mixed with other cleaning products as chemical reactions can occur. The choice of disinfectant to be used depends on the particular situation. Some disinfectants have a wide spectrum (kill many different types of microorganisms), while others kill a smaller range of disease-causing organisms but are preferred for other properties (they may be non-corrosive, non-toxic, or inexpensive). There are arguments for creating or maintaining conditions that are not conducive to bacterial survival and multiplication, rather than attempting to kill them with chemicals. Bacteria can increase iumber very quickly, which enables them to evolve rapidly. Should some bacteria survive a chemical attack, they give rise to new generations composed completely of bacteria that have resistance to the particular chemical used. Under a sustained chemical attack, the surviving bacteria in successive generations are increasingly resistant to the chemical used, and ultimately the chemical is rendered ineffective. For this reason, some question the wisdom of impregnating cloths, cutting boards and worktops in the home with bactericidal chemicals

Antisepsis is the application of a liquid antimicrobial chemical to skin or living tissue to inhibit or destroy microorganisms. It includes swabbing an injection site on a person or animal and hand washing with germicial solutions. Although some chemicals may be utilized as either a disinfectant or an antiseptic, adequacy for one application does not guarantee adequacy for the other. Manufacturer’s recommendations for appropriate use of germicides should always be followed.

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 iewspaper 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 caever 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 cottoapkin 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 nitrogeutrients 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.

 

Air disinfectants

Air disinfectants are typically chemical substances capable of disinfecting microorganisms suspended in the air. Disinfectants are generally assumed to be limited to use on surfaces, but that is not the case. In 1928, a study found that airborne microorganisms could be killed using mists of dilute bleach.^[5] An air disinfectant must be dispersed either as an aerosol or vapour at a sufficient concentration in the air to cause the number of viable infectious microorganisms to be significantly reduced.

In the 1940s and early 1950s, further studies showed inactivation of diverse bacteria, influenza virus, and Penicillium chrysogenum (previously P. notatum) mold fungus using various glycols, principally propylene glycol and triethylene glycol.In principle, these chemical substances are ideal air disinfectants because they have both high lethality to microorganisms and low mammalian toxicity.^[Although glycols are effective air disinfectants in controlled laboratory environments, it is more difficult to use them effectively in real-world environments because the disinfection of air is sensitive to continuous action. Continuous action in real-world environments with outside air exchanges at door, HVAC, and window interfaces, and in the presence of materials that adsorb and remove glycols from the air, poses engineering challenges that are not critical for surface disinfection. The engineering challenge associated with creating a sufficient concentration of the glycol vapours in the air have not to date been sufficiently addressed.

 

Pelton & Crane Delta AE Autoclave Sterilizer (Surgery, Medical, Dental, Tattoo)

METHODS

There are four main categories of physical and chemical means of decontamination. They are heat, liquid disinfection, vapors and gases, and radiation. Each category is discussed briefly below.

1. Heat

Wet heat is the most dependable method of sterilization. Autoclaving (saturated steam under pressure of approximately 15 psi to achieve a chamber temperature of at least 250 degrees F for a prescribed time) rapidly achieves destruction of microorganisms, decontaminates infectious waste and sterilizes laboratory glassware, media, and reagents. For efficient heat transfer, steam must flush the air out of the autoclave chamber. Before using the autoclave, check the drain screen at the bottom of the chamber and clean it, if blocked. If the sieve is blocked with debris, a layer of air may form at the bottom of the autoclave, preventing efficient operation. Prevention of entrapment of air is critical to achieving sterility. Material to be sterilized must come in contact with steam and heat.

Chemical indicators, e.g., autoclave tape, must be used with each load placed in the autoclave. The use of autoclave tape alone is not an adequate monitor of efficacy. Autoclave sterility monitoring should be conducted on a regular basis (at least monthly) using appropriate biological indicators (B.stearothermophilus spore strips) placed at locations throughout the autoclave. The spores, which can survive 250 degrees F for 5 minutes but are killed at 250 degrees F in 13 minutes, are more resistant to heat than most, thereby providing an adequate safety margin when validating decontamination procedures. Each type of container employed should be spore tested because efficacy varies with the load, fluid volume, etc.

Decontaminate all infectious materials and all contaminated equipment or labware before washing, storage or discard as infectious waste. Autoclaving is the preferred method. Never leave an autoclave in operation unattended (do not start a cycle prior to leaving for the evening). Log sheets should be available at each autoclave to record the name of the user, time of run, and amount being autoclaved.

All personnel using autoclaves must be adequately trained by their PI or lab manager. Never allow untrained personnel to operate an autoclave.

View the Recommended Procedures for Autoclaving.

Dry heat is less efficient than wet heat and requires longer times and/or higher temperatures to achieve sterilization. It is suitable for the destruction of viable organisms on impermeable non-organic surfaces such as glass, but it is not reliable in the presence of shallow layers of organic or inorganic materials which may act as insulation. Sterilization of glassware by dry heat can usually be accomplished at 160-170 degrees C for periods of 2-4 hours. Dry heat sterilizers should be monitored on a regular basis using appropriate biological indicators [B.subtilis (globigii) spore strips].

The Dry-Heat sterilization process is accomplished by conduction; that is where heat is absorbed by the exterior surface of an item and then passed inward to the next layer. Eventually, the entire item reaches the proper temperature needed to achieve sterilization. The proper time and temperature for Dry-Heat sterilization is 160°C (320°F) for 2 hours or 170°C (340°F) for 1 hour.[citatioeeded] Instruments should be dry before sterilization since water will interfere with the process. Dry-heat destroys microorganisms by causing coagulation of proteins.

Dry heat sterilization is probably the least used and most misunderstood method of sterilization.

It is usually used for heat safe items that caot be disassembled or may rust. Sterilizing by dry heat is accomplished by conduction. The heat is absorbed by the outside surface of the item, then passes towards the centre of the item, layer by layer. The entire item will eventually reach the temperature required for sterilization to take place. Death of microorganisms occurs in dry heat by the process called oxidation. In other words, the slow process of coagulating the protein of the cell. The sterilization process is accomplished in HOT AIR OVENS, and it is longer than in steam sterilizers, due to the lack of moisture.

There are two types of hot air ovens used in dry heat sterilization. Those that use gravity convection and those that use mechanical convection. Convection is the term used for the circulation of the heated air within the chamber of the oven.

In Gravity Convention Ovens, air is heated and rises. As you know, heated air expands and possesses less density, thus less weight than cooler air. Therefore heated air rises. The cooler air falls as it is displaced by the rising heated air. Due to this rising and falling of heated and cool air the temperature tends to be uneven in the chamber. Therefore, sterilizing in a gravity convection oven is sometimes difficult, because you cannot be sure that you will uniformly achieve the required minimum temperature for the required minimum time. Due to this temperature variation, monitoring a gravity convection oven is often difficult. Examples of gravity convection ovens are a regular kitchen oven, or a toaster oven.

Mechanical Convection Ovens, are the most effective type. The oven contains a fan or blower which continually circulates the heated air to maintain a uniform temperature throughout the chamber. Most commercially available dry heat sterilizers are of this type. An example is the home convection oven.

Like other methods of sterilization there are both advantages and disadvantages for using dry heat as a method of sterilization. Advantages:

 

Dry heat can sterilize items that caot be sterilized in steam or chemical sterilizers, such as powders and oils, or those that are prone to rust.

Dry heat can be used for glassware, as it will not score or erode the surface as, steam might do.

Dry heat will not corrode or rust instruments or needles.

Dry heat will sterilize instruments containing many parts that caot be disassembled. Disadvantages:

Dry heat penetrates slowly and unevenly.

Dry heat requires long exposure times to effectively achieve sterility.

Dry heat requires higher temperatures that many items cannot be safely exposed to.

Dry heat requires specialized packaging materials that can sustain integrity under high heat conditions.

Dry heat may require different temperature and exposure times, depending on the type of item being sterilized.

As in all methods of sterilization, all items must be clean and free of all types of visible soil. Due to its high temperature, specific packaging materials must be used. Only four types of packaging materials may be used in hot air ovens. They are glass such as petri dishes, test tubes and small jars, stainless steel trays or pans with lids. Cotton wrappers or aluminum foil may be used, if the temperature within the chamber does not exceed 204°C. , (or 400°F.)

In order to sterilize effectively, the hot air must circulate freely throughout the chamber. Packages must be kept away from the walls, from each other, and should be of a similar nature, size and thickness. Never overload the oven.

The manufacturer’s recommendations as to the preparation, packaging and loading of the sterilizer must always be followed. Exposure times will vary depending on the how the items are packaged, the temperature, types of items being sterilized, and depth of substances in the container. The most common temperatures used are 162.8°C (325°F) for 90 minutes, and 160°C (320°F) for 120 minutes.

When monitoring biologically, a spore test must be done at least weekly. When a gravity convection oven is used, it is recommended that a spore test be done with each load. The spore used for testing dry heat sterilizers is Bacillus Subtilus. As in all spore testing, a negative result means that the sterilization cycle has been successful.

Periodic inspection, cleaning and maintenance should be done according to the manufacturer’s recommendations. Cleaning and preventative maintenance reduces possible equipment malfunctions that could lead to sterilization failures. However, there is usually very little preventative maintenance required for hot air ovens. Monitoring the accuracy of the thermostats, the motor for the fan, the electrical cords and plugs, is really all that is required.

Dry heat sterilization can be an effective method of sterilization, using gravity or mechanical convection ovens. As in all sterilizers, correct time, temperature, packaging, routine monitoring, cleaning and preventative maintenance, will assure that the items that you sterilize, will be sterile.

The presence of moisture, such as in steam sterilization, significantly speeds up heat penetration.

Portable dry heat autoclave sterilizer medical dental veterinary tattoo 110vPortable dry heat autoclave sterilizer medical dental veterinary tattoo 110v

Sterilizer Prestige Medical Dental/Veterinary/Tattoo Steam Autoclave 120V

 

As air is heated, it expands and possesses less density (weight per unit volume) than cooler air. Therefore, the heated air rises and displaces the cooler air (the cooler air descends). The method of Dry-heat gravity convection produces inconsistent temperatures within the chamber and has a very slow turn over.

See also: Moist heat sterilization

There are two types of Hot-Air convection (Convection refers to the circulation of heated air within the chamber of the oven) sterilizers:

 

Gravity convection

Mechanical convection

Incineration is another effective means of decontamination by heat. As a disposal method, incineration has the advantage of reducing the volume of the material prior to its final disposal. However, local and federal environmental regulations contain strigent requirements and permits to operate incinerators are increasingly more difficult to obtain. There are no incinerators here at USciences.

2. Liquid Disinfection

The most practical use of liquid disinfectants is for surface decontamination and, when used in sufficient concentration, as a decontaminate for liquid wastes prior to final disposal in the sanitary sewer. (A one time approval from EHRS must be obtained for sink disposal.) If liquid disinfectants are used, they must have been shown to be effective against the organism(s) present.

Liquid disinfectant are available under a wide variety of trade names. In general, these can be classified as: halogens, acids, alkalis, heavy metal salts, quaternary ammonium compounds, phenolic compounds, aldehydes, ketones, alcohols and amines. The more active a compound is, the more likely it is to have undesirable characteristics such as corrosivity. No liquid disinfectant is equally useful or effective under all conditions and for all viable agents.

Concentrations and exposure times vary depending on the formulation and the manufacturer’s instructions for use. Therefore, always follow the manufacturer’s recommended instructions for use to ensure proper decontamination. View Activity Levels of Selected Liquid Germicides for additional information.

3. Vapors and Gases

A variety of vapors and gases possess decontamination properties. Vapors and gases are primarily used to decontaminate biological safety cabinets and associated systems, bulky or stationary equipment not suited to liquid disinfectants, instruments or optics which might be damaged by other decontamination methods, and rooms, buildings and associated air-handling systems. Agents included in this category are glutaraldehyde and formaldehyde vapor, ethylene oxide gas, peracetic acid and hydrogen peroxide vapor.

When used in closed systems and under controlled conditions of temperature and humidity, excellent disinfection can be obtained. Great care must be taken during use because of the hazardous nature of many of these compounds. Vapor and gas decontamination should not be attempted by research personnel.

4. Radiation

Although ionizing radiation will destroy microorganisms, it is not a practical tool for laboratory use. Non-ionizing radiation in the form of ultraviolet radiation (UV) is used for inactivating viruses, bacteria and fungi. It will destroy airborne microorganisms and inactivate microorganisms on exposed surfaces or in the presence of products of unstable composition that cannot be treated by conventional means.

Because of the low penetrating power of UV, microorganisms inside dust or soil particles will be protected from its action, limiting its usefulness. UV is used in air locks, animal holding areas, ventilated cabinets and laboratory rooms to reduce levels of airborne microorganisms and maintain good air hygiene. Because UV can cause burns to the eyes and skin of people exposed for even a short period of time, proper shielding should be maintained when it is in use. UV lamps that are used for space decontamination should be interlocked with the general room or cabinet illumination, so that turning on the lights extinguishes the UV.

The Centers for Disease Control and the National Institutes of Health agree that UV lamps are not recommended nor required in biological safety cabinets. If UV lamps are installed and used, they must be properly maintained. They must be cleaned weekly to remove any dust and dirt that may block the germicidal effectiveness of the UV light. Additionally, because UV lamp intensity or destructive power decreases with time, it should be checked with a UV meter yearly. If the UV lamp must be used, it should be used when areas are not occupied. If the cabinet has a sliding sash, also close the sash when operating the UV lamp.

 

Facultative anaerobe: An organism that can grow well both in the absence of oxygen and in the presence of a level of oxygen equivalent to that in an air atmosphere (21% oxygen). Some are capable of growing aerobically by respiring with oxygen and anaerobically by fermentation [anaerobic respiration is also possible]; others have a strictly fermentative type of metabolism and do not respire with oxygen. [We form the “aerotolerant anaerobe” category with the latter type; see below.]

 

Microaerophile: An organism that is capable of oxygen-dependent growth but cannot grow in the presence of a level of oxygen equivalent to that present in an air atmosphere (21% oxygen). Oxygen-dependant growth [i.e., aerobic respiration] occurs only at low oxygen levels. In addition to being able to respire with oxygen, some microaerophiles may be capable of respiring anaerobically with electron acceptors other than oxygen.

 

[Strict or Obligate] Anaerobe: An organism that is incapable of oxygen-dependent growth and cannot grow in the presence of an oxygen concentration equivalent to that present in an air atmosphere (21% oxygen). Some anaerobes may have a fermentative type of metabolism; others may carry out anaerobic respiration in which a terminal electron acceptor other than oxygen is used. [The primary consideration for defining an organism as a strict anaerobe is its total intolerance of oxygen.]

 

With these Bergey’s Manual definitions, phototrophs would be categorized with difficulty if at all. As one example, the purple non-sulfur photosynthetic bacteria can respire and can also grow anaerobically, but anaerobic growth is associated with the organisms’ use of energy derived from light, not (except for certain exceptional strains and species) from fermentation or anaerobic respiration.

 

 

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

 

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.

 

 

 

Isolation and Identification of Pure Culture of Anaerobic Bacteria

 

Methods of obtaining anaerobic conditions. Taking into account that free molecular oxygen is toxic for obligate anaerobic bacteria, the main condition of such microorganisms cultivation is limitation of its access. There are some methods (mechanical, physical, biological) which allow providing it.

Toxic forms of oxygen

•  Certain oxygen derivatives are toxic to microorganisms.

•  Oxygen in its ground state is triplet oxygen (3O2).

•  Toxic forms of oxygen include singlet oxygen 1O2,

(superoxide anion) O2-, hydrogen peroxide H2O2 and hydroxyl radical (OH-).

As molecules have an unpaired electron, they are very reactive and cause destruction.

 

Enzymes that destroy toxic oxygen

•                                             Enzymes are present in cells that caeutralise most toxic forms of oxygen.

•                                             Catalase

•                                             Peroxidase

•                                             Superoxide dismutase

 

Physical methods. 1. Before inoculation of bacteria on/iutrient media it is necessarily to regenerate them for deletion of surplus oxygen (boiling them for 15-20 min in water bath, quickl cooling to the necessary temperature).

2.      For warning oxygen penetration into nutrient medium it must be covered with the layer of sterile vaseline oil or paraffin (for liquid media).

3.      A column of nutrient media in test tubes must be quite high (10-12 cm). Oxygen, as a rule, penetrates into the column of medium on a depth up to 2 cm, that is why favourable conditions for cultivation of anaerobic microbes create below.

 

 

4.      An evacuation and replaceable method foresees the use of anaerobic jar. They are hermetically sealed metallic or plastic jars from which it is possible to pump out oxygen and replace it by special gases (helium, nitrogen, argon). Triple gas mixture which consists of nitrogen 80 %, carbon dioxide 10 %, and hydrogen 10 % is used. Sometimes natural gas may be used. For a deoxygenation in the jar palladic catalysts are used. For absorption of aquatic steams calcium chloride, silicagel and others substances are used in the jars.

 

 

5. Place the burning candle into the flask or jar with Petri plates.

 

Chemical methods foresee the use of substances absorb an oxygen (alkaline solution of pyrogallol, sodium hydrosulphite (Na2S2O4).

There may be used special reduced substances: cysteine (0,03-0,0,5 %), thioglycolic acid or sodium thioglycolate (0,01-0,02 %), sodium sulphide, ascorbic acid (0,1 %), different sugars.

Such functions have pieces of animals parenchymatous organs (liver, kidneys, heart) or even plants (potato).

The degree of deoxygenation or degree of nutrient medium reduction may be measured by indicators (rezazurine, neutral red, phenosafranine).

3. Use of the special gas generating systems which allow to create oxygen-free conditions in the jars, transport plastic packages and so on. One of most widespread there is the system of “Gas Generating Box”.

The GasPak™ EZ Gas Generating Pouch Systems are single-use systems that produce atmospheres suitable to support the primary isolation and cultivation of anaerobic, microaerophilic, or capnophilic bacteria by use of gas generating sachets inside single-use resealable pouches. The GasPak EZ Gas Generating Sachet consists of a reagent sachet containing inorganic carbonate, activated carbon, ascorbic (citric) acid and water. When the sachet is removed from the outer wrapper, the sachet becomes activated by exposure to air. The activated reagent sachet and specimens are placed in the GasPak EZ Incubation Container and the container is sealed. The sachet rapidly reduces the oxygen concentration within the container. At the same time, inorganic carbonate produces carbon dioxide.

Anaerobic environment-action: The gas generator envelope is activated by the addition of water; Hydrogen generated from a sodium borohydride tablet combines with the oxygen in the jar in the presence of the palladium catalyst to form water, removing the oxygen.

Anaerobic conditions are achieved rapidly, generally within 1 hour of incubation; the carbon dioxide concentration is approximately 4-10%. At 35 C, the Gas Pak methylene blue anaerobic indicator becomes decolorized at 4-6 hours.

 

 

 

Gas Pak with  indicator strip and  CO2 generator pack

 

Biological methods. 1. Fortner’s method. A method includes general cultivation outrient medium an aerobic and an anaerobic microorganisms. At first part of nutrient medium in Petri plate aerobic bacteria  (Serratia marcescens) are inoculated, at second – tested material with anaerobic bacteria. The edges of cup are closed hermetically (e.g. with paraffin). In a few days the colonies both aerobic and anaerobic microbes grow. Serratia marcescens forms pink or colourless colonies, and when there are violations of hermetic conditions – bright red ones. The colonies of anaerobic microbes grow on other half of Petri plate.

2. Hennel’s technique (“watch glasses technique”). There is original modification of previous one. Tested material with anaerobic bacteria is inoculated on the square 2-2,5 cm in diameter. Later it is covered by special convex glass where is nutrient medium and Serratia marcescens on it. Aerobic microbes (Serratia spp.) taking an oxygen create favourable conditions for anaerobes growth.

Now the stationary anaerobic boxes for cultivation of anaerobic bacteria are made.

 

 

One of the main requirements in cultivating anaerobic bacteria is removal of oxygen from the nutrient medium. The content of oxygen can be reduced by a great variety of methods: immersing of the sur¬face of the nutrient medium with petrolatum, introduction of micro¬organisms deep into a solid nutrient medium, the use of special anaerobic jars.

 

First day. Inoculate the studied material into Kitt-Tarozzi medi¬um (nutrient medium): concentrated meat-peptone broth or Hottinger’s broth, glucose, 0.15 per cent agar (pHl 7.2-7.4).

To adsorb oxygen, place pieces of boiled liver or minced meat to form a 1-1.5 cm layer and pieces of cotton wool on the bottom of the test tube and pour in 6-7 mi of the medium. Prior to inoculation place the medium into boiling water for 10-20 min in order to remove air oxygen contained in it and then let it cool. Upon isolation of spore forms of anaerobes the inoculated culture is reheated at 80 ‘”C for 20-30 min to kill non-spore-forming bacteria. The cultures are immersed with petrolatum and placed into an incubator. Apart from Kitt-Tarozzi medium, liquid media containing 0.5-1 per cent glucose and pieces of animal organs, casein-acid and casein-mycotic hydrolysates can also be employed.

Casein-acid medium’, casein-acid hydrolysate, 0.5 1; 10 per cent yeast extract, 0.35 1: 20 per cent corn extract, 0.15 1; millet, 240 g; cotton wool, 25 g. The me¬dium is poured into flasks with millet and cotton wool and sterilized for 30 min at 110 0C. Use casein-mycotic hydrolysate to obtain casein-mycotic me¬dium.

Second day. Take note of changes in the enrichment medium, namely, the appearance of opacification or opacification in combination with gas formation. Take broth culture with a’ Pasteur pipette and transfer it through a layer of petrolatum onto the bottom of the test tube. Prepare smears on a glass slide in the usual manner, then flame fix and Gram-stain them.During microscopic examination record the presence of Gram-positive rod forms (with or without spores). Streak the culture from the enrichment medium onto solid nutrient media. Isolated colonies are prepared by two methods.

1. Prepare three plates with blood-sugar agar. To do it, melt and cool to 45 °C 100 ml of 2 per cent agnr on llottinger’s broth, then add 10-15 ml of deftbrinated sheep or rabbit blood and 10 ml of 20 per cent sterile glucose. Take a drop of the medium witli microorgan¬isms into the first plate and spread it along the surface, using a glass spatula. Use the same spatula to streak tlic culture onto tlie second and then third plates and place them into an anaerobic jar or other similar devices at 37 ”C for 24-48 hrs (Zoisslcr’s method).

2, Anaerobic microorganisms are grown deep in a solid nutrient medium (Veinherg’s method of sequential dilutions). The culture from the medium is taken with a Pasteur pipette with a soldcd tip and transferred consecutively into the 1st, 2nd, and 3rd test tubes with 10 ml of isotonic sodium chloride solution. Continue to dilute^ transferring the material into the 4th, 5th. and 6th thin-walled test tubes (0.8 cm in diameter and 18 cm in height) with melted and cooled to 50 °C meat-peptone agar or Wilson-Blair medium (to 100 ml of melted meat-peptone agar with 1 per cent glucose add 10 ml of 20 per cent sodium sulphite solution and 1 ml of 8 per cent ferric chloride). Alter agar has solidified, place the inoculated culture into an incubator.

On the third day, study the isolated colonies formed in tlie plates and make smears from the most typical ones. The remainder is in¬oculated into Kitt-Tarozzi medium. The colonies in the test tubes are removed by means of a sterile Pasteur pipette or the agar column may be pushed out of the tube by steam generated upon warming the bottom of the test tube. Some portion of the colony is used to prepare smears, while its remainder is inoculated into Kitt-Tarozzi medium to enrich pure culture to be later identified by its morpholo¬gical, cultural, biochemical, toxicogenic, antigenic, and other properties.

The Vinyale-Veyone’s method is used for mechanical protection from oxygen. The seeding are made into tube with melting and cooling (at 42 0C) agar media.

 

 

 

III. Thioglycollate Medium – which we utilize in our Bacteriology laboratory courses – is a “standard” medium for the determination of oxygen relationships, and it will support the growth of common, easily-grown chemoheterotrophic bacteria. The observed growth patterns of organisms in this medium determine their oxygen relationship designations (strict aerobe, facultative anaerobe, etc.) which correlate with such physiological abilities as respiration, fermentation and the catalase reaction and also whether there is an inhibitory effect on the organism in the presence of air. See the table under the photo below. Thus, a description of a chemoheterotrophic organism as a “strict aerobe” can imply a number of associated characteristics that may be unnecessary to specify separately (able to respire, unable to ferment, catalase-positive, azide-sensitive, etc.).

The amino acids and glucose in the medium can be respired, and glucose is the only fermentable energy source in the medium except for those exceptional organisms such as certain species of Clostridium which can ferment amino acids.

With Thioglycollate Medium, we are able to differentiate two distinct patterns of growth for those classified in the Bergey’s Manual definitions (above) as “facultative anaerobes”:

Those which are indifferent to oxygen and have a strictly fermentative type of metabolism grow evenly throughout the medium. We term such an organism an aerotolerant anaerobe and set that off as an additional category of oxygen relationship (added to the list of four above).

Those left in the facultative anaerobe category show greater concentration of growth at the top of the medium where oxygen is present and aerobic respiration is then possible. Comparing the degree of growth under aerobic vs. anaerobic conditions can be a good demonstration of the relative efficiencies of aerobic respiration and fermentation when it comes to generation of cell mass.

The terms “facultative” and “aerotolerant” are always meant to modify another term such as “anaerobe” and they should not be used by themselves. Describing an organism as simply “facultative” may mean “facultative anaerobe,” “facultative phototroph,” or a variety of other things.

One must consider the following limitations of Thioglycollate Medium:

Many organisms (including a lot of chemoheterotrophs) cannot grow in this medium for one reason or another.

No allowance is given in the medium or method for anaerobic growth (1) with alternate electron acceptors (such as nitrate) or (2) in light (such as what is seen with the anoxygenic photosynthetic bacteria). Thus, an organism which may be termed a “strict anaerobe” in the more general sense – i.e., one which cannot tolerate oxygen and can only obtain energy by reactions which do not involve O2 – would only show anaerobic growth in this test if it were capable of fermentation of the glucose in this medium.

The results in Thioglycollate Medium can be difficult to read. As shown in the table below, an organism’s oxygen relationship designation can be determined by a combination of other methods which can be used as a check to see if the medium is showing the correct results – i.e., (1) testing for fermentation in Glucose Fermentation Broth, (2) performing the catalase test, and (3) testing if the organism can grow in the presence of oxygen. These methods tend to be quite reliable and can be utilized if Thioglycollate Medium is not available or even specified for use in the identification process. With that in mind, Thioglycollate Medium could be considered redundant.

The results we see in Thioglycollate Medium are shown below. (Note that microaerophiles are not included.) The accompanying table gives related information.

 

 

 

Corresponding tube no. above         1       2       3       4

Oxygen relationship designation     STRICT

(OBLIGATE)

AEROBE   FACULTATIVE

ANAEROBE                AEROTOLERANT

ANAEROBE       STRICT

(OBLIGATE)

ANAEROBE

 

Aerobic respiration*               +       +       –       –

Fermentation*     –       +       +       +

Ability to grow aerobically

(oxygen tolerance)         +       +       +       –

Ability to grow anaerobically –       +       +       +

Catalase reaction +       +       –       –

Reaction in Glucose O/F Medium

(for those able to grow well in medium)    O or –         F                

Response to sodium azide in a growth medium

         SENSITIVE         SENSITIVE (under aerobic conditions)    RESISTANT         RESISTANT

 

*  These are the basic things tested for in this medium. Whether or not any organism can obtain energy by anaerobic respiration or phototrophy is not relevant to these designations of oxygen relationships.

 

So, in becoming a practicing bacteriologist, one will see that there is more to this concept than whether bacteria simply “like” or “don’t like” oxygen – which, unfortunately, is the extent to which oxygen relationships are too-often and unconscionably taught.

 

IV. Rather than (or in addition to) using “oxygen relationships” as descriptive terms – however they may be determined or defined – we can characterize and classify bacteria more consistently and comprehensively by applying the method(s) of energy generation of which an organism is capable:

aerobic respiration

anaerobic respiration

fermentation

anoxygenic phototrophy

oxygenic phototrophy

 

Remember that Thioglycollate Medium tests for an organism’s ability to perform aerobic respiration and/or fermentation – the results of which give us the “oxygen relationship” categories for those organisms which can grow in the medium under the incubation conditions provided. Anaerobic growth in this medium is only associated with fermentation.

 

V. The following summary may help to explain how media formulations can allow anaerobic growth for organisms capable of doing so for one reason or another. The same organism – a typical strain of E. coli – was inoculated into tubes 1, 2 and 3, and a “facultative phototroph” was inoculated into tube 4.

 

In Tube #1, we have a medium containing peptone and agar plus other nutrients a “typical organism” (i.e., a commonly-found, easy-to-grow chemo- or photoheterotroph) might require for metabolism and replication – except that nothing is included which would support anaerobic growth such as glucose (or something else that could be fermented) or nitrate (or some other electron acceptor/”oxygen substitute” that could be used in anaerobic respiration). After inoculation of this medium and incubation in the dark, any growth would be due to aerobic respiration with the growth only at the top of the medium. There would be no anaerobic growth except for some rare, exceptional organisms which can ferment amino acids.

 

 

Tube #2 is the same medium as in #1, but glucose has been added. After incubation (in the dark), any anaerobic growth would be due to fermentation of the glucose. Thus the medium can be used to detect whether or not an organism can respire (aerobically) or ferment. An example of such a medium is the Thioglycollate Medium we use to test common chemoheterotrophs for “oxygen relationships” (discussed above).

 

Tube #3 is the same medium as in #1, but potassium nitrate has been added. After incubation (in the dark), any anaerobic growth would be due to anaerobic respiration where the organism is using nitrate as the electron acceptor. In Bact. 102 (Exp. 7), we do a test in a broth medium for nitrate reduction; with reagents we can detect nitrite formation, and with the Durham tube we can detect N2 gas formation. One can probably see why we would not want to include nitrate in the Thioglycollate Medium above.

 

Tube #4 is the same medium as in #1, but we have incubated the tube in the presence of light. With light as the ultimate energy source, anaerobic growth would be due to anoxygenic phototrophy. This is the basis for the test we do in Bact. 102 (Exp. 11.1) to see if our isolates of purple non-sulfur bacteria are either “strict phototrophs” (just anaerobic growth in the light) or “facultative phototrophs” (anaerobic growth in the light, plus aerobic growth due to aerobic respiration whether in the dark or the light). Click here for a summary of this test.

 

 

As genotypic characterization (determination of the DNA and RNA characteristics of our bacteria) is becoming more widely practiced, we may soon be back to one standard of characterizing and identifying bacteria. This time it will be universally applicable as all bacterial genera and species become uniformly defined according to genotypic uniqueness. We hope that the results of the phenotypic tests we run will correlate with the genotypic characteristics and bring about accurate and useful identification of our organisms.

 

In the table below, a few commonly-found and easily-grown chemoheterotrophic genera are sorted out based on various “primary tests” which include the use of Both Glucose Fermentation Broth and Glucose O/F Medium include. The benzidine test which has been used effectively for the presence of iron-porphyrin compounds such as cytochromes and the true catalase enzyme. Some organisms possess the enzyme cytochrome a3 oxidase as part of the electron transport system in respiration; this enzyme is responsible for a positive reaction in the oxidase test where the dye tetramethyl-p-phenylenediamine is reduced to a purple compound.

Further tests (not indicated) would then be done to determine positively the genus identification and also the likely species. You can go where the experts are and consult the latest editions of Bergey’s Manual of Systematic Bacteriology and Bergey’s Manual of Determinative Bacteriology for more information. Bergey’s Manual of Systematic Bacteriology is a multi-volume set, and the first volume of the new, 2nd edition is out now but may not be specifically helpful for the organisms listed in the table below. Bergey’s Manual of Determinative Bacteriology is mainly used for identification, but the present 9th edition has become quite dated in that respect.

The idea for the format of the following table comes from the classic Cowan and Steel’s Manual for the Identification of Medical Bacteria, 2nd edition, revised by S. T. Cowan (1974, Cambridge University Press). This table of often-isolated chemoheterotrophic bacteria was put as a guide in targeting likely names of genera to pin on the “nature isolates.” An X marks the place where a certain pattern of characteristics matches up with a possible genus. Considering additional characteristics of the isolate, one can consult Bergey’s Manual or The Prokaryotes for this genus and related genera (oearby pages) for a more definitive identification.

Gram reaction

(young culture)    +       +       +       +       +       +       +       +       +       +       –       –       –         –       –       –

shape          coccus

(clusters)    coccus

(clusters)    coccus

(chains)      coccus

(tetrads)      rod    rod    irreg.

rod    rod    rod    rod    rod    rod    rod    rod    rod    coccus

(pairs)

aerobic growth     +       +       +       +       +       +       +       –       +       +       +       +       +         +       +       +

anaerobic growth –       +       +       +       +       –       –       +       +       –       –       –       +         +       +       –

endospores –       –       –       –       –       –       –       +       +       +       –       –       –       –         –       –

motility

(Motility Medium)        –       –       –       –       –       +       –       +

or –   +

or –   +

or –   +

or –   +

or –   –       +       +       –

catalase reaction  +       +       –       –       –       +       +       –       +       +       +       +       +         +       +       +

benzidine reaction         +       +       –       –       –       +       +       –       +       +       +       +         +       +       +       +

oxidase reaction   +       –       –       –       –       –       –       –       +

or –   +

or –   +       +       –       –       +       +

glucose fermentation to acid or to acid+gas        –       +       +       +       +       –       –       + (or

–)      +       –       –       –       +       +       +       –

Glucose O/F Medium                                                                                             –       O         F       F       F       O

Micrococcus        X                                                                                                                                      

Staphylococcus           X                                                                                                                             

Streptococcus                       X                                                                                                                    

Lactococcus                          X                                                                                                                    

Enterococcus                        X                                                                                                                    

Leuconostoc                         X                                                                                                                    

 

Physical and Environmental Requirements for Microbial Growth

The procaryotes exist iature 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 proteiature, 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 outrient 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

 

Sterilization (or sterilisation) is a term referring to any process that eliminates (removes) or kills all forms of microbial life, including transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.) present on a surface, contained in a fluid, in medication, or in a compound such as biological culture media.[1][2] Sterilization can be achieved by applying heat, chemicals, irradiation, high pressure, and filtration or combinations thereof.

 

The term has evolved to include the disabling or destruction of infectious proteins such as prions related to Transmissible Spongiform Encephalopathies (TSE)

Medicine and surgery[edit]

 

 

Joseph Lister was a pioneer of antiseptic surgery.

In general, surgical instruments and medications that enter an already aseptic part of the body (such as the bloodstream, or penetrating the skin) must be sterilized to a high sterility assurance level, or SAL. Examples of such instruments include scalpels, hypodermic needles and artificial pacemakers. This is also essential in the manufacture of parenteral pharmaceuticals.

Heat (flame) sterilization of medical instruments is known to have been used in Ancient Rome, but it mostly disappeared throughout the Middle Ages resulting in significant increases in disability and death following surgical procedures.

Preparation of injectable medications and intravenous solutions for fluid replacement therapy requires not only a high sterility assurance level, but also well-designed containers to prevent entry of adventitious agents after initial product sterilization.

Sterilization as a definition terminates all life; whereas sanitization and disinfection terminates selectively and partially. Both sanitization and disinfection reduce the number of targeted pathogenic organisms to what are considered “acceptable” levels – levels that a reasonably healthy, intact, body can deal with. An example of this class of process is Pasteurization.

Steam sterilization[edit]

 

Front-loading autoclaves

A widely used method for heat sterilization is the autoclave, sometimes called a converter. Autoclaves commonly use steam heated to 121–134 °C (250–273 °F). To achieve sterility, a holding time of at least 15 minutes at 121 °C (250 °F) at 100 kPa (15 psi), or 3 minutes at 134 °C (273 °F) at 100 kPa (15 psi) is required. Additional sterilizing time is usually required for liquids and instruments packed in layers of cloth, as they may take longer to reach the required temperature (unnecessary in machines that grind the contents prior to sterilization). Following sterilization, liquids in a pressurized autoclave must be cooled slowly to avoid boiling over when the pressure is released. Modern converters operate around this problem by gradually depressing the sterilization chamber and allowing liquids to evaporate under a negative pressure, while cooling the contents.

Proper autoclave treatment will inactivate all fungi, bacteria, viruses and also bacterial spores, which can be quite resistant. It will not necessarily eliminate all prions.

For prion elimination, various recommendations state 121–132 °C (250–270 °F) for 60 minutes or 134 °C (273 °F) for at least 18 minutes. The prion that causes the disease scrapie (strain 263K) is inactivated relatively quickly by such sterilization procedures; however, other strains of scrapie, as well as strains of CJD and BSE are more resistant. Using mice as test animals, one experiment showed that heating BSE positive brain tissue at 134–138 °C (273–280 °F) for 18 minutes resulted in only a 2.5 log decrease in prion infectivity. (The initial BSE concentration in the tissue was relatively low). For a significant margin of safety, cleaning should reduce infectivity by 4 logs, and the sterilization method should reduce it a further 5 logs.

To ensure the autoclaving process was able to cause sterilization, most autoclaves have meters and charts that record or display pertinent information such as temperature and pressure as a function of time. Indicator tape is often placed on packages of products prior to autoclaving. A chemical in the tape will change color when the appropriate conditions have been met. Some types of packaging have built-in indicators on them.

Biological indicators (“bioindicators”) can also be used to independently confirm autoclave performance. Simple bioindicator devices are commercially available based on microbial spores. Most contain spores of the heat resistant microbe Geobacillus stearothermophilus (formerly Bacillus stearothermophilus), among the toughest organisms for an autoclave to destroy. Typically these devices have a self-contained liquid growth medium and a growth indicator. After autoclaving an internal glass ampule is shattered, releasing the spores into the growth medium. The vial is then incubated (typically at 56 °C (133 °F)) for 24 hours. If the autoclave destroyed the spores, the medium will retain its original color. If autoclaving was unsuccessful the B. sterothermophilus will metabolize during incubation, causing a color change during the incubation.

For effective sterilization, steam needs to penetrate the autoclave load uniformly, so an autoclave must not be overcrowded, and the lids of bottles and containers must be left ajar. Alternatively steam penetration can be achieved by shredding the waste in some Autoclave models that also render the end product unrecognizable. During the initial heating of the chamber, residual air must be removed. Indicators should be placed in the most difficult places for the steam to reach to ensure that steam actually penetrates there.

For autoclaving, as for all disinfection or sterilization methods, cleaning is critical. Extraneous biological matter or grime may shield organisms from the property intended to kill them, whether it physical or chemical. Cleaning can also remove a large number of organisms. Proper cleaning can be achieved by physical scrubbing. This should be done with detergent and warm water to get the best results. Cleaning instruments or utensils with organic matter, cool water must be used because warm or hot water may cause organic debris to coagulate. Treatment with ultrasound or pulsed air can also be used to remove debris.

Heat sterilization of foods[edit]

See also: Food safety

Although imperfect, cooking and canning are the most common applications of heat sterilization. Boiling water kills the vegetative stage of all common microbes. Roasting meat until it is well done typically completely sterilizes the surface. Since the surface is also the part of food most likely to be contaminated by microbes, roasting usually prevents food poisoning. Note that the common methods of cooking food do not sterilize food – they simply reduce the number of disease-causing micro-organisms to a level that is not dangerous for people with normal digestive and immune systems.

Pressure cooking is analogous to autoclaving and when performed correctly renders food sterile. However, some foods are notoriously difficult to sterilize with home canning equipment, so expert recommendations should be followed for home processing to avoid food poisoning.

Other heat sterilization methods[edit]

Other heat methods include flaming, incineration, boiling, tindalization, and using dry heat.

Flaming is done to loops and straight-wires in microbiology labs. Leaving the loop in the flame of a Bunsen burner or alcohol lamp until it glows red ensures that any infectious agent gets inactivated. This is commonly used for small metal or glass objects, but not for large objects (see Incineration below). However, during the initial heating infectious material may be “sprayed” from the wire surface before it is killed, contaminating nearby surfaces and objects. Therefore, special heaters have been developed that surround the inoculating loop with a heated cage, ensuring that such sprayed material does not further contaminate the area. Another problem is that gas flames may leave residues on the object, e.g. carbon, if the object is not heated enough.

A variation on flaming is to dip the object in 70% ethanol (or a higher concentration) and merely touch the object briefly to the Bunsen burner flame, but not hold it in the gas flame. The ethanol will ignite and burn off in a few seconds. 70% ethanol kills many, but not all, bacteria and viruses, and has the advantage that it leaves less residue than a gas flame. This method works well for the glass “hockey stick”-shaped bacteria spreaders.

Incineration will also burn any organism to ash. It is used to sanitize medical and other biohazardous waste before it is discarded with non-hazardous waste.

Boiling in water for fifteen minutes will kill most vegetative bacteria and inactivate viruses, but boiling is ineffective against prions and many bacterial and fungal spores; therefore boiling is unsuitable for sterilization. However, since boiling does kill most vegetative microbes and viruses, it is useful for reducing viable levels if no better method is available. Boiling is a simple process, and is an option available to most people, requiring only water, enough heat, and a container that can withstand the heat; however, boiling can be hazardous and cumbersome.

Tindalization[6] /Tyndallizationnamed after John Tyndall is a lengthy process designed to reduce the level of activity of sporulating bacteria that are left by a simple boiling water method. The process involves boiling for a period (typically 20 minutes) at atmospheric pressure, cooling, incubating for a day, boiling, cooling, incubating for a day, boiling, cooling, incubating for a day, and finally boiling again. The three incubation periods are to allow heat-resistant spores surviving the previous boiling period to germinate to form the heat-sensitive vegetative (growing) stage, which can be killed by the next boiling step. This is effective because many spores are stimulated to grow by the heat shock. The procedure only works for media that can support bacterial growth – it will not sterilize plain water. Tindalization/tyndallization is ineffective against prions.

Dry heat sterilizer

Dry heat can be used to sterilize items, but as the heat takes much longer to be transferred to the organism, both the time and the temperature must usually be increased, unless forced ventilation of the hot air is used. The standard setting for a hot air oven is at least two hours at 160 °C (320 °F). A rapid method heats air to 190 °C (374 °F) for 6 minutes for unwrapped objects and 12 minutes for wrapped objects.[8][9] Dry heat has the advantage that it can be used on powders and other heat-stable items that are adversely affected by steam (for instance, it does not cause rusting of steel objects).

Prions can be inactivated by immersion in sodium hydroxide (NaOH 0.09N) for two hours plus one hour autoclaving (121 °C or 250 °F). Several investigators have shown complete (>7.4 logs) inactivation with this combined treatment. However, sodium hydroxide may corrode surgical instruments, especially at the elevated temperatures of the autoclave.

Glass bead sterilizer, once a common sterilization method employed in dental offices as well as biologic laboratories,[10] is not approved by the U.S. Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC) to be used as inter-patients sterilizer since 1997.[11] Still it is popular in European as well as Israeli dental practice although there are no current evidence-based guidelines for using this sterilizer.[10]

Chemical sterilization[edit]

 

Chemiclav

Chemicals are also used for sterilization. Although heating provides the most reliable way to rid objects of all transmissible agents, it is not always appropriate, because it will damage heat-sensitive materials such as biological materials, fiber optics, electronics, and many plastics. Low temperature gas sterilizers function by exposing the articles to be sterilized to high concentrations (typically 5 – 10% v/v) of very reactive gases (alkylating agents such as ethylene oxide, and oxidizing agents such as hydrogen peroxide and ozone). Liquid sterilants and high disinfectants typically include oxidizing agents such as hydrogen peroxide and peracetic acid and aldehydes such as glutaraldehyde and more recently o-phthalaldehyde. While the use of gas and liquid chemical sterilants/high level disinfectants avoids the problem of heat damage, users must ensure that article to be sterilized is chemically compatible with the sterilant being used. The manufacturer of the article can provide specific information regarding compatible sterilants. In addition, the use of chemical sterilants poses new challenges for workplace safety. The chemicals used as sterilants are designed to destroy a wide range of pathogens and typically the same properties that make them good sterilants makes them harmful to humans. American employers have a duty to ensure a safe work environment (Occupational Safety and Health Act of 1970, section 5 for United States) and work practices, engineering controls and monitoring should be employed appropriately.

Ethylene oxide[edit]

Ethylene oxide

Chemical formula:C2H4O

(EO or EtO) gas is commonly used to sterilize objects sensitive to temperatures greater than 60 °C and / or radiation such as plastics, optics and electrics. Ethylene oxide treatment is generally carried out between 30 °C and 60 °C with relative humidity above 30% and a gas concentration between 200 and 800 mg/l, and typically lasts for at least three hours. Ethylene oxide penetrates well, moving through paper, cloth, and some plastic films and is highly effective. EtO can kill all known viruses, bacteria and fungi, including bacterial spores and is compatible with most materials (e.g. of medical devices), even when repeatedly applied. However, it is highly flammable, toxic and carcinogenic with a potential to cause adverse reproductive effects. Ethylene oxide sterilizers requires biological validation and testing of every load, after sterilization installation, repairs or process failure. Biological testing or spore testing are paper filter saturated in millions of Bacillus atropheus known as Bacillus subtilis.

A typical process consists of a preconditioning phase, the actual sterilization run and a period of post-sterilization aeration to remove toxic residues, such as ethylene oxide residues and by-products such ethylene glycol (formed out of EtO and ambient humidity) and ethylene chlorohydrine (formed out of EtO and materials containing chlorine, such as PVC). Besides moist heat and irradiation, ethylene oxide is the most common sterilization method, used for over 70% of total sterilizations, and for 50% of all disposable medical devices.

The two most important ethylene oxide sterilization methods are: (1) the gas chamber method and (2) the micro-dose method. To benefit from economies of scale, EtO has traditionally been delivered by flooding a large chamber with a combination of EtO and other gases used as dilutants (usually CFCs or carbon dioxide). This method has drawbacks inherent to the use of large amounts of sterilant being released into a large space, including air contamination produced by CFCs and/or large amounts of EtO residuals, flammability and storage issues calling for special handling and storage, operator exposure risk and training costs.

Ethylene oxide is still widely used by medical device manufacturers for larger scale sterilization (e.g. by the pallet), but while still used, EtO is becoming less popular in hospitals. Since EtO is explosive from its lower explosive limit of 3% all the way to 100%, EtO was traditionally supplied with an inert carrier gas such as a CFC or halogenated hydrocarbon. The use of CFCs as the carrier gas was banned because of concerns of ozone depletion [12] and halogenated hydrocarbons are being replaced by so-called 100% EtO systems because of the much greater cost of the blends. In hospitals, most EtO sterilizers use single use cartridges (e.g. 3M’s Steri-Vac line,[13] or Steris Corporation’s Stericert sterilizers[14]) because of the convenience and ease of use compared to the former plumbed gas cylinders of EtO blends. Another 100% method is the so-called micro-dose sterilization method, developed in the late 1950s, using a specially designed bag to eliminate the need to flood a larger chamber with EtO. This method is also known as gas diffusion sterilization, or bag sterilization. This method minimizes the use of gas.[15]

Another reason for the decrease in use of EtO are the well known health effects. In addition to being a primary irritant, EtO is now classified by the IARC as a known human carcinogen.[16] The US OSHA has set the permissible exposure limit (PEL) at 1 ppm calculated as an eight hour time weighted average (TWA) [29 CFR 1910.1047] and 5 ppm as a 15 minute TWA. The NIOSH Immediately dangerous to life and health limit for EtO is 800 ppm.[17] The odor threshold is around 500 ppm[18] and so EtO is imperceptible until concentrations well above the OSHA PEL. Therefore, OSHA recommends that some kind of continuous gas monitoring system be used to protect workers using EtO for sterilization.[19] While the hazards of EtO are generally well known, it should be noted that all chemical sterilants are designed to kill a broad spectrum of organisms, by exposing them to high concentrations of reactive chemicals. Therefore, it is no surprise that all the common chemical gas sterilants are toxic and adequate protective measures must be taken to protect workers using these materials.

Employees health records must be maintained during employment and after termination of employment for 30 years.

Nitrogen dioxide[edit]

Nitrogen dioxide

Chemical Formula: NO2

Nitrogen Dioxide NO2 gas is a rapid and effective sterilant for use against a wide range of microorganisms, including common bacteria, viruses, and spores. The unique physical properties of NO2 gas allow for sterilant dispersion in an enclosed environment at room temperature and ambient pressure. The mechanism for lethality is the degradation of DNA in the spore core through nitration of the phosphate backbone, which kills the exposed organism as it absorbs NO2. This degradation occurs at even very low concentrations of the gas.[20] NO2 has a boiling point of 21°C at sea level, which results in a relatively high saturated vapor pressure at ambient temperature. Because of this, liquid NO2 may be used as a convenient source for the sterilant gas. Liquid NO2 is often referred to by the name of its dimer, dinitrogen tetroxide (N2O4). Additionally, the low levels of concentration required, coupled with the high vapor pressure, assures that no condensation occurs on the devices being sterilized. This means that no aeration of the devices is required immediately following the sterilization cycle.[21] NO2 is also less corrosive than other sterilant gases, and is compatible with most medical materials and adhesives.[22]

The most-resistant organism (MRO) to sterilization with NO2 gas is the spore of Geobacillus stearothermophilus, which is the same MRO for both steam and hydrogen peroxide sterilization processes. The spore form of G. stearothermophilus has been well characterized over the years as a biological indicator in sterilization applications. Microbial inactivation of G. stearothermophilus with NO2 gas proceeds rapidly in a log-linear fashion, as is typical of other sterilization processes. Noxilizer, Inc. has commercialized this technology to offer contract sterilization services for medical devices at its Baltimore, MD facility.[23] This has been demonstrated in Noxilizer’s lab in multiple studies and is supported by published reports from other labs. These same properties also allow for quicker removal of the sterilant and residuals through aeration of the enclosed environment. The combination of rapid lethality and easy removal of the gas allows for shorter overall cycle times during the sterilization (or decontamination) process and a lower level of sterilant residuals than are found with other sterilization methods.[24]

Ozone[edit]

Ozone is used in industrial settings to sterilize water and air, as well as a disinfectant for surfaces. It has the benefit of being able to oxidize most organic matter. On the other hand, it is a toxic and unstable gas that must be produced on-site, so it is not practical to use in many settings.

Ozone offers many advantages as a sterilant gas; ozone is a very efficient sterilant because of its strong oxidizing properties (E = 2.076 vs SHE, CRC Handbook of Chemistry and Physics, 76th Ed, 1995–1996) capable of destroying a wide range of pathogens, including prions without the need for handling hazardous chemicals since the ozone is generated within the sterilizer from medical grade oxygen. The high reactivity of ozone means that waste ozone can be destroyed by passing over a simple catalyst that reverts it back to oxygen and also means that the cycle time is relatively short. The downside of using ozone is that the gas is very reactive and very hazardous. The NIOSH immediately dangerous to life and health limit for ozone is 5 ppm, 160 times smaller than the 800 ppm IDLH for ethylene oxide. Documentation for Immediately Dangerous to Life or Health Concentrations (IDLH): NIOSH Chemical Listing and Documentation of Revised IDLH Values (as of 3/1/95)[25] and OSHA has set the PEL for ozone at 0.1 ppm calculated as an 8 hour time weighted average (29 CFR 1910.1000, Table Z-1). The Canadian Center for Occupation Health and Safety provides an excellent summary of the health effects of exposure to ozone. The sterilant gas manufacturers include many safety features in their products but prudent practice is to provide continuous monitoring to below the OSHA PEL to provide a rapid warning in the event of a leak and monitors for determining workplace exposure to ozone are commercially available.

Bleach[edit]

Chlorine bleach is another accepted liquid sterilizing agent. Household bleach consists of 5.25% sodium hypochlorite. It is usually diluted to 1/10 immediately before use; however to kill Mycobacterium tuberculosis it should be diluted only 1/5, and 1/2.5 (1 part bleach and 1.5 parts water) to inactivate prions. The dilution factor must take into account the volume of any liquid waste that it is being used to sterilize.[26] Bleach will kill many organisms immediately, but for full sterilization it should be allowed to react for 20 minutes. Bleach will kill many, but not all spores. It is also highly corrosive.

Bleach decomposes over time when exposed to air, so fresh solutions should be made daily.

Glutaraldehyde and formaldehyde[edit]

Glutaraldehyde and formaldehyde solutions (also used as fixatives) are accepted liquid sterilizing agents, provided that the immersion time is sufficiently long. To kill all spores in a clear liquid can take up to 22 hours with glutaraldehyde and even longer with formaldehyde. The presence of solid particles may lengthen the required period or render the treatment ineffective. Sterilization of blocks of tissue can take much longer, due to the time required for the fixative to penetrate. Glutaraldehyde and formaldehyde are volatile, and toxic by both skin contact and inhalation. Glutaraldehyde has a short shelf life (<2 weeks), and is expensive. Formaldehyde is less expensive and has a much longer shelf life if some methanol is added to inhibit polymerization to paraformaldehyde, but is much more volatile. Formaldehyde is also used as a gaseous sterilizing agent; in this case, it is prepared on-site by depolymerization of solid paraformaldehyde. Many vaccines, such as the original Salk polio vaccine, are sterilized with formaldehyde.

Phthalaldehyde[edit]

Ortho-phthalaldehyde (OPA) is a chemical sterilizing agent that received Food and Drug Administration (FDA) clearance in late 1999. Typically used in a 0.55% solution, OPA shows better myco-bactericidal activity than glutaraldehyde. It also is effective against glutaraldehyde-resistant spores. OPA has superior stability, is less volatile, and does not irritate skin or eyes, and it acts more quickly than glutaraldehyde. On the other hand, it is more expensive, and will stain proteins (including skin) gray in color. Some side effects from equipment sterilized using this reagent have been reported. For example, two cases of anaphylaxis following cystoscopy with endoscopes sterilized with OPA were reported by Cooper, et al., (J Endourol. 2008 Sep;22(9):2181-4), and four cases of ortho-phthalaldehyde-induced anaphylaxis after laryngoscopy with the detection of specific IgE in serum were reported by Suzukawa, et al., (Allergol Int. 2007 Sep;56(3):313-6. Epub 2007 Jul 1; J Allergy Clin Immunol. 2006 Jun;117(6):1500-1. Epub 2006 Mar 31).

Hydrogen peroxideedit

Hydrogen peroxide is another chemical sterilizing agent. It is relatively non-toxic when diluted to low concentrations, such as the familiar 3% retail solutions although hydrogen peroxide is a dangerous oxidizer at high concentrations (> 10% w/w). Hydrogen peroxide is strong oxidant and these oxidizing properties allow it to destroy a wide range of pathogens and it is used to sterilize heat or temperature sensitive articles such as rigid endoscopes. In medical sterilization hydrogen peroxide is used at higher concentrations, ranging from around 35% up to 90%. The biggest advantage of hydrogen peroxide as a sterilant is the short cycle time. Whereas the cycle time for ethylene oxide (discussed above) may be 10 to 15 hours, the use of very high concentrations of hydrogen peroxide allows much shorter cycle times. Some hydrogen peroxide modern sterilizers, such as the Sterrad NX have a cycle time as short as 28 minutes.

Hydrogen peroxide sterilizers have their drawbacks. Since hydrogen peroxide is a strong oxidant, there are material compatibility issues and users should consult the manufacturer of the article to be sterilized to ensure that it is compatible with this method of sterilization. Paper products cannot be sterilized in the Sterrad system because of a process called cellulostics, in which the hydrogen peroxide would be completely absorbed by the paper product. The penetrating ability of hydrogen peroxide is not as good as ethylene oxide and so there are limitations on the length and diameter of lumens that can be effectively sterilized and guidance is available from the sterilizer manufacturers.

While hydrogen peroxide offers significant advantages in terms of throughput, as with all sterilant gases, sterility is achieved through the use of high concentrations of reactive gases. Hydrogen peroxide is primary irritant and the contact of the liquid solution with skin will cause bleaching or ulceration depending on the concentration and contact time. The vapor is also hazardous with the target organs being the eyes and respiratory system. Even short term exposures can be hazardous and NIOSH has set the Immediately Dangerous to Life and Health Level (IDLH) at 75 ppm. less than one tenth the IDLH for ethylene oxide (800 ppm). Prolonged exposure to even low ppm concentrations can cause permanent lung damage and consequently OSHA has set the permissible exposure limit to 1.0 ppm, calculated as an 8 hour time weighted average (29 CFR 1910.1000 Table Z-1). Employers thus have a legal duty to ensure that their personnel are not exposed to concentrations exceeding this PEL. Even though the sterilizer manufacturers go to great lengths to make their products safe through careful design and incorporation of many safety features, workplace exposures of hydrogen peroxide from gas sterilizers are documented in the FDA MAUDE database. When using any type of gas sterilizer, prudent work practices will include good ventilation (10 air exchanges per hour), a continuous gas monitor for hydrogen peroxide as well as good work practices and training. Further information about the health effects of hydrogen peroxide and good work practices is available from OSHA[29] and the ATSDR.[30]

Hydrogen peroxide can also be mixed with formic acid as needed in the Endoclens device for sterilization of endoscopes. This device has two independent asynchronous bays, and cleans (in warm detergent with pulsed air), sterilizes and dries endoscopes automatically in 30 minutes. Studies with synthetic soil with bacterial spores showed the effectiveness of this device.

Vaporized hydrogen peroxide (VHP) is used to sterilize large enclosed and sealed areas such as entire rooms and aircraft interiors.

Dry sterilization process[edit]

Dry sterilization process (DSP) uses hydrogen peroxide at a concentration of 30-35% under low pressure conditions. This process achieves bacterial reduction of 10−6…10−8. The complete process cycle time is just 6 seconds, and the surface temperature is increased only 10-15 °C (18 to 27 °F). Originally designed for the sterilization of plastic bottles in the beverage industry, because of the high germ reduction and the slight temperature increase the dry sterilization process is also useful for medical and pharmaceutical applications.

Peracetic acid[edit]

Peracetic acid (0.2%) is used to sterilize instruments in some STERIS Corporation systems.

Silver[edit]

Silver ions and silver compounds show a toxic effect on some bacteria, viruses, algae and fungi, typical of heavy metals like lead or mercury, but without the high toxicity to humans that is normally associated with these other metals. Its germicidal effects kill many microbial organisms in vitro, but testing and standardization of silver products is yet difficult.[31] In the antique Greek Hippocratic Corpus it is written that silver has beneficial healing and anti-disease properties,[32] and the Phoenicians used to store water, wine, and vinegar in silver bottles to prevent spoiling. In the early 1900s people would put silver dollars in milk bottles to prolong the milk’s freshness. The exact process of silver’s germicidal effect is still not well understood. One of the explanations is the oligodynamic effect, which accounts for the effect on microorganisms but not on viruses.

Silver compounds were used to prevent infection in World War I before the advent of antibiotics. Silver nitrate solution was a standard of care but was largely replaced by silver sulfadiazine cream (SSD Cream), which was generally the “standard of care” for the antibacterial and antibiotic treatment of serious burns until the late 1990s. Now, other options, such as silver-coated dressings (activated silver dressings), are used in addition to SSD cream. However, the evidence for the use of such silver-treated dressings is mixed and although the evidence on if they are effective is promising, it is marred by the poor quality of the trials used to assess these products. Consequently a major systematic review by the Cochrane Collaboration found insufficient evidence to recommend the use of silver-treated dressings to treat infected wounds.

The widespread use of silver went out of fashion with the development of antibiotics. However, recently there has been renewed interest in silver as a broad-spectrum antimicrobial. In particular, silver is being used with alginate, a naturally occurring biopolymer derived from seaweed, in a range of products designed to prevent infections as part of wound management procedures, particularly applicable to burn victims. In 2007, AGC Flat Glass Europe introduced the first antibacterial glass to fight hospital-caught infection: it is covered with a thin layer of silver.[38] In addition, Samsung has introduced washing machines with a final rinse containing silver ions to provide several days of antibacterial protection in the clothes.[39] Kohler has introduced a line of toilet seats that have silver ions embedded to kill germs. A company called Thomson Research Associates has begun treating products with Ultra Fresh, an anti-microbial technology involving “proprietary nano-technology to produce the ultra-fine silver particles essential to ease of application and long-term protection.”[40] The U.S. Food and Drug Administration (FDA) has recently approved an endotracheal breathing tube with a fine coat of silver for use in mechanical ventilation, after studies found it reduced the risk of ventilator-associated pneumonia.[41]

It has long been known that antibacterial action of silver is enhanced by the presence of an electric field. Applying a few volts of electricity across silver electrodes drastically enhances the rate that bacteria in solution are killed. It was found recently that the antibacterial action of silver electrodes is greatly improved if the electrodes are covered with silver nanorods.[42] Note that enhanced antibacterial properties of nanoparticles compared to bulk material is not limited to silver, but has also been demonstrated on other materials such as ZnO[43]

Potential for chemical sterilization of prions[edit]

Prions are highly resistant to chemical sterilization. Treatment with aldehydes (e.g., formaldehyde) have actually been shown to increase prion resistance. Hydrogen peroxide (3%) for one hour was shown to be ineffective, providing less than 3 logs (10−3) reduction in contamination. Iodine, formaldehyde, glutaraldehyde and peracetic acid also fail this test (one hour treatment). Only chlorine, phenolic compounds, guanidinium thiocyanate, and sodium hydroxide (NaOH) reduce prion levels by more than 4 logs. Chlorine and NaOH are the most consistent agents for prions. Chlorine is too corrosive to use on certain objects. Sodium hydroxide has had many studies showing its effectiveness.[44]

Radiation Sterilization[edit]

Methods of sterilization exist using radiation such as electron beams, X-rays, gamma rays, or subatomic particles.[45]

Non Ionizing Radiation Sterilization[edit]

•        Ultraviolet light irradiation (UV, from a germicidal lamp) is useful only for sterilization of surfaces and some transparent objects. Many objects that are transparent to visible light absorb UV, glass for example completely absorbs all UV light. UV irradiation is routinely used to sterilize the interiors of biological safety cabinets between uses, but is ineffective in shaded areas, including areas under dirt (which may become polymerized after prolonged irradiation, so that it is very difficult to remove). It also damages some plastics, such as polystyrene foam if exposed for prolonged periods of time.

Further information: Ultraviolet germicidal irradiation

Ionizing Radiation Sterilization[edit]

The safety of irradiation facilities is regulated by the United Nations International Atomic Energy Agency and monitored by the different national Nuclear Regulatory Commissions. The incidents that have occurred in the past are documented by the agency and thoroughly analyzed to determine root cause and improvement potential. Such improvements are then mandated to retrofit existing facilities and future design.

 

Illustration of the penetration properties of the different radiation technologies (electron beam, X-ray, gamma rays)

•        Gamma rays are very penetrating and are commonly used for sterilization of disposable medical equipment, such as syringes, needles, cannulas and IV sets, and food. The gamma radiation is emitted from a radioisotope (usually Cobalt-60 or caesium-137). Caesium-137 is used in small hospital units to treat blood before transfusion to prevent Graft-versus-host disease. Use of a radioisotope requires shielding for the safety of the operators while in use and in storage as these radioisotopes continuously emits gamma rays (cannot be turned off). With most designs the radioisotope is lowered into a water-filled source storage pool (the water in the pool absorbs the radiation) to allow maintenance personnel to enter the radiation shield. One variant of gamma irradiators keeps the radioisotope under water at all times and lowers the product to be irradiated under water in hermetic bells. No further shielding is required for such designs. Other uncommonly used designs feature dry storage by providing movable shields that reduce radiation levels in areas of the irradiation chamber. An incident in Decatur, Georgia where water soluble caesium-137 leaked into the source storage pool requiring NRC intervention[46] has led to near elimination of this radioisotope; it has been replaced by the more costly, non-water soluble cobalt-60. Cobalt-60 gammas also has about twice the energy and therefore normally greater penetrating range than Caesium-137 gammas.

 

Efficiency illustration of the different radiation technologies (electron beam, X-ray, gamma rays)

•        Electron beam processing is also commonly used for sterilization. Electron beams use an on-off technology and provide a much higher dosing rate than gamma or x-rays. Due to the higher dose rate, less exposure time is needed and thereby any potential degradation to polymers is reduced. A limitation is that electron beams are less penetrating than either gamma or x-rays. Facilities rely on substantial concrete shields to protect workers and the environment from radiation exposure.

•        X-rays, High-energy X-rays (bremsstrahlung) are a form of ionizing energy allowing to irradiate large packages and pallet loads of medical devices. Their penetration is sufficient to treat multiple pallet loads of low-density packages with very good dose uniformity ratios. X-ray sterilization is an electricity based process not requiring chemical nor radio-active material. High energy and high power X-rays are generated by an X-ray machine that can be turned off for wheot in use, and therefore does not require any shielding when in storage. X-rays are generated by colliding accelerated electrons with a dense material (target) such as tantalum or tungsten in a process known as bremsstrahlung-conversion. These systems generally have low energetic efficiency during the conversion of electron energy to photon radiation requiring much more electrical energy than other systems.

•        Subatomic particles may be more or less penetrating, and may be generated by a radioisotope or a device, depending upon the type of particle.

Irradiation with X-rays or gamma rays does not make materials radioactive. Irradiation with particles may make materials radioactive, depending upon the type of particles and their energy, and the type of target material: neutrons and very high-energy particles can make materials radioactive, but have good penetration, whereas lower energy particles (other thaeutrons) cannot make materials radioactive, but have poorer penetration.

Sterlization by irradiation with gamma rays may however in some cases affect material propertiesIrradiation is used by the United States Postal Service to sterilize mail in the Washington, DC area. Some foods (e.g. spices, ground meats) are irradiated for sterilization (see food irradiation).

Sterile filtration[edit]

Fluids that would be damaged by heat (such as those containing proteins like large molecule drug products, but also wine and beer) irradiation or chemical sterilization, can be only sterilized by Microfiltration using membrane filters[citation needed]. This method is commonly used for heat labile pharmaceuticals and protein solutions in medicinal drug processing. Usually, a filter with pore size 0.2 µm (microfiltration) will effectively remove microorganisms.[48] In the processing of Biologics, viruses must be removed or inactivated. Nanofilters with a smaller pore size of 20 -50 nm (nanofiltration) are used. The smaller the pore size the lower the flow rate. To achieve higher total throughput or to avoid premature blockage, pre-filters might be used to protect small pore membrane filters. In some studies[which?] it has been shown that Prions can be removed or reduced by filtration[citatioeeded].

Membrane filters used in production processes are commonly made from materials such as mixester cellulose or polyethersulfone (PES). The filtration equipment and the filters themselves may be purchased as pre-sterilized disposable units in sealed packaging, or must be sterilized by the user, generally by autoclaving at a temperature that does not damage the fragile filter membranes. To ensure proper functioning of the filter, the membrane filters are integrity tested post-use and in occasions pre-use. The non-destructive integrity test assures the filter is undamaged, it also is a regulatory requirement enforced by agencies like FDA, EMA etc. For best results, final or terminal pharmaceutical sterile filtration is performed in cleanroom classes A

 

 

Additional materials

http://www.innvista.com/health/microbes/bacteria/classif.htm

http://www.earthlife.net/prokaryotes/phyla.html

http://web.uct.ac.za/depts/mmi/lectures/bactax/ppframe.html

http://www.gsbs.utmb.edu/microbook/ch003.htm

http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/dental/tutorials/classification/introduction.html

http://www.microbiol.org/WPaper.Gram.htm

your bunny has an infection of any kind–from an upper respiratory infection, to a jaw abscess to a urinary tract infection–it’s critical to know which antibiotics will be effective against the particular pathogen (i.e., disease-causing agent) causing the problem. This means that (1) the species (and strain) of bacteria (or other pathogen) must be identified and (2) the drugs most effective at inhibiting their growth must be determined. The only reliable way this can be done is a culture and sensitivity test.

In modern laboratories, bacteria are usually identified by characterization of the genome: identifying the characteristics of the DNA and RNA of a sample species. This type of testing is generally considered more reliable (and soon, less expensive) than actually growing bacterial cultures and exposing them to various types of antibiotics to see which drugs kill or inhibit the bacterial growth. But if more than identification is required, and if an antibiotic that usually works against a particular bacterial strain is ineffective, then it may be necessary to actually grow the bacteria and perform an “old fashioned” culture and sensitivity test.

How is a Culture and Sensitivity Test Done?Your rabbit-experienced vet will take a sample of infected tissue or discharge from the infected area (the capsule of an abscess is the best location from which to take a sample, as the internal pus often contains only dead bacteria that will not grow in culture), and send it in a special culture tube to a licensed laboratory for testing.

In the lab, technicians will spread a sample of the infective material onto a plate of nutrient substance (usually agar, a type of gel made from algae) and allow to grow whatever species of bacteria were in the bunny’s infected area.

With a sufficient population of bacteria grown on the plate in the form of a “lawn”, the technicians will perform two main operations:

1. IDENTIFY THE SPECIES OF BACTERIA.

• This is done with various techniques, including examination of lawn characteristics (color, texture, growth pattern, etc.) gram-staining, microscopic examination, metabolic requirement “footprints” and even DNA sequencing.

• Bacterial species commonly isolated from rabbit infections include Pasteurella multocida, Pseudomonas aeruginosa, Bordetella bronchiseptica, Staphylococcus aureus, and several others, though just about anything might turn up, depending on the location and cause of the infection.

2. DETERMINE THE BACTERIAL POPULATIONS SENSITIVITY TO A RANGE OF ANTIBIOTICS.

• This can be done by placing small disks of filter paper or agar impregnated with various types of antibiotics onto the bacterial lawn. The bacteria are allowed to incubate for a day or two, and then the plate is examined to see whether the bacterial growth is inhibited (or not) by the antibiotics on each disk.

• SENSITIVE: In this case, a clear, circular “halo” (technically known as a “plaque,” or zone of inhibition) will appear around the antibiotic disk, indicating an absence of bacteria. The antibiotic has inhibited their growth and/or killed them, meaning that this particular antibiotic should be effective against the infection your rabbit has.

• INTERMEDIATE: A somewhat cloudy plaque indicates that not all the bacteria in the area around the disk have been killed. This means that there are some members of the bacterial population that are sensitive to this particular antibiotic, but others that are genetically immune to its effects. If an antibiotic to which the bacteria show “intermediate” sensitivity is used, it is likely that the sensitive members of the bacterial population will be killed, and the resistant ones will survive, resulting in the selection of a population resistant to that particular antibiotic.

• RESISTANT: In this case, the filter paper will have no discernable plaque around it, meaning that the bacteria are growing normally, even in the presence of the antibiotic. An antibiotic producing no plaque will most likely be ineffective against the bacteria causing your bunny’s infection.

The Petri dish in the image above (shamelessly borrowed from the University of Wisconsin at Madison online Textbook of Bacteriology, which includes a more detailed explanation of the appearance of the halos used in bacterial identification), shows bacteria being strongly and moderately inhibited by most of the antibiotics (impregnated on circles of filter paper), but unaffected by the antibiotics on the disks located at 5 o’clock and 9 o’clock on the dish.

In three to seven days after the sample is taken, your vet will receive the results from the lab, including the species of bacteria and the range of antibiotics to which the bacteria are sensitive (S), resistant (R) and intermediate (I). Again, “sensitive” means that the bacteria were inhibited or killed by that particular antibiotic, and this is what you want to hear.

Choosing and Using the Appropriate AntibioticNote that not all antibiotics are safe for rabbits! Any oral penicillins (e.g., amoxycillin, ampicillin, penicillin) or lincosamines (e.g., clindamycin) should be avoided, as they can cause fatal cecal dysbiosis by killing normal, beneficial intestinal microorganisms and allowing dangerous ones to proliferate. Be sure your bunny is seen by a veterinarian who is familiar with the special medical needs of rabbits. If you don’t already have such a vet, you can find one via the list linked at the House Rabbit Society’s Veterinary Referral Page.

Commonly used antibiotics that are safe for rabbits include the fluoroquinolones (e.g., Baytril and ciprofloxacin), sulfas of various types, chloramphenicol, aminoglycosides (e.g., gentamycin, tobramycin, amikacin–though these are not a first choice as they can be toxic to the kidneys), and injectible Penicillin-G Procaine. It’s critical that the appropriate rabbit-safe antibiotic for the particular infection be prescribed and administered for a course long enough to allow the bunny’s immune system to conquer the infection (with a little bit of help from the antibiotics).

It can take several weeks of antibiotics (sometimes a combination of two different ones!) to get the problem under control. Don’t delay having your bunny properly diagnosed and treated. Almost any infection can develop into a much worse problem if left to its own devices.

It is also extremely important to

• give the full dose

• not miss any doses

• and continue the treatment for the full time period your vet prescribes, even if the symptoms subside.

If you stop treatment early, or give too low a dose, you will risk breeding resistant strains of bacteria by killing off only those most sensitive to the drug(s) you are using, and leaving only the more resistant individuals behind to be the progenitors of the next generation, and to share their genetic resistance with the sensitive members of the bacterial population. It’s not hard to see that misuse of antibiotics can cause real problems.

Why Bother with a Culture and Sensitivity Test?One cautionary note. Some veterinarians who are not as experienced with rabbits as they are with cats and dogs will take one look at a rabbit with “snuffles” or other infection and proclaim that the problem is caused by Pasteruella multocida. Although this bacterial species is not uncommonly carried by rabbits, please do not let anyone convince you that your rabbit’s problem is caused by Pasteurella unless that diagnosis is confirmed via culture and sensitivity test! Not only are some strains of Pasteurella resistant to commonly prescribed antibiotics such as Trimethoprim sulfa, Baytril (enrofloxacin) and even ciprofloxacin, but infections in rabbits also can be caused by even more resilient strains of bacteria, such as Pseudomonas aeruginosa, Bordetella bronchiseptica, Staphylococcus aureus, and others. Without a culture and sensitivity test to positively I.D. the pathogen, you could not only delay your rabbit’s return to good health, but also be throwing good money after bad by treating with an antibiotic that is not effective against the particular strain of bacteria your bunny has.

If no bacteria grow at all, then it’s possible that the bunny has a fungal infection. If this is the case, antibiotics will likely make the problem worse, not better. Hence, it’s very important to check for fungal species if the culture and sensitivity test comes back negative for bacteria. Completely different medications are needed to control infections caused by fungi.

Followup: Backtracking to the CauseOnce an infection is under control, it’s wise to do a bit of detective work and seek possible causes, especially if the condition is chronic. For example, runny eyes and nose or jaw abscesses can be caused by dental problems such as molar spurs or molar roots extending into the sinuses. This is much more common in older rabbits, but all rabbits should routinely have their molars checked for spurs, which are not only painful, but potentially dangerous. A tear duct flush will sometimes temporarily stop runny eye problems, but ultimately it is best to do a complete check for molar problems including visual inspection for spurs and even radiographs to detect molar root infections.

Good care, healthy diet, a happy, calm environment, and your constant vigilance for problems are your bunny’s best bets for a long, healthy, infection-free life. But when even those things fail, it’s good to know there are medications that can help, as long as they’re used wisely, appropriately, and always under the supervision of a good, rabbit-savvy veterinarian.