3 DISINFECTION AND STERILIZATION
第3章 消毒与灭菌

INTRODUCTION TO THE CONTROL OF MICROORGANISMS

Control of microorganisms is essential in order to prevent the transmission of diseases and infection, stop decomposition and spoilage, and prevent unwanted microbial contamination. Microorganisms are controlled by means of physical agents and chemical agents. Physical agents include such methods of control as high or low temperature, desiccation, osmotic pressure, radiation, and filtration. Control by chemical agents refers to the use of disinfectants, antiseptics, antibiotics, and chemotherapeutic antimicrobial chemicals抗菌化学药品.
Basic terms used in discussing the control of microorganisms include:
1. Sterilization 灭菌
The process of destroying all microbial forms. A sterile object is one free of all microbial forms, including bacterial spores.
2. Disinfection 消毒
The reduction or elimination of pathogenic microorganisms in or on materials, so they are no longer a health hazard.
3. Antisepsis 防腐
Use of chemical agents on skin or other living tissue to inhibit or eliminate microbes; no sporicidal杀芽胞的 action is implied.
4. Bacteriostasis抑菌
Inhibits the growth of microorganisms.
5. Asepsis 无菌
No living microorganisms exists.

CONTROLLING MICROORGANISMS BY PHYSICAL AGENTS

A. High Temperature
Microorganisms have a minimum, an optimum, and a maximum temperature for growth. Temperatures below the minimum usually have a static action on microorganisms抑制细菌生长的效应. They inhibit microbial growth by slowing down metabolism but do not necessarily kill the organism. Temperatures above the maximum usually have a cidal action杀菌作用, since they denature microbial enzymes and other proteins. Temperature is a very common and effective way of controlling microorganisms.
Vegetative microorganisms can generally be killed at temperatures from 50°C to 70°C with moist heat. Bacterial endospores, however, are very resistant to heat and extended exposure to much higher temperature is necessary for their destruction. High temperature may be applied as either moist heat or dry heat.
1. Dry heat干热
Dry heat kills microorganisms through a process of protein oxidation氧化 rather than protein coagulation凝固. Examples of dry heat include:
a. Hot air sterilization
Microbiological ovens employ very high dry temperatures: 171°C for 1 hour; 160°C for 2 hours or longer; or 121°C for 16 hours or longer depending on the volume. They are generally used only for sterilizing glassware, metal instruments, and other inert materials like oils and powders that are not damaged by excessive temperature.
b. Incineration焚化， red heat赤热 and flaming烧灼
Incinerators are used to destroy disposable or expendable materials by burning. We also sterilize our inoculating wires, and loops by holding them in the flame until they are red hot. Flaming is exposure of the neck of flasks to flame for a few seconds, but this dose not produce a sufficiently high temperature for sterilization.
2. Moist heat湿热
Moist heat is generally more effective than dry heat for killing microorganisms because of its ability to penetrate microbial cells. Moist heat kills microorganisms by denaturing their proteins (causes proteins and enzymes to lose their three-dimensional functional shape). It also may melt lipids in cytoplasmic membranes.
a. Autoclaving高压灭菌
Autoclaving employs steam under pressure高压蒸气. During autoclaving, the materials to be sterilized are placed under 15 pounds per square inch of pressure in a pressure-cooker type of apparatus. Under 15 pounds of pressure, the boiling point of water is raised to 121°C, a temperature sufficient to kill bacterial endospores. The time the material is left in the autoclave varies with the nature and amount of material being sterilized. Given sufficient time (generally 15-45 minutes), autoclaving is cidal for both vegetative organisms and endospores, and is the most common method of sterilization for materials not damaged by heat.
b. Boiling water煮沸灭菌
Boiling water (100°C) will generally kill vegetative cells after about 10 minutes of exposure. However, certain viruses, such as the hepatitis viruses, may survive exposure to boiling water for up to 30 minutes, and endospores of certain Clostridium梭菌 and Bacillus杆菌 species may survive even hours of boiling.
c. Pasteurization巴氏消毒法
Pasteurization is the mild heating of milk and other materials to kill particular spoilage organisms or pathogens. It does not, however, kill all organisms. Milk is usually pasteurized by heating to 71.6°C for at least 15 seconds in the flash method瞬间法 or 62.9°C for 30 minutes in the holding method持续法.
B. Radiation 辐射
a. Ultraviolet Radiation紫外线辐射
The ultraviolet portion of the light spectrum includes all radiations with wavelengths from 100 nm to 400 nm. The microbicidal activity of ultraviolet (UV) light depends on the length of exposure: the longer the exposure the greater the cidal activity. It also depends on the wavelength of UV used. The most cidal wavelengths of UV light lie in the 260 nm - 270 nm range where it is absorbed by nucleic acid.
In terms of its mode of action, UV light is absorbed by microbial DNA and causes adjacent thymine bases胸腺嘧啶碱基 on the same DNA strand to covalently bond together, forming what are called thymine-thymine dimmers胸腺嘧啶二聚体. As the DNA replicates, nucleotides核苷do not pair complementary base 互补碱基with the thymine dimers and this terminates the replication of that DNA strand.
The effect of this improper base pairing may be reversed to some extent by exposing the bacteria to strong visible light immediately after exposure to the UV light. The visible light activates an enzyme that breaks the bond that joins the thymine bases, thus enabling correct complementary base pairing to again take place. This process is called photoreactivation光复活作用.
UV lights are frequently used to reduce the microbial populations in hospital operating rooms and sinks, aseptic filling rooms of pharmaceutical companies, in microbiological hoods通风橱, and in the processing equipment used by the food and dairy industries.
An important consideration when using UV light is that it has very poor penetrating power很弱的穿透力. Only microorganisms on the surface of a material that are exposed directly to the radiation are susceptible to destruction. UV light can also damage the eyes, cause burns, and cause mutation in cells of the skin.
b. Ionizing Radiation 电离辐射
Ionizing radiation, such as X-rays and gamma rays, has much more energy and penetrating power than ultraviolet radiation. It ionizes water and other molecules to form radicals基团 (molecular fragments with unpaired electrons) that can break DNA strands. It is often used to sterilize pharmaceuticals and disposable medical supplies such as syringes, surgical gloves, catheters导尿管, and sutures缝合线. It can also be used to retard spoilage in seafoods, meats, poultry, and fruits.
C. Filtration 过滤
Microbiological membrane filters provide a useful way of sterilizing materials such as vaccines, antibiotic solutions, animal sera, enzyme solutions, vitamin solutions, and other solutions that may be damaged or denatured by high temperatures or chemical agents. The filters contain pores small enough to prevent the passage of microbes but large enough to allow the organism-free fluid to pass through. The liquid is then collected in a sterile flask. Filters with a pore diameter from 25 nm to 0.45 ?m are usually used in this procedure. Filters can also be used to remove microorganisms from water and air for microbiological testing.
D. Low Temperature
Low temperature inhibits microbial growth by slowing down microbial metabolism. Examples include refrigeration and freezing. Refrigeration at 5°C slows the growth of microorganisms and keeps food fresh for a few days. Freezing at -10°C stops microbial growth, but generally does not kill microorganisms, and keeps food fresh for several months.
E. Desiccation 干燥
Desiccation, or drying, generally has a static effect on microorganisms. Lack of water inhibits the action of microbial enzymes. Dehydrated and freeze-dried foods, for example, do not require refrigeration because the absence of water inhibits microbial growth.

USING DISINFECTANTS AND ANTISEPTICS TO CONTROL MICRO- ORGANISMS

A. Antimicrobial modes of action for disinfectants and antiseptics消毒剂和防腐剂的抗菌作用:
1. They may damage the lipids and/or proteins of the semipermeable cytoplasmic membrane 半透性的细胞膜of microorganisms resulting in leakage of cellular materials needed to sustain life.
2. They may denature microbial enzymes and other proteins, usually by disrupting the hydrogen and disulfide bonds破坏氢键和二硫键 that give the protein its three-dimensional functional shape. This blocks metabolism.
B. Different categories of such chemical agents:
1. Phenol and phenol derivatives
Phenol (5-10%) was the first disinfectant commonly used. However, because of its toxicity and odor, phenol derivatives, e.g., chlorhexidine洗必泰are now generally used. These agents kill most bacteria, most fungi, and some viruses, but are usually ineffective against endospores. They alter membrane permeability and denature proteins.
2. Soaps and detergents去垢剂
Soaps are only mildly microbicidal. Their use aids in the mechanical removal of microorganisms by breaking up the oily film on the skin (emulsification乳化作用) and reducing the surface tension表面张力 of water so it spreads and penetrates more readily. Many cosmetic soaps also contain added disinfectants to increase antimicrobial activity.
Detergents may be anionic or cationic. Anionic (negatively charged) detergents, such as laundry powders, mechanically remove microorganisms and other materials but are not very microbicidal. Cationic (positively charged) detergents alter membrane permeability and denature proteins. They are effective against many vegetative bacteria, some fungi, and some viruses. However, endospores, Mycobacterium tuberculosis结核分枝杆菌, and Pseudomonas species假单胞菌 are usually resistant.
3. Alcohols
70% solutions of ethyl or isopropyl alcohol乙醇或异丙醇 are effective in killing vegetative bacteria, enveloped viruses, and fungi. However, they are usually ineffective against endospores and non-enveloped viruses. Once they evaporate, their cidal activity will cease. Alcohols denature membranes and are often combined with other disinfectants, such as iodine碘, mercurials汞制剂, and cationic detergents for increased effectiveness.
4.Acids and alkalies
Acids and alkalies alter membrane permeability and denature proteins and other molecules. Salts of organic acids, such as calcium propionate丙酸钙, are commonly used as food preservatives. Undecylenic acid十一烯酸 (Desenex?) is used for dermatophyte皮肤真菌 infections of the skin. An example of an alkali is lye碱液 (sodium hydroxide氢氧化钠).
5. Heavy metals
Heavy metals, such as mercury, silver, and copper, denature proteins. Mercury compounds (mercurochrome红汞, merthiolate硫柳汞) are only bacteriostatic and are not effective against endospores. Silver nitrate 硝酸银(1%) is sometimes put in the eyes of newborns to prevent gonococcal ophthalmia淋菌性眼炎.
6. Chlorine
Chlorine gas reacts with water to form hypochlorite次氯酸盐 ions, which in turn denature microbial enzymes. Chlorine is used in the chlorination氯化 of drinking water, swimming pools, and sewage. Calcium hypochlorite次氯酸钙, sodium hypochlorite, and chloramines氯胺 (chlorine plus ammonia) are used to sanitize glassware, eating utensils, dairy and food processing equipment, and hemodialysis血液透析 systems.
7. Iodine and iodophores
Iodine also denatures microbial proteins and is usually dissolved in an alcohol solution to produce a tincture酊剂. Iodophores碘附 are a combination of iodine and an anionic detergent (such as polyvinylpyrrolidone, PVP聚乙烯吡咯烷酮) which reduces surface tension and slowly releases the iodine. Iodophores are less irritating than iodine and do not stain. They are generally effective against vegetative bacteria, Mycobacterium tuberculosis, fungi, some viruses, and some endospores.
8. Aldehydes醛
Aldehydes, such as formaldehyde甲醛 and glutaraldehyde戊二醛, denature microbial proteins. Formalin 福尔马林(37% aqueous solution of formaldehyde gas) is extremely active and kills most forms of microbial life. It is used in embalming尸体防腐, preserving biological specimens, and in preparing vaccines. Alkaline glutaraldehyde (Cidex?), acid glutaraldehyde (Sonacide?), and glutaraldehyde phenate酚盐 solutions (Sporocidin?) kill vegetative bacteria in 10-30 minutes and endospores in about 4 hours. A 10-hour exposure to a 2% glutaraldehyde solution can be used for cold sterilization of materials.

FACTORS INFLUENCING ANTIMICROBIAL ACTIVITY

A. The concentration and kind of a chemical agent used;
B. The intensity and nature of a physical agent used;
C. The length of exposure to the agent;
D. The temperature at which the agent is used;
E. The number of microorganisms present;
F. The species or strain of microorganism;
G. The nature of the material bearing the microorganism;
H. The presence of organic or other interfering substances.

(袁建平)