Basic knowledge of microbiological testing and summary of common disinfection and sterilization methods
Basic knowledge of microbiological testing and summary of common disinfection and sterilization methods
First, the concept and classification of microorganisms
In nature, many naked eyes cannot be seen directly, and microscopic organisms must be observed by means of a microscope. These tiny organisms are collectively called microbes.
The classification of microorganisms is currently recognized to include seven major categories. That is, virus → rickettsia → mycoplasma → bacteria → actinomycetes → spirochetes → fungi (yeast, mold). The small and low rankings in the front, the larger and higher in the back, the content of this article only focuses on three categories.
Second, the characteristics of microorganisms
In addition to the basic characteristics of small individuals and simple structures, microbes have some other characteristics that are different from many living things.
(1) a wide variety
The number and variety of microorganisms is amazing. A drop of water, a soil is often a microbial "society." Among them, the density of soil is the largest, and 1 gram of fertile soil often contains hundreds of millions to billions of microorganisms; poor soil contains millions to tens of millions of gram per gram, a dirty fly, full body energy Carry more than 500 million bacteria.
Three pairs of feet can adhere to 7 million to 10 million bacteria, showing the denseness of microorganisms. The variety of microorganisms is also considerable, and there are about 100,000 kinds of microorganisms known in nature.
(2) Wide distribution
Microorganisms are the most widely distributed species on Earth. In the vast natural world, there are a large number of microorganisms in soil, water, air, and animals, plants, and humans. Some microorganisms can survive under abnormal conditions. For example, acid-resistant bacteria can live in 10% sulfuric acid solution. Thermophilic bacteria can live in hot springs with temperatures above 98 °C, and some even deep sea volcanoes up to 200-300 °C. It can also be active near the mouth. The halophilic bacteria survive in the Dead Sea with a salt content of 23% to 25%, and even 30% of the extracted sodium solution and the salt block. Obviously this is beyond the reach of ordinary organisms.
(3) Rapid reproduction
The rate of microbial reproduction is equally astonishing. It is much faster than higher plants and animals. Some bacteria can breed every 20 minutes under suitable conditions, such as E. coli. One bacteria divides into two in 20 minutes. It can be split 3 times, so that 8 bacteria will produce 8 bacteria after 3 generations of bacteria. which is:
1 hour = 8
2 hours 8 × 23 = 64
3 hours 64 × 23 = 512
4 hours 512 × 23 = 4096
24 hours 1 × 272 = 4.7 * 1021
Breeding at this speed, 72 generations can be reproduced in 24 hours, the number is about 4.7 * 1021. Therefore, the bacteria can be seen in the culture for 24 hours. Its proliferation rate is quite amazing. However, due to various conditions (nutrition), this rate of reproduction is not sustainable. Despite this, the rapid propagation ability of microorganisms is applied to industrial fermentation, and it is also important to obtain a large amount of proliferation and harvest more products in a short period of time.
Third, common microorganisms
(a) bacteria
Bacteria are single-celled organisms that can grow and multiply independently. They come in a variety of sizes and sizes, with an average size of about 1.0 μm. They are magnified 1000 times with a microscope and are only 1.0 mm in size, so they are invisible to the naked eye.
The morphology of the bacteria is not exactly the same, and can generally be classified into three types:
1. When the cocci are present alone, they are spherical. According to their splitting and post-splitting arrangement, they can be divided into single cocci, dicocci, streptococci, tetracytococcus, octacocci, staphylococcus, etc., and the diameter of the cells is about 0.5~. 1.0 μm.
2. Bacillus bacteria are rod-shaped or approximately rod-shaped. Size, length, thickness are very inconsistent, some are short and short, similar to cocci, called cocci; some bacilli are swollen like a rod, called coryneform bacteria; some are arranged in a chain, called streptococci; some grow into side branches , called mycobacteria. Most of the bacilli are about 2.0 to 3.0 μm in length and 0.5 to 1.0 μm in width, and the most common types of bacilli are the pathogenic bacteria.
3. Vibrio and spirulina are curved or spiral, only one curved, comma-like Vibrio, such as Vibrio cholerae; there are several snails that are more tough, such as the snail.
(2) Virus
The virus is the smallest, the simplest structure, has no cell structure, and can only be parasitized in the cells of other organisms, using the host cell's enzyme system and nutrients for the growth and reproduction of the original organism. The shape of the virus is mostly spherical, a few are rod-shaped or brick-shaped, and the bacterial viruses (phage) are mostly scorpion-like, with an average size of only 0.1 μm, about 10 times smaller than bacteria, and the smallest virus is only 0.01 μm, close to protein molecules. Most of the viruses can pass through the bacteria filter, so they are commonly called viral viruses.
Swine flu virus
(three) fungi
Fungi are the largest and more complex microorganisms. Fungi are widely found in nature, such as molds that make clothes, food molds, alcoholic or alcoholic flour, and fungi. Pathogenic fungi are also more common, such as superficial sputum. The fungus is not very resistant to heat and is killed by heating at 60 ° C for 1 hour. Fungi are divided into single-cell fungi and multi-cell fungi; single-cell fungi and multi-cell fungi; single-cell fungi are commonly referred to as yeast. Multicellular fungi are commonly referred to as molds.
Yeast
Yeast
Mold
Aspergillus niger
Fourth, the special structure of bacterial cells
In addition to the above basic structure, some bacteria have some special structures, such as capsules, spores, flagella, and pili.
Capsule
Some bacteria secrete a mucous substance to the surface of the cell wall under certain nutrient conditions, forming a thicker membrane (about 0.2 μm) called a capsule. The capsule contains a lot of water, and the capsule has a protective effect on the bacteria, which can protect the bacteria against drying. The capsule of bacteria is related to the pathogenicity of bacteria. The bacteria with capsules are not easily swallowed by white blood cells, so they can grow and reproduce in the body and cause infection.
Spore
When some cells grow to a certain period or when the external conditions change, the cytoplasm of the bacteria is dehydrated and concentrated, and gradually forms a circular or elliptical small body, which is located at the center or the end of the bacteria, called a spore. As shown in the figure below, the spores are dormant bodies of bacteria, and one propagule can only form one spore. There are several thick and dense membranes on the outside of the spores, which can resist the bad environment of the outside world. It is more resistant to high temperature, dryness, light, chemicals, etc. than the propagules. The bacteria that do not form spores gradually die above 70 °C. The spores are resistant to temperatures of 100 ° C or higher. Therefore, it is much more difficult to kill the spores than to kill the bacteria. Some spores survive for many years without losing their vitality. When they meet suitable conditions, they can grow and reproduce. Therefore, the effect of sterilization should be based on killing spores. .
Bacterial spore
5. Bacterial growth and reproduction and metabolites
1. How the bacteria multiply
The way in which bacteria are propagated is a simple asexual two-splitting method, in which one bacterium is divided into two, two are divided into four, and so on. Cocci are in a state of chain, grape, diplococcus, tetracysococcus, octacocci, etc. because they are along a plane or planes; bacilli generally divide along the horizontal axis.
Most bacteria breed very fast. For example, E. coli can breed for one generation every 20 minutes under suitable conditions. If you keep breeding at this speed for 10 hours, one bacteria can multiply one billion. Although bacterial reproduction is so rapid, it is restricted by various factors and must not be reproduced indefinitely.
2. Conditions for growth and reproduction
1) The growth and reproduction of water bacteria is to absorb nutrients from the outside, and through a number of enzyme systems contained in it, a series of biochemical reactions are decomposed and converted into simple substances, which are then absorbed and utilized. Water is a substance that is indispensable for the life of bacteria.
2) Carbon and nitrogen compounds, inorganic salts, etc. are essential substances for bacterial nutrition.
3) Gas oxygen, carbon dioxide and nitrogen are closely related to the growth and reproduction of bacteria. Some bacteria must have sufficient oxygen supply to live, called aerobic bacteria, such as Mycobacterium tuberculosis; the other is the opposite, in order to live in an oxygen-deficient environment, the presence of oxygen is not good for their growth, called anaerobic Oxygen bacteria, such as tetanus; most but pathogenic bacteria are in between, aerobic and anaerobic life is called facultative anaerobic bacteria, such as E. coli.
(4) pH The most suitable pH for growth of most pathogenic bacteria is pH 7.2-7.6.
(5) Temperature can be divided into psychrophilic bacteria, mesophilic bacteria and thermophilic bacteria according to the temperature requirements of bacteria. The suitable temperature of the general pathogen is basically the same as the human body temperature, so the laboratory culture bacteria are used at 37 °C. (Mold 25 ° C)
3. Metabolites
In the growth and reproduction of bacteria, a large number of metabolites can be produced, some are secreted outside the bacteria, some are contained in the bacteria, and some metabolites are beneficial to the human body, such as antibiotics, vitamins, etc.; however, some metabolites are Harmful, such as pyrogens, these products can cause illness or increase body temperature once they enter the body.
6. Factors affecting microbial growth in the external environment
1. Temperature and chemical factors Temperature and chemical factors have a great influence on the growth and reproduction of microorganisms, and will be described in physical sterilization and chemical sterilization.
2. The effect of pH value on microbial growth is the fastest in neutral reproduction.
3. Humidity temperature can promote bacterial growth, especially for mold breeding. Therefore, preventing excessive humidity has a certain effect on avoiding mildew.
The importance of water activity to bacterial growth increases the ability of food ingredients to bind water and thereby alter water activity.
4. The cells of the osmotic microorganism have a semi-permeable membrane property, which is permeable to water, but is selective for the permeation of other substances. The hypotonic solution can swell the cell membrane, but the high permeability liquid can cause the water to ooze out inside the cell. It is in a dehydrated state. Therefore, the general microorganisms are not easy to grow and multiply in the concentrated salt solution or the concentrated sugar solution, which is because the water in the bacterial cells is exuded and dehydrated. For example, sucrose is a good nutrient for microorganisms; single syrup contains 85% sucrose, which is more than 9 times its isotonic concentration of 9.25%, but it can keep no bacteria; if the concentration is not enough, it is easy to grow bacteria.
Seven, related terms
Sterilization refers to the killing or removal of propagules and spores of all microorganisms to make them completely sterile.
Disinfection means killing only pathogenic microorganisms so that they do not become a source of infection.
Sterilization refers to the use of chemicals or physical factors to kill microorganisms in a short period of time.
Bacteriostatic inhibition inhibits the growth and reproduction of microbial propagules.
Antiseptic means a method of preventing or inhibiting the growth and reproduction of microorganisms.
Sterility means that no living microorganisms are present.
Eight, physical sterilization
(1) Heat sterilization
Heating can destroy enzymes, proteins and nucleic acids in microorganisms, leading to the death of microorganisms. Heat sterilization is divided into dry heat sterilization and moist heat sterilization. At the same temperature, the effect of moist heat sterilization is better than dry heat sterilization. Mainly due to the presence of moisture during sterilization by moist heat, the protein is easily denatured. Moisture is also easy to wet the microbial membrane wall, and the penetration of moist heat is greater than dry heat.
Dry heat sterilization methods are commonly used in flame sterilization and dry hot air sterilization.
(2) UV sterilization
It refers to a method of killing microorganisms by ultraviolet irradiation. The ultraviolet wavelength generally used for sterilization is 200 to 300 nm, and the wavelength with the strongest sterilization power is 253.7 nm. Ultraviolet light acts on nucleic acid proteins to cause denaturation, and at the same time, the air is exposed to ultraviolet rays to generate trace amounts of ozone, thereby co-sterilizing. Ultraviolet light travels in a straight line and can be reflected by different surfaces. The penetration is weak, but it is easier to penetrate clean air and pure water. Therefore, the method is applicable to the sterilization of the surface of the irradiated object, the sterilization of the air and water in the sterile room; the sterilization of the liquid medicine and the sterilization of the deep solid material; the ordinary glass absorbs the ultraviolet light, so it is installed in the glass container. The drugs in this medicine cannot be sterilized by this method. Ultraviolet rays are exposed to the human body for a long time, and conjunctivitis, erythema and skin burning may occur. Therefore, the ultraviolet light is usually turned on for 1 to 2 hours before the person enters the room, and the talent enters the clean room after being closed. If ultraviolet disinfection must be carried out after the person has entered, the human skin and eyes should have effective protective measures. Generally, a 30-watt UV lamp can be installed in a space of 6 to 15 m3, preferably 2.5 to 3 m from the ground.
Pay attention to the following problems when sterilizing with ultraviolet radiation:
1. The bactericidal power of ultraviolet light decreases with the increase of the use time. When the normal use time reaches 70% of the rated time, the ultraviolet lamp should be replaced to ensure the sterilization effect. The average life of domestic UV lamps is generally 2000h.
2, the bactericidal effect of ultraviolet light varies with the strain, the amount of mold killing is 40 to 50 times larger than the bacillus.
3. Ultraviolet radiation is usually designed on the basis of a relative humidity of 60%. When the indoor humidity increases, the exposure should be increased accordingly.
4, the effect of ultraviolet sterilization is related to the length of the irradiation, which requires verification to determine the irradiation time.
5. The installation form and height of the UV irradiation lamp should be determined according to the actual situation and with reference to the instructions for use.
The validation of the sterilization method uses a biological indicator challenge test, and the biological indicator uses a Bacillus subtilis.
(3) Microwave sterilization
Microwave refers to electromagnetic waves with a frequency between 30 and 3000 MHz. Water can strongly absorb microwaves, rotate its polar molecules, generate heat by friction between molecules, and heat up quickly, sterilizing by heat. It can reach 100-150 ° C in tens of seconds to several minutes, and all kill microorganisms in the liquid, suitable for sterilization of water-soluble injection. In addition, the solid medicine decoction pieces and solid preparations (pills, powders, capsule powders, etc.) also contain a small amount of water, and the microwave energy can penetrate into the solid interior, and is uniformly heated from the surface to the inside, and functions as a drying and sterilization. It is mostly used for the sterilization of oral liquid preparations and is expected to be used for sterilization of injection liquids. However, it may affect the PH value, content and color of certain drugs.
(4) Radiation sterilization method
Radiation sterilization is a method of killing bacteria by using γ-rays and β-rays. It is also called ionizing radiation. The former is emitted by cobalt-60 or cesium-137, and has strong penetrating power. The latter is produced by an electron accelerator with charge and penetrating power. Weak, poor sterilization effect.
Nine, chemical sterilization
Chemical sterilization refers to the use of chemicals to kill microorganisms. The same chemical exhibits a bacteriostatic effect at a low concentration and a bactericidal action at a high concentration. The bactericidal mechanism may be: denaturation of the microbial protein, or interaction with the enzyme system to affect metabolism, or change the permeability of the membrane wall to cause microbial death. Commonly used methods include disinfectant disinfection and chemical gas sterilization.
(1) Disinfectant disinfection method
Disinfection refers to the method of killing pathogenic microorganisms. However, most chemical disinfectants can only kill the propagules of microorganisms and cannot kill spores, and can control a certain range of sterility. The disinfectant can be formulated into a suitable concentration, and the materials, environment, appliances, etc. can be disinfected by spraying, rubbing or soaking. Commonly used chemical disinfectants are 0.1% to 0.2% benzalkonium bromide solution, 3% to 5% phenol or coal phenol soap solution, 75% ethanol, and the like. Commonly used for surface sterilization of objects. However, be careful not to over-concentrate it to prevent chemical corrosion. Clean room walls, ceilings, doors and windows, machinery and equipment, instruments, consoles, cars, tables, chairs and other surfaces as well as human hands (gloves) should be cleaned regularly and sprayed with disinfectant during environmental verification and daily production. The disinfectant used must be in a laminar flow table and filtered with a 0.22 μm filter before it can be used.
(2) Chemical gas sterilization
It refers to a method of killing microorganisms by using a chemical gas or generated steam.
Ethylene oxide sterilization
The ethylene oxide sterilization method is a method of sterilizing using ethylene oxide gas. It is a traditional sterilization method, which can be applied to the sterilization of garment sterilization, medicines, facilities, equipment and the like which are not resistant to heat sterilization.
The ethylene oxide sterilization system mainly has the following four important factors that restrict each other:
1, temperature; 2, humidity; 3, gas concentration; 4, sterilization time.
Because ethylene oxide is a flammable and explosive substance, an open flame can cause combustion, and at the same time, it may cause an explosion due to gas decomposition. Safety concerns should be paid attention to in the sterilization of ethylene oxide.
2. Formaldehyde and other steam fumigation methods
The use of formaldehyde, propylene glycol or peracetic acid and other chemicals, steam generated by heating for air environment sterilization.
(1) Calculate the volume of the room and weigh out the formaldehyde at a rate of 10 g/m3.
(2) Pour formaldehyde into the formaldehyde generator or heating plate or beaker, and put the humidification water, if necessary, add potassium permanganate (2~3g/m3), then heat (the formaldehyde generator is heated by steam, The heating plate or beaker is heated with water therein to evaporate it into a gas.
(3) Sterilization process: the air conditioner stops running → start the formaldehyde gas generator or heat the formaldehyde in the heating plate → let the formaldehyde gas spread for about 30 minutes → start the air conditioner to let the formaldehyde gas circulate for about 30 minutes → stop the air conditioner, the room Fumigation, no less than 8 hours → Room exhaust, replaced with fresh air for about 2 hours → resume normal operation.
When the relative humidity is above 65% and the temperature is between 24 and 40 °C, the disinfection effect of formaldehyde gas is the best.
3. Ozone disinfection method
The principle of ozone (O3) disinfection is: ozone has unstable molecular structure at normal temperature and pressure, and quickly decomposes itself into oxygen (O2) and single oxygen atom (O). The latter has strong activity and is extremely polar to bacteria. Strong oxidation, ozone oxidation decomposes the enzymes necessary to oxidize glucose inside the bacteria, destroying its cell membrane and killing it. The excess oxygen atoms recombine themselves into universal oxygen molecules (O2) without any harmful residues, so they are called non-polluting disinfectants. It not only has strong killing ability against various bacteria (including hepatitis virus, Escherichia coli, Pseudomonas aeruginosa and bacteria), but also is effective for mold.
Ten, aseptic method
The aseptic operating room should meet the GMP requirements, and comprehensive measures can be taken to meet the requirements for sterility:
1 should use laminar clean air technology;
2 can be sterilized by air fumigation by steam fumigation and ultraviolet lamp sterilization;
3 indoor appliances, walls, tables and other exposed surfaces are sprayed and wiped with disinfectant;
4 other items can be hot pressed or dry heat sterilization;
5 Operators clothing, caps, shoes, hands, etc. disinfection and sterilization.
Colony inspections should be performed regularly in the aseptic processing room. Generally, the culture dish is exposed for 20~30min, and cultured at 37°C for 48h, and no more than 3 colonies in each dish are qualified.
11. Antiseptic and preservatives for food
Microorganisms can be propagated by moisture and other suitable conditions. Many foods are easily contaminated by microorganisms. Therefore, in addition to sterilization by appropriate means, preservatives can be added to inhibit and kill microorganisms.
Preservatives, also known as bacteriostatic agents, are substances that inhibit the growth and reproduction of microorganisms. Commonly used preservatives are:
1. Benzoic acid and sodium benzoate are commonly used effective preservatives, and the general dosage is 0.1% to 2.25%. Benzoic acid has poor water solubility, while sodium benzoate has good water solubility, is easy to dissolve, and is convenient to use.
2. Parabens (parabens) have four esters of A, B, C and D. The antibacterial effect is strong, and the general dosage is 0.01% to 0.25%.
3. The molecular structure of sorbic acid and potassium sorbate sorbic acid is CH3—CH=CH—CH=CH—COOH, and the usual concentration is 0.15% to 0.05%. It has strong antibacterial effect on bacteria and mold.
4. Others containing more than 20% ethanol, containing more than 30% glycerol, many volatile oils in traditional Chinese medicine and 0.1 dehydrated acetic acid have antiseptic effect.
Pet Housewares,Slicker Brush For Dogs,Cat Detangler Brush,Pet Comb For Cats
DongGuan Lucky Pet Products Co., Ltd. , https://www.dgluckypet.com