Food microbiology laboratory staff must have a strict sterility concept, many tests require to be performed under sterile conditions, the main reasons are:
The first is to prevent artificial contamination of samples in test operations;
The second is to ensure the safety of workers and prevent personal contamination caused by detected pathogens due to improper operation.
I. Aseptic requirements
1. Wear uniforms and work caps when inoculating bacteria.
2. When inoculating food samples, special work clothes, caps, and slippers must be worn. They should be placed in a sterile room buffer and used after being disinfected by ultraviolet light before work.
3. When inoculating food samples, wash hands with soap before entering the sterile room, then wipe off the hands with a 75% alcohol swab.
4. The pipettes, plates, and culture media used for inoculation must be sterilized and sterilized. Unopened containers should be opened and not used after being placed. Metalware should be autoclaved or burned with 95% alcohol for three times before use.
5. When the pipette is removed from the package, the tip of the pipette tip cannot touch the exposed part. When using a pipette to inoculate a test tube or plate, the tip of the pipette tip must not touch the side of the tube or the plate.
6. Inoculation of the sample, transfer of bacteria must be in front of the alcohol lamp operation, inoculation of bacteria or samples, after the suction tube removed from the package and open the tube plug must be sterilized by the flame.
7. Inoculate the rings and needles before the inoculation of bacteria. All flames shall be cauterized. If necessary, the ring and the connection between the needle and the rod are to be burned. The inoculation loop for inoculating TB and strong bacteria should be boiled in boiling water for 5 minutes. Burned by flames.
8. When the pipette sucks the bacterium solution or sample, it should be sucked with the corresponding rubber head and it should not be sucked directly.
Two. Aseptic room use requirements
1. The window leading to the outside of the sterile room shall be double glazed and shall be sealed. It shall not be opened arbitrarily and shall be provided with a buffer room and a sliding door corresponding to the size of the sterile room. There shall be a small window of 0.5-0.7 m2. Prepare to transfer items into the sterile room.
2. The sterile room should be kept clean. After work, it should be disinfected with 2%-3% coal phenol soap solution. Wipe the work surface and store no items not related to the experiment.
3.Before and after use, the door should be closed and the UV light should be turned on. If using an indoor suspension UV lamp, a 30W UV lamp is required. The distance is 1.0m and the irradiation time is not less than 30min. The use of UV lamp should be avoided. Directly under ultraviolet light to avoid damage, the lamp tube needs to be wiped gently with alcohol cotton ball every two weeks to remove the dust and grease, so as to reduce the effect of ultraviolet light penetration.
4. When handling and inoculating food specimens, enter the sterile room and do not enter or leave the area at will. If you need to transfer items, you can pass them through the small window.
5. In the sterile room, if you need to install air conditioning, you should have a filter device.
Three disinfection and sterilization requirements
Glassware, metal utensils and culture media for microbial detection, contaminated and inoculated cultures must be sterilized before they can be used.
(I) Dry heat and damp heat autoclave sterilization methods
1. Preparation before sterilization
(1) All items that need to be sterilized should be cleaned and dried first. Glassware such as straws and plates should be packed tightly. If a metal tube is used, the upper vent hole should be opened.
(2) The triangle cork of the culture medium is packed with paper, the test tube is covered with a lid, and the syringe is pulled out from the tube and wrapped with gauze.
(1) Dry heat sterilizer: The loading and unloading items must not be over-squeezed and cannot contact the four walls of the box.
(2) Large-scale high-pressure steam cooker: Place the sterilized articles separately and wrap them directly into the sterilizing drum.
3. Equipment inspection
(1) Check whether the switch of the door is flexible, and whether the rubber band is damaged or not.
(2) Check whether the pressure gauge stays in the zero position when the steam is exhausted, close the door and cover, pass the steam or heat, observe whether there is leakage, whether the pressure gauge and the condition marked by the thermometer are consistent, and whether the pipeline is blocked.
(3) For sterilizers with automatic electronic program control devices, check the prescribed procedures before use, and check whether they meet the requirements for sterilization.
(1) Dry heat sterilization:
This method is applicable to the sterilization of items that are not damaged, deteriorated, and does not evaporate under the conditions of dry heat, and are commonly used for glassware, metal products, ceramic products, and the like.
The instrument utensils should be cleaned and then dried to prevent the carbon attached to the surface from charring.
When sterilizing, the objects should not be over-squeezed. Do not touch the bottom or the wall of the tank directly. There should be gaps between the items.
When bacteria close the door, connect the power supply, first open the exhaust hole for about 30 minutes, eliminate the cold air in the sterilizer, temperature rise to 160 °C, adjust the indicator, maintain 1.5 ~ 2h.
After the sterilization is completed or the temperature is raised, the door must be opened below 60°C.
(2) The use of portable pressure cookers or vertical pressure steam sterilizers should be carried out as follows:
The portable pressure cooker is filled with 3L of water in the main body and the vertical pressure cooker is filled with 16L of water (refill the amount of water when it is used repeatedly, and the water must be replaced when it becomes cloudy);
The portable pressure cooker inserts the exhaust pipe on the top cover into the square pipe on the inner wall of the disinfection barrel (the sterilizer without hose or hose corrosion cracking shall not be used);
Tighten the top cover tightly, do not leak; set the sterilizer on the fire source to heat, the vertical pressure cooker is connected to the power supply, and open the exhaust valve on the top cover and put the air-conditioning (after the water is boiled exhaust for 10-15min) ;
Close the exhaust valve to increase the vapor pressure to the specified requirement and maintain the prescribed time (depending on the nature of the sterilized article and the relevant circumstances);
After reaching the prescribed time, immediately open the exhaust valve to vent the vapors. When the pressure is restored to zero, naturally cool to 60°C and then open the lid. If it is a liquid object, do not open the exhaust valve. Immediately remove the heat source from the pan, wait until it cools naturally, restore the pressure to zero, and then lower the temperature to less than 60°C and then open the lid to prevent sudden violent decompression of the liquid and boiling or explosion of the container;
(3) Use of horizontal pressure cooker steam sterilizers according to the following steps:
Close the pot door, open the inlet valve, and introduce steam into the sandwich to preheat. The cold air in the sandwich is automatically discharged by the gas trap. After the interlayer reaches the predetermined temperature, the inlet valve of the pot chamber is opened to introduce the steam into the pot chamber. The cold air in the pot chamber is automatically discharged through the gas block of the pot chamber.
When the pot room reaches the specified pressure and temperature, adjust the intake valve so that it remains constant
Natural or artificial cooling to 60 °C and then open the door to take things, shall not use the rapid discharge of steam law to prevent a sudden pressure drop, violent liquid boiling or container explosion.
Use an automatic program-controlled pressure steam sterilizer. After closing the door with items placed, press the appropriate switch according to the type of the item to automatically perform sterilization according to the required procedure. When sterilizing, use the attached instrument to record the temperature and time for future reference. The operating requirements should be strictly in accordance with the manufacturer's instructions.
5. Sterilization temperature and time
(1) Dry heat sterilizer sterilization temperature 160 °C, 1.5-2h.
(2) Pressure Steam Sterilizer Sterilization Temperature and Time
(b) Intermittent sterilization methods
1. Sterilization method:
Using steam sterilization without pressure, certain substances are easily sterilized by autoclaving and can be sterilized by this method.
(1) Put the items to be sterilized into the pot, cover the top cover, open the drain outlet, and drain the remaining water in the device.
(2) Close the drain, open the intake valve and sterilize for 10-20 minutes as required.
(3) After the sterilization is completed, close the intake valve, take out the items until they are cooled to room temperature, and put them into the 37°C incubator overnight. Disinfect according to the above method on the next day. In this way, the sterilization can be achieved three times.
2. How to use the serum coagulator:
When the medium contains special components of serum or eggs, the high heat will destroy the nutrients, so the use of low temperature can make the serum coagulate, and can achieve the purpose of sterilization:
(1) When aliquots sterilized using this method are to be aliquoted, aseptic processing must be strictly observed, and test tubes and plates are also used after sterilization. (2) The culture medium is made to be beveled or high-level as required. After adding enough water, connect the power supply, warm up 75-90°C for 1 hour, put it in a 37°C incubator overnight, and sterilize it three times.
3. Boiling disinfection:
Cooker or boiling sterilizer can be used, the water boiled and then cook 5 ~ 15 min, can also be added in the water 2% carbolic acid boiled 5min, add 0.02% formaldehyde, 80 °C cook 60min can achieve the purpose of sterilization, but the choice of boiling disinfection In the case of an enhancer, care should be taken with the corrosion of the article.
The sterilized items are aseptic under normal conditions, and should be carefully removed from the sterilizer to avoid re-contamination.
(1) The item is removed and the integrity of the package is checked immediately. If there is damage or the tampon is removed, it cannot be used as a sterile item;
(2) The items taken out are not sterilized items if they are packaged with obvious flooding;
(3) Culture media or reagents, etc., should be checked for compliance with the color or state after sterilization; those that have not been reached should be discarded;
(4) Open-closed containers should be closed when they are removed;
(5) The items taken out are dropped on the ground or misplaced, or stained with water, are considered to be contaminated and cannot be used as sterile items;
(6) The qualified sterilized articles taken out shall be stored in the storage room or dustproof cabinet, and shall not be mixed with non-sterile items.
(7) All qualified items should be marked with a sterilization date and expiration date;
(8) Record the name, quantity, temperature, time, and operator of the sterilization product after each batch of sterilization.
IV. Toxic and contaminated soil treatment requirements
The laboratory equipment, cultures, etc. used in the microbiological experiments shall not be taken out of the laboratory without being disinfected.
1. Contaminated materials and wastes that have been cultivated should be placed in tight containers or wire baskets and stored centrally in designated locations until autoclave sterilization.
2. Microbial contaminated cultures must be autoclaved at 121°C for 30 minutes.
3. Sterile straw after use, put into 5% coal phenol soap solution or carbolic acid solution after use, soaking for a minimum of 24h (disinfection liquid shall not be lower than the soaking height) and then autoclaving at 121°C for 30 minutes.
4. Smear-dyeing The rinse liquid can generally be flushed directly into the sewer. Violent broth must be washed in a beaker. After autoclaving, it can be poured into the sewer. The dyed slide is put into a 5% coal phenol soap solution. After soaking for 24h, boil and wash. Slides or plates for agglutination tests must be autoclaved and washed.
5. Shatter the culture, immediately spray and soak the contaminated area with 5% coal phenol soap solution or carbolic acid solution, soak for half an hour and wipe clean.
Contaminated work clothes or work clothes, caps, masks, etc. worn during the intense test should be placed in a special sterilization bag and then washed after autoclaving.
V. Medium preparation requirements
The quality of the culture medium preparation will directly affect the growth of microorganisms. Because various microorganisms do not have the same nutrient requirements and different culture purposes, various media preparation requirements are as follows:
1. According to the composition of the medium formulation, the ingredients are weighed and then dissolved in distilled water. The quality of the applied reagent drugs should be checked before use.
2. Determination and adjustment of pH: pH measurement should be carried out when the medium is cooled to room temperature, because there is a certain difference in pH when it is hot or cold. When the measurement is good, add alkali or acid after calculation, Should be tested again. The pH of the culture medium must be accurate, otherwise it will affect the growth of microorganisms or affect the observation of the results. However, attention should be paid to the fact that autoclaving can affect the pH of some of the culture media. Therefore, it is not recommended that the sterilization pressure be too high or too many times so as not to affect the quality of the culture medium. The indicator, sodium deoxycholate, agar, etc. Add after adjusting the pH.
3. The culture medium should be kept clarified to observe the growth of the bacteria. After the medium is heated and boiled, it can be filtered with absorbent cotton or flannel to remove the sediment. If necessary, the egg white protein can be used for clarification. The agar strips used should be washed and dried beforehand. After use, avoid affecting transparency due to impurities in agar.
4. It is better not to use iron, copper or other containers for holding the culture medium. It is better to use washed neutral hard glass containers.
5. Sterilization of the culture medium must achieve the purpose of complete sterilization, but also pay attention to not reduce its nutritional value due to heating, generally 121 °C, 15min can be, such as containing high-heat intolerant medium such as sugar, serum Gelatin, etc., should be used low-temperature sterilization or intermittent sterilization, some can not be heated reagents such as potassium tellurite, yolk, TTC, antibiotics, etc., until the base agar after autoclaving cool to about 50 °C and then added;
6. After each batch of culture medium is prepared, aseptic growth test and growth test of the tested strains should be performed. If it is a biochemical culture medium, use standard strains to inoculate and culture, observe the biochemical reaction results, it should be normal reaction, and the medium should not be stored for too long. If necessary, store in a 4°C refrigerator.
7. At present, there are many kinds of drying media. Each batch needs to use standard strains for growth test or biochemical reaction observation. Each kind of medium can be applied after the growth test of the corresponding strains is good, and newly purchased or stored for too long. The culture medium should also be tested for pH when it is formulated, and it should be used according to the dosage and method of the product instructions.
8. The chemical reagents, sterilization conditions, growth test results, and production personnel used in each batch of culture media should be well documented for future reference.
Six sample collection and processing requirements
1. The collected test samples must be representative. When sampling, firstly conduct a detailed investigation on the raw materials, processing, transportation, storage method conditions, and sanitary conditions of the surrounding environment, and check whether there is any pollution source.
2. According to the type and quantity of food, the sampling quantity and method should be carried out according to the requirements of the standard inspection method.
3. Sampling should pay attention to aseptic operation, the container must be sterilized, to avoid microbial contamination in the environment, the container must not use phenol soap solution, sterilization with benzalkonium, alcohol and other disinfecting drugs, but can not contain such disinfectant or antibiotics, In order to avoid killing the microorganisms in the sample, the shears, knives and spoons used must also be sterilized.
4. Samples should be sent to the inspection room immediately after they are collected for inspection. Generally, the inspection process should not exceed 3 hours. For example, if the distance is long, it can be stored in a 1 to 5°C environment. For those who need to freeze, they should be frozen. Inspection.
5.After receiving the sample in the inspection room, register (sample name, inspection unit, quantity, date, serial number, etc.) and observe the sample
The appearance, if you find one of the following conditions, you can refuse inspection:
(1) If the sample is sterilized by special high pressure, boiling or other methods, it will lose its significance on behalf of the original food inspection;
(2) Those who have opened the bottle or packaged food, cooked meat and its products, cooked poultry and other foods have been broken and incomplete, that is to lose the original food shape (except food poisoning samples);
(3) Insufficient number of samples as required;
For samples submitted to meet the requirements, the inspection room shall be inspected immediately after it is received. If the conditions are not met, it shall be stored in a refrigerator at 4°C, ready to create conditions, and then tested.
6. When the samples are tested, they are treated appropriately according to their different traits.
(1) When the liquid sample is inoculated, it should be thoroughly mixed and inoculated by the amount.
(2) Solid samples: Use a sterilized knife and cut out 25 g of different parts and place them in 225 mL of sterile saline or other solution. Mix and homogenize with a homogenizer.
(3) Bottles and bags of foods should be sterilized by application of sterilizing operations, and inoculated after treatment according to the traits.
Seven sample inspection, recording and reporting requirements
1. After the sample is received in the inspection room, it firstly undergoes appearance inspection and timely inspection according to the national standard inspection method. During the inspection process, serious, responsible, and strict aseptic operations must be performed to avoid microbial contamination in the environment.
2. The method used in the sample inspection process, the phenomena and results that appear, etc., should be written in the text to record the test records as a basis for the analysis and determination of the results. The record requirements are detailed, clear, true, objective, and must not be altered or forged.
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