Summary of microbiological testing requirements

2024-01-24 15:14:33

The requirements and steps for microbiological testing are as follows:

Aseptic operation requirements:

1.You must wear work clothes and a work cap when inoculating bacteria.
2. When inoculating food samples, special work clothes, caps and slippers must be worn, which should be placed in the buffer room of the sterile room and used after UV disinfection before work.
3. When inoculating food samples, wash your hands with soap before entering the sterile room, and then wipe your hands clean with a 75% alcohol cotton ball.
4. The straws, flat dishes and culture media used for inoculation must be sterilized and sterilized. Unused containers should not be left unused after opening the package. Metal utensils should be autoclaved or burned three times with 95% alcohol before use.
5. When taking the straw out of the package, the tip of the straw must not touch the exposed parts. When using the straw to inoculate the test tube or the plate, the tip of the straw must not touch the edge of the test tube or the plate.
6. Inoculating samples and transferring bacteria must be done in front of an alcohol lamp. When inoculating bacteria or samples, the straw must be sterilized by flame after taking it out of the package and opening the test tube stopper.
7. Before inoculating bacteria, all metal wires of the inoculation loop and needle should be burned with a flame, and if necessary, the connection between the loop, needle and rod should be burned. The inoculation loop for inoculating tuberculosis bacteria and virulent bacteria should be boiled in boiling water for 5 minutes, and then Burned by flame.
8. When sucking bacterial liquid or samples with a straw, use the corresponding rubber tip to suck it, and do not suck it directly with your mouth.

Sterile room usage requirements:

1. The windows leading to the outside of the sterile room should be double-glazed and sealed. They should not be opened at will. There should be a buffer room and sliding door corresponding to the size of the sterile room. There should also be a small window of 0.5~0.7m2. In preparation for passing items after entering the sterile room.

2. The sterile room should be kept clean. After work, it should be disinfected with 2% to 3% cresol soap solution, the work surface should be wiped, and items unrelated to the experiment should not be stored.

3. The door of the sterile room should be closed tightly before and after use, and the ultraviolet lamp should be turned on. If an indoor suspended ultraviolet lamp is used for disinfection, a 30W ultraviolet lamp is required. The distance is 1.0m, and the irradiation time is not less than 30 minutes. When using ultraviolet lamps, attention should be paid to Operate directly under ultraviolet light to avoid damage. The lamp needs to be gently wiped with an alcohol cotton ball every two weeks to remove dust and grease to reduce the impact of ultraviolet penetration.

4. When processing and inoculating food specimens, enter the sterile room for operation and are not allowed to enter or exit at will. If items need to be passed on, they can be passed through the small window.

5. If air conditioning needs to be installed in the sterile room, there should be a filtering device.

​Disinfection and Sterilization Requirements

Glassware, metal utensils and culture media used for microbiological testing, contaminated and inoculated cultures, etc. must be sterilized before use. Dry heat and moist heat pressure steam sterilization methods.
1. Preparation before sterilization
(1) All items that need to be sterilized should be washed and dried first. Glassware such as straws and flat dishes should be tightly packed with paper. If metal tubes are used, the ventilation holes on them should be opened.
(2) Wrap the stopper of the triangular flask containing the culture medium in paper, cover the test tube, and pull out the core of the syringe, and wrap it with gauze.
2. install
(1) Dry heat sterilizer: The items must not be overcrowded and cannot touch the four walls of the box.
(2) Large high-pressure steam cooker: Place sterilized items, wrap them separately, and put them directly into the sterilization cylinder. Do not overcrowd the items.
3. Equipment inspection
(1) Check whether the door switch is flexible, whether the rubber ring is damaged and whether it is flat.
(2) Check whether the pressure gauge stays at the zero position when the steam is exhausted, close the door and cover, ventilate steam or heat, and observe whether there is air leakage, whether the conditions marked by the pressure gauge and the thermometer match, and whether the pipeline is blocked.
(3) For sterilizers with automatic electronic program control devices, the specified program should be checked before use to see whether it meets the requirements for sterilization.
4. Sterilization treatment
(1) Dry heat sterilization method:
This method is suitable for items that do not damage, deteriorate, or evaporate under dry heat conditions. It is more commonly used for sterilizing glassware, metal products, ceramic products, etc.
①Instruments and utensils should be washed before dry-baking to prevent the dirt attached to the surface from being carbonized.
② During sterilization, the items must not be overcrowded, do not directly contact the bottom and wall of the box, and leave gaps between items.
③ During sterilization, close the door tightly, connect the power supply, open the exhaust hole for about 30 minutes to remove the cold air in the sterilizer, adjust the indicator light when the temperature rises to 160°C, and maintain it for 1.5 to 2 hours.
④After sterilization is completed or during the temperature rise process, the door must be opened below 60°C.
(2) The following steps should be followed when using a portable pressure cooker or vertical pressure steam sterilizer:
① Add 3L of water to the main body of the portable pressure cooker, and 16L of water to the vertical pressure cooker (the amount of water should be replenished when reused, and the water needs to be replaced if it becomes turbid);
② For a portable pressure cooker, insert the exhaust pipe on the top cover into the square tube on the inner wall of the sterilization barrel (sterilizers without hoses or those with corroded and cracked hoses must not be used);
③ Cover the top cover and tighten it tightly to prevent air leakage; place the sterilizer on the fire source to heat, turn on the power of the vertical pressure cooker, and open the exhaust valve on the top cover to let out the air (exhaust air for 10 to 15 minutes after the water boils) );
④Close the exhaust valve to raise the steam pressure to the specified requirements and maintain it for the specified time (depending on the nature of the sterilized items and relevant circumstances);
⑤ After the specified time is reached, for items that need to be dried, immediately open the exhaust valve to discharge the steam. When the pressure returns to zero, cool it naturally to 60°C and then open the lid to take out the items. If it is a liquid item, do not open the exhaust valve. The pot should be removed from the heat source immediately and allowed to cool naturally until the pressure returns to zero and the temperature drops below 60°C before opening the lid to take out the contents to prevent the sudden decompression of the liquid from violent boiling or explosion of the container.
(3) Follow the following steps to use the horizontal pressure cooker steam sterilizer:
①Close the pot door tightly, open the air inlet valve, introduce steam into the mezzanine for preheating, and the cold air in the mezzanine will be automatically discharged through the air barrier;
②After the interlayer reaches the predetermined temperature, open the air inlet valve of the pot chamber, introduce the steam into the pot chamber, and the cold air in the pot chamber will be automatically discharged through the pot chamber air arrester;
③When the pressure and temperature in the pot chamber reach the specified pressure, adjust the air inlet valve to keep it constant;
④ Cool the temperature naturally or artificially to 60℃ before opening the door to take out the items. Do not use the rapid steam discharge method to prevent sudden pressure drop, violent boiling of the liquid or explosion of the container;
⑤ When using an automatic program-controlled pressure steam sterilizer, after placing the items and closing the door tightly, the corresponding switch should be pressed according to the category of the items so that the sterilization can be automatically carried out according to the required procedures. During sterilization, the attached instrument must be used 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) Sterilization temperature of dry heat sterilizer is 160℃, 1.5~2h.
(2) Sterilization temperature and time of pressure steam sterilizer

 Intermittent sterilization method

1. Sterilization method:
Use steam sterilization without pressure. Some substances are easily destroyed by high-pressure steam sterilization, so this method can be used for sterilization.
(1) Place the items to be sterilized in the pot, cover the top cover, open the drain outlet, and drain away the remaining water in the pot.
(2) Close the drain outlet, open the air inlet door, and disinfect for 10 to 20 minutes as needed.
(3) After sterilization is completed, close the air inlet door, take out the items and wait to cool to room temperature, put them in a 37°C incubator overnight, and continue to sterilize according to the above method the next day. Do this three times to achieve the purpose of sterilization.

2. How to use the serum coagulator:
When the culture medium contains special ingredients such as serum or eggs, high heat will destroy its nutrients. Therefore, low temperature can be used to coagulate the serum and achieve sterilization purposes:
(1) When using serum sterilized by this method to aliquot, aseptic operations must be strictly followed, and test tubes and plates must also be sterilized before use;
(2) Make the culture medium into a slope or high layer as required. After adding enough water, connect the power supply, raise the temperature to 75~90°C for one hour and then sterilize it. Place it in a 37°C incubator overnight, and then sterilize it three times.

3. Boil and disinfect:
You can use a boiling pot or boiling sterilizer. After the water boils, boil it for 5 to 15 minutes. You can also add 2% carbolic acid to the water and boil it for 5 minutes. Add 0.02% formaldehyde and boil it at 80°C for 60 minutes to achieve the purpose of sterilization. However, boiling sterilization can be achieved. When using enhancers, attention should be paid to the corrosiveness of items.

4. Sterilization treatment:
After sterilization, items are already sterile under normal circumstances. They should be carefully inspected and placed when taken out of the sterilizer to avoid re-contamination;
(1) Take out the items and check the integrity of the packaging immediately. If there is damage or the tampon is removed, it cannot be used as sterile items;
(2) The items taken out cannot be used as sterile items if their packaging is obviously soaked in water;
(3) The culture medium or reagents should be checked to see if they meet the color or state after sterilization. Those that do not meet the requirements should be discarded;
(4) For open-close containers, the screen holes should be closed when taking them out;
(5) If the removed items fall to the ground, are misplaced in an unclean place, or are stained with water, they are considered contaminated and cannot be used as sterile items;
(6) The qualified sterilized items taken out should be stored in the storage room or dust-proof cabinet, and it is strictly prohibited to mix them with unsterilized items;
(7) All qualified items should be marked with the sterilization date and expiry date;
(8) After each batch of sterilization processing is completed, record the name, quantity, temperature, time, and operator of the sterilized products.

 Requirements for the treatment of toxic and bacterial waste

Experimental equipment and cultures used in microbiological experiments must not be taken out of the laboratory without disinfection.
1. Cultured contaminated materials and waste should be placed in tight containers or wire baskets and stored centrally in designated locations until they are uniformly autoclaved.

2. Cultures contaminated by microorganisms must be autoclaved at 121°C for 30 minutes.

3. After use, the contaminated straws should be placed in 5% creol soap solution or carbolic acid solution, soaked for at least 24 hours (the disinfectant liquid must not be lower than the soaking height), and then sterilized by high pressure at 121°C for 30 minutes.

4. The liquid for staining and rinsing smears can generally be flushed directly into the sewer. The rinsing fluid for virulent bacteria must be flushed in a beaker and sterilized by high pressure before being poured into the sewer. The stained slides are put into 5% cresol soap solution. After soaking in water for 24 hours, boil and wash. The slides or dishes used for agglutination testing must be autoclaved and then washed.

5. After breaking the culture, immediately spray and soak the contaminated parts with 5% cresol soap solution or carbolic acid solution, soak for half an hour and then wipe clean.

Contaminated work clothes or work clothes, caps, masks, etc. worn for violent tests should be placed in special disinfection bags and sterilized by high pressure before being washed.
 
Medium preparation requirements

The quality of culture medium preparation will directly affect the growth of microorganisms, because various microorganisms have different nutritional requirements and different culture purposes. The preparation requirements for various culture media are as follows:
1. Weigh the ingredients according to the medium formula, and then dissolve them in distilled water. The quality of the applied reagents and drugs should be inspected before use.

2. pH measurement and adjustment: pH measurement should be carried out when the culture medium is cooled to room temperature, because there is a certain difference in pH under hot or cold conditions. When the measurement is completed, add alkali or acid according to the calculated amount and mix well. Should be tested again. The pH value of the culture medium must be accurate, otherwise it will affect the growth of microorganisms or affect the observation of results. However, it should be noted that high-pressure sterilization can affect the decrease or increase of the pH of some culture media, so it is not advisable to sterilize at too high a pressure or too many times to avoid affecting the quality of the culture medium. Indicators, sodium deoxycholate, agar, etc. are generally Add it after adjusting the pH.

3. The culture medium needs to be kept clear to facilitate observation of bacterial growth. After the culture medium is heated and boiled, it can be filtered with absorbent cotton or flannel to remove sediment. If necessary, it can be clarified with egg white. The agar strips used must be washed and dried in advance. Before use, avoid affecting the transparency due to impurities in the agar.

4. Iron, copper and other containers should not be used to hold the culture medium. It is better to use clean neutral hard glass containers.

5. The sterilization of the culture medium must not only achieve the purpose of complete sterilization, but also pay attention not to reduce its nutritional value due to heating. Generally, 121°C for 15 minutes is sufficient. For culture media containing substances that are intolerant to high heat such as sugars and serum , gelatin, etc., should be sterilized by low temperature or intermittent method. Some reagents that cannot be heated, such as potassium tellurite, egg yolk, TTC, antibiotics, etc., should be cooled to about 50°C after the basic agar is autoclaved and then added;

6. After each batch of culture media is prepared, a sterile growth test and a growth test of the tested strains should be performed. If it is a biochemical culture medium, use standard bacterial strains for inoculation and culture, and observe the biochemical reaction results. It should show a normal reaction. The culture medium should not be stored for too long. If necessary, it can be stored in a 4°C refrigerator.

7. At present, there are many kinds of dry culture media. Each batch needs to use standard strains for growth test or biochemical reaction observation. Various culture media can be used only after the growth test of the corresponding strains is good. Newly purchased or dried ones that have been stored for a long time can be used. The pH of the culture medium should also be measured when preparing it, and the dosage and method should be followed according to the product instructions.

8. The chemical reagents, sterilization conditions, strain growth test results, and production personnel used in each batch of prepared culture media should be recorded for inquiry.

 Sample collection and processing requirements

1. The inspection samples collected must be representative. When sampling, the raw materials, processing, transportation, storage methods and conditions of the batch of food, the surrounding environmental health conditions, etc. should be investigated in detail to check whether there are any sources of contamination.

2. According to the type and quantity of food, the sampling quantity and method should be carried out in accordance with the requirements of standard inspection methods.

3. Pay attention to aseptic operation when sampling, and the container must be sterilized to avoid microbial contamination in the environment. The container must not be sterilized with creosal soap solution, sterilization with chlormethionine, alcohol and other disinfectants, and it must not contain such disinfectants or antibiotics. In order to avoid killing microorganisms in the sample, the scissors, knives, and spoons used must also be sterilized before use.

4. Samples should be sent to the inspection room for inspection immediately after collection. The inspection process generally does not exceed 3 hours. If the distance is long, it can be stored in an environment of 1~5℃. If it needs to be frozen, it should be kept in a frozen state. Submit for inspection.

5. After receiving the sample, the inspection room will register it (sample name, unit for inspection, quantity, date, number, etc.) and observe the appearance of the sample. If any of the following conditions are found, the inspection may be refused:
(1) Samples that have been sterilized by special high pressure, boiling or other methods will lose their significance in representing the original food;
(2) Bottles and bags of food have been opened, cooked meat and its products, cooked poultry and other foods have been broken and incomplete, that is, the original food shape has been lost (except for food poisoning samples);
(3) The sampling quantity is insufficient as required;
For samples submitted for inspection that meet the requirements, the inspection room should immediately conduct inspection after receiving them. If the conditions are not met, they should be stored in a 4°C refrigerator, prepare to create conditions in time, and then conduct inspection.

6. When inspecting samples, perform appropriate processing according to their different properties.
(1) When inoculating liquid samples, they should be thoroughly mixed and inoculated according to the amount.
(2) For solid samples, use a sterilized knife to cut out 25g of different parts, place them in 225mL of sterilized saline or other solutions, crush and mix with a homogenizer, and inoculate according to the amount.
(3) Bottles and bags of food should be opened by sterilization, and the above methods should be selected according to the characteristics before inoculation.

 Sample inspection, recording and reporting requirements

1. After receiving the sample, the inspection room will first conduct an appearance inspection and promptly conduct the inspection in accordance with national standard inspection methods. During the inspection process, aseptic operations must be performed carefully, responsibly, and strictly to avoid
microbial  test result contamination in the environment.

2. The methods used during the sample inspection process, the phenomena and results that occurred, etc. must be written in written test records as a basis for analysis and judgment of the results. The records must be detailed, clear, authentic, objective, and must not be altered or forged.


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