Food microbiological laboratory operation requirements
The staff of food microbiology laboratory must have strict aseptic concept, and many test requirements are carried out in aseptic conditions, the main reason:
first , to prevent the artificial contamination samples in the test operation;
Second, to ensure the safety of staff, to prevent the detection of pathogenic bacteria caused by improper operation caused personal pollution.
FIRST . ASEPTIC OPERATION REQUIREMENT
1. You must wear work clothes and a working cap when you are inoculated with bacteria.
2. When preparing food samples, special work clothes, hats and slippers must be worn. They should be placed in the buffer room of the sterile room and used after ultraviolet disinfection before work.
3. When receiving food samples, wash your hands with soap before entering the sterile room and wipe your hands with 75% alcohol cotton ball.
4. Used for inoculation of straws, AGAR and medium must be disinfection sterilization, such as open the packing vessel has not used up, can't placed before using, metal utensils should be high pressure sterilization or used after burning for three times with 95% alcohol.
5. When removing the straw from the packaging, the tip of the straw can't touch the exposed part. When using straw to inoculate the tube or the plate, the tip of the straw should not touch the test tube or the edge of the plate.
6. The inoculation sample and the transfer bacteria must be operated before the alcohol lamp, when the bacteria or samples are inoculated, the straw is removed from the packaging and the tube is opened to be disinfected by the flame.
7. Inoculation loops and needle before inoculation of bacteria should be fully wire by the flame burning, burn when necessary to ring and pin and the joint of rod, n/med tuberculosis bacterium and virulent bacteria inoculated ring should be in boiling water boil 5 min, then through the flame burning.
8. When sucking the bacteria or samples from the straw, apply the corresponding rubber head to absorb, and do not suck directly with the mouth.
SECOND . ASEPTIC ROOM USE REQUIREMENTS
1. Sterile between the outside of the window should be double deck glass, and I will seal, do not get optional open and offers corresponding to the size of the buffer between sterile room and sliding door, the other is equipped with 0.5 0.7 m2 small window, for after entering between sterile items.
2. The sterile room should be kept clean. After work, it should be disinfected with 2% to 3% of the coal phenol soap solution, and wipe the work surface, and should not store anything unrelated to the experiment.
3. Sterile between before and after use should be shut the door, open the uv lamp, such as using indoor hanging lamp uv disinfection, need 30 w uv lamp, the distance at 1.0 m, the irradiation time of not less than 30 min, the use of uv lamp, may not directly under ultraviolet light should be paid attention to the operation, lest cause damage, lamp every two weeks need alcohol sponge wiped gently, remove dirt and oil dirties, above in order to reduce the influence of ultraviolet rays penetrate.
4. When handling and inoculating food samples, enter the sterile room, and shall not go in and out at will. If you need to pass the goods, you can pass through the small window.
5. When air conditioning is required in the sterile room, the filter device should be installed.
THIRD . DISINFECTION AND STERILIZATION REQUIREMENTS
The glassware, metal utensils and culture medium used for microbiological testing, and the culture of contaminated and inoculated, must be used after sterilization.
(1) sterilization method of dry heat and hot and humid high pressure steam pot.
1. Preparation before sterilization.
(1) all items that need to be sterilized should be cleaned and dried first, and the glassware such as a straw and a plate paper is tightly packed, such as a metal cylinder should be opened.
(2) the triangle bottle with the medium is wrapped in paper and the tube is covered with a lid. The syringe must be pulled out and wrapped in gauze.
(1) hot and dry sterilizer: it is not too crowded to pack and put, and can not reach the walls of the box.
(2) large pressure steam pot: place the sterilized items separately and put them into the sterilizer, and the items should not be crowded.
3. Equipment inspection
(1) whether the switch of inspection door is flexible, whether the rubber band is damaged or not.
(2) check the pressure gauge when the steam exhaust all stay at zero, and door cover, after the steam or heat, to observe whether the leak, pressure gauge and thermometer marked it fits the situation, pipe with and without clogging.
(3) for sterilizer with automatic electronic program control device, the prescribed procedure should be checked before use, and whether it conforms to the requirement of sterilization treatment.
4. Sterilization treatment
(1) dry heat sterilization method:
This method is suitable for the sterilization of non-damaged, unspoilt, non-evaporative items and more commonly used in glassware, metal products and ceramic products in dry and hot conditions.
The equipment should be cleaned and then dried to prevent the surface contamination from carbonizing.
When sterilizing, the goods should not be crowded, do not contact the bottom and box wall directly, there is space between the articles.
(3) when the bacteria will shut the door, turn on the juice and open the vent about 30 min first, eliminate the cold air in the sterilizer, rose to a temperature of 160 ℃ to adjust light, maintain 1.5-2 h.
(4) after sterilization or in the process of temperature rising, under 60 ℃ before the door is opened.
(2) the use of portable pressure cooker or vertical pressure steam sterilizer should be carried out according to the following steps:
In the main body, a portable pressure cooker is added with 3L water, and the vertical pressure cooker is filled with water 16L (the water should be filled when reused, and the water will be replaced by turbid water).
A portable pressure cooker shall insert the exhaust pipe of the top cover into the square pipe of the inner wall of the disinfection bucket (no hose or hose corrosion cracking sterilizer shall not be used);
Tighten the top cover tightly, do not allow the leakage;The sterilizer is heated on the fire source, the vertical pressure cooker is connected to the power supply, and the exhaust valve on the top cover is released with air conditioning (10-15min after boiling);
It closes the exhaust valve, causes the vapor pressure to rise to the specified requirements, and maintains the specified time (according to the nature of the sterilized goods and the relevant situation);
(5) after reaching time, to need to dry goods, immediately open the discharge valve discharge steam, pressure recovery to zero, after natural cooling to 60 ℃ open content, such as liquid items, do not open the exhaust valve, and shall immediately remove pan heat source, natural cooling, pressure buildup to zero, the temperature dropped below 60 ℃ to open cover fetch, in case of sudden decompression violent boiling liquid or container demolition;
(3) horizontal pressure cooker steam sterilizer is used in the following steps:
The door is closed, the inlet valve is opened, the steam is introduced into the interlayer for preheating, and the cold air in the interlayer is automatically discharged through the choke.After the interlayer reaches the predetermined temperature, open the boiler room inlet valve and introduce the steam into the boiler room. The cold air in the pot is automatically discharged through the boiler chamber.
When the pressure and temperature are reached in the boiler room, adjust the inlet valve to keep it constant.
(4) natural or artificial cooling and 60 ℃ and then open the door, quick discharge steam method, shall not be used in case of sudden depressurization, violent boiling liquid containers or blasting.
(5) the use of automatic program control type pressure steam sterilizer, after put good items, shut the door, should be made according to the category, press the corresponding switch so that the required program automatically sterilization, sterilization must be used for instrument to record the temperature and time for future reference, operation requirements shall be in strict accordance with the manufacturer instructions.
5. Sterilization temperature and time.
(1) hot air sterilizer sterilization temperature 160 ℃, 1.5 2 h.
(2) pressure steam sterilization pot sterilization temperature and time.
(2) intermittent sterilization method.
1. Sterilization method:
The sterilization of certain substances can be easily destroyed by high-pressure steam sterilization.
(1) put the sterilized items into the pot, cover the top cover, open the drain, and drain the remaining water.
(2) close the drain, open the intake valve, and sterilize for 10-20min as needed.
Close the inlet valve (3) sterilization, remove the items to be cold temperature to room temperature, in 37 ℃ warm box for the night, the next day, still according to the above method of disinfection, so three times, can achieve sterilization.
2. Method of using serum coagulator:
When the culture medium contains serum or egg special composition, because high heat can destroy its nutrition composition, use low temperature, can make serum coagulate, can achieve sterilization purpose again:
(1) when using the antibacterial serum of the method, the sterile operation should be strictly observed, and the test tube and the petri dish should also be used after sterilization.(2) the medium escarp as required or top, add enough water, turn on the juice and 1 h sterilization temperature 75 ~ 90 ℃, with 37 ℃ temperature box for the night, and so the sterilization three times.
3. Boil and disinfect:
Can use the pot or boiling sterilizer, cook for 5 ~ 15 min after the water boils, also 5 min 2% carbolic acid can be added in the water is boiling, add 0.02% formaldehyde, 80 ℃ boiled 60 min all can achieve sterilization, but when you boil disinfection of elimination agent should pay attention to corrosion of the item.
4. Sterilization treatment:
After sterilization, the products should be sterilized according to normal conditions, and should be carefully checked and placed to avoid re-contamination.
(1) when the goods are removed, check the integrity of the packaging. If any damage or cotton plugs are taken off, it shall not be used as a sterile article;
(2) items taken out, such as those with obvious water leaching for the packaging, shall not be used as sterile articles;
(3) the medium or reagent should be checked for the color or condition after the sterilization, and the unfulfilled person should be discarded;
(4) open and closed container should be closed when taking out;
(5) the items taken out fall in the ground or in the wrong place, or are contaminated with water, which are deemed to be contaminated and cannot be used as sterile articles;
(6) the qualified sterilized items taken out shall be stored in the storeroom or dustproof cabinet and are strictly prohibited to be mixed with unsterilized items;
(7) all qualified articles shall be marked with the date of sterilization and the expiry date;
(8) after each batch of sterilization treatment is completed, the name, quantity, temperature, time and operator of the sterilized product are recorded.
FOURTH . TOXIC AND CONTAMINATED MATERIAL HANDLING REQUIREMENTS
The laboratory equipment and culture materials used in microbiological experiments shall not be taken out of the laboratory without sterilization.
1. The cultivated contaminated materials and waste should be placed in a strict container or wire basket and stored in a designated place for high pressure sterilization.
2. The microbial contamination of cultures, must after 121 ℃, 30 min autoclave.
3. The dyed bacteria after straws, after using acid into 5% lysol or carboniferous, at least for 24 h (height) soaking disinfection liquid shall not be lower than 121 ℃, and then 30 min autoclave.
4. Smear dyeing washing liquid, generally can be directly into the sewer, virulent bacteria rinses must be charged in a beaker, via high pressure sterilization rear can pour into the sewer, stained glass in the 5% lysol 24 h after soaking, boiling washing.The glass or plate for agglutination test must be washed after high pressure sterilization.
5. For the broken culture, immediately spray and soak the contaminated site with 5% coal phenol soap solution or carbonic acid solution, and then wipe clean after soaking for half an hour.
The working clothes of the pollution or the work clothes, caps and masks worn by the test shall be put into the special sterilizing bag, which can be washed after high pressure sterilization.
FIFTH . MEDIUM PREPARATION REQUIREMENTS
The quality of the culture medium will directly affect microorganism growth.Because the nutritional requirements of various microorganisms are not identical, different culture media preparation requirements are as follows:
1. According to the ingredients according to the formula of the culture medium, and then dissolve in distilled water, the reagent drugs used before use should be tested for quality.
2. PH measurement and adjust: pH measurement to cool to room temperature in the medium, as in the case of hot or cold, its pH have certain differences, when good determination, according to the amount of calculation in acid or alkali, after blending should be test again.The pH value of the culture medium must be accurate, otherwise it will affect the growth of microorganisms or the observation of the results.But need to pay attention to because of the high pressure sterilization can affect some of the culture medium PH lower or higher, therefore, should not be sterilized pressure too high or too many times, so as not to affect the quality of the medium, indicator, deoxidation cholic acid sodium, AGAR, etc in commonly after adjust the PH to join again.
3. Medium needs to keep clear, easy to observe the growth of bacteria, medium heat to boil after, can use absorbent cotton or flannelette filtering, to remove sediment, egg white clarification if necessary processing, the article AGAR to wash to dry after use, to avoid affect transparency by AGAR containing impurities.
4. It is not suitable to use iron, copper and other containers for the medium. It is good to use the clean and neutral hard glass container.
Sterilization of the culture medium to achieve complete sterilization, and pay attention to not because of the heat and reduce its nutritional value, generally 121 ℃, 15 min, such as culture medium containing no heat resistant material such as sugar, serum, gelatin, etc., should adopt low temperature sterilization or intermittent sterilization method, some can't heating reagents such as potassium tellurite, egg yolk, TTC, antibiotics, etc., after being based AGAR autoclave cooler to 50 ℃ or so and then join;
6. After each batch of medium is prepared, sterile growth test and strain growth test should be done.If it is a biochemical culture medium, the use of standard strain inoculation training, observing biochemical reactions as a result, should be a normal reaction, medium should not be stored for too long, can buy 4 ℃ refrigerator storage when necessary.
7. At present, all kinds of drying medium is more, each batch need standard strain growth test or biochemical reactions observed, all kinds of culture medium with corresponding growth test is good application, new purchases or for too long drying medium, should also be measured PH during preparation, when used to dosage and method according to product specifications.
8. The chemical reagents, sterilization conditions and strain growth test results and production personnel of each batch of culture media shall be recorded for the purpose of inquiry.
SIXTH . SAMPLE COLLECTION AND PROCESSING REQUIREMENTS
1. The inspection samples must be representative, collected samples should be for the first batch of food raw materials, processing, transportation, storage conditions, environment sanitation detailed investigation, and check whether there are sources of pollution.
2. According to the type and quantity of food, the sampling quantity and method shall be carried out according to the standard test method.
3. The sample should be paid attention to aseptic operation, the container must be sterilized, avoid microbial contamination in the environment, the container shall not use lysol, with new disinfection sterilization, such as jie er die, alcohol cannot contain more such disinfection or antibiotic drugs, the samples in order to avoid kill microorganisms, scissors, knife, spoon used utensils sterilization is required to the application.
4. After the samples should be taken immediately to the test in the checkout room, generally not more than 3 h, in the process of inspection, such as, distance far away, can be stored in the 1 ~ 5 ℃ environment, such as refrigerated, is in freeze state censorship.
5. After receiving the sample, the inspection room shall register (sample name, unit of inspection, quantity, date, number, etc.) and observe the sample.
If one of the following circumstances is found, the inspection may be refused:
(1) if the sample is sterilized by special pressure, boiling or other methods, it loses the meaning of the original food inspection;
(2) bottles and bagged food have been opened, and the cooked meat and its products, cooked poultry and other foods have been broken and incomplete, that is, the original food shape is lost (except for food poisoning samples);
(3) if the quantity of samples is not sufficient according to the regulations;
After you receive the samples for inspection to meet the requirements, the checkout room, should immediately, if the conditions do not have, should buy 4 ℃ refrigerator storage, prepare to create conditions, and then tested.
6. When the sample is inspected, appropriate treatment shall be carried out according to its different characters.
(1) when the liquid sample is inoculated, it should be fully mixed and inoculated.
(2) solid samples, 25g with sterilization knife and scissors, were placed in 225mL sterilized saline or other solution, mixed with homogenizer, and inoculated with the quantity.
(3) the sterilization operation of the bottle and bagged food was opened, and the above methods were selected according to the characters.
SEVENTH . REQUIREMENTS FOR SAMPLE INSPECTION,RECORDING AND REPORTING
1. The checkout room, after receipt of the samples first appearance inspection, inspection in a timely manner in accordance with the national standard test methods, test process to serious, responsible, strict aseptic operation, avoid microbial contamination in the environment.
2. In the process of sample testing method, phenomenon and the results are used to the test record in writing, as the basis of analysis, judgement, the result records requirements detailed, clear, real, objective and shall not be altered and forged.