How to properly prepare the microbial detection medium <br> The preparation steps of the microbial culture are divided into nine steps, how is each step operated? Next, Xiaobian will give you a breakdown.
1. Weighing <br> Weigh the medicine needed for the medium with an electronic balance with an accuracy of 1/100. First, according to the formula, the amount of the various ingredients required to prepare a certain amount of the medium is calculated, and then accurately weighed with the balance. When weighing, weigh the paper into a scorpion to hold the medicine. The weighing paper folding method is shown in the figure.
Second, dissolve <br> The medium is placed in a beaker, slowly add a small amount of water, and stir with a glass rod while adding. If the medium does not contain agar, the medium does not need to be heated; if it contains agar, it needs to be heated and boiled with Bunsen burner or induction cooker. After completely dissolving, fill the required water and stir evenly (as shown in Figure 3). . If the amount of the culture medium is large, it can be heated and melted in a stainless steel pot. It can be heated with warm water and stirred at any time to prevent coking. If there is coking, the prepared medium cannot be used and must be reconstituted.
When the medium is prepared, it is not possible to dissolve the copper or iron container. Because the copper or iron container may exceed the content of copper and iron in the medium, the experiment is affected. (The copper content in the medium is more than 0.3 mg/L, and the bacteria should not grow. The iron content exceeds 0.14 mg/L, which hinders the bacteria from producing toxins. For drugs that are prone to reaction and produce precipitation, they should be dissolved separately, and then added to the medium such as dipotassium hydrogen phosphate and magnesium sulfate.
Third, adjust the pH
Although the medium contains a buffer substance component, the pH of the medium can be kept as much as possible within the required range, but if the medium to be dispensed does not meet the requirements, necessary adjustments are made. If you have a calibrated pH meter, you can use a pH meter. If not, use a precision pH test paper, and if necessary, use 1 mol/L sodium hydroxide or 1 mol/L hydrochloric acid (finely adjust 0.1-NaOH or 0.1 mol/L hydrochloric acid) Modulate the desired pH.
The pH of the base is generally 7.4 to 7.6, and it is also acidic or basic. The medium to be autoclaved with sodium hydroxide should be adjusted to 0.1 to 0.2 units higher when the pH is adjusted. When adjusting with sodium hydroxide, the pH of the medium should be lowered after autoclaving. 0.1~0.2. If the medium contains calcium carbonate, the pH may not be adjusted.
4. Filtration <br> The prepared medium can be omitted if there is no special requirement. If there is precipitation or turbidity, the liquid medium that needs clarification can be filtered with oil paper. The solid medium can be filtered with a double layer of gauze sandwiched between a thin layer of absorbent cotton. If the filtration method can not meet the requirements of clarification, the egg white clarification method can be used, that is, the medium is heated and cooled to 50-60 ° C, and is placed in a triangular bottle (not more than one-half of the capacity), and one is added according to 1000 mL. Egg white of 2 eggs, shake vigorously for 3-5min, autoclave at 121 ° C, 20 min, remove the hot filter.
5. Packing <br> The prepared medium is packed in a triangular bottle, test tube and other containers according to different purposes, and the test tube is divided. If the test tube is large, an automatic liquid dispenser can be used. If the test tube is small, the funnel can be used. Dispense.
The amount of dispensing does not exceed two-thirds of the volume of the container. The triangular bottle should not exceed one-half of the volume; the agar slope should not exceed one-fifth of the length of the test tube. After sterilization, the inclined surface is one-third of the medium amount, and the bottom layer is three. The amount of the second is suitable; the semi-solid agar is one-third of the length of the test tube; the high-grade agar used for inoculation or preservation is divided into one quarter or one third of the length of the test tube. To inoculate anaerobic bacteria to achieve two-thirds; agar plates, the inner diameter of 90mm is 13mL ~ 15mL is appropriate, the inner diameter of 70mm plate is 8mL ~ 10mL, the agar plate produced if the surface moisture is more, The plate was inverted and placed in a 37 ° C incubator for 30 min, dried and reused. Each batch of medium should be separately dispensed with a small glass bottle (about 20 mL) and sterilized simultaneously with the batch medium to determine the final pH of the batch.
6. Sterilization <br> The packaged medium should be sterilized immediately. The types of sterilization methods are as follows:
1. High-pressure steam sterilization method Most of the heat-resistant medium can be used in this method. When the small amount is dispensed, use 121 ° C, 15 min; when the sterilization amount is large, use 121 ° C, 30 min to sterilize; the sugar-containing medium can only Sterilize at 113~115°C for 15min to avoid damage to sugar.
2. Boiling sterilization method This method can be used for a medium containing a substance that is not resistant to high temperature.
3. Filter sterilization and sterilization by filtration, and add the medium quantitatively by aseptic technique. Blood and antibiotics can be extracted by aseptic technique and added to the medium cooled to about 50 °C. This method can be used when the medium contains a substance that is not heat resistant.
When the LST medium is sterilized, there may be bubbles in the fermentation tube. In order to prevent bubbles in the fermentation tube, the following measures can be taken:
1. The small inverted tube is immersed in the medium (no air bubbles are left) and then added to the test tube containing LST.
2. Before the sterilizing pot closes the venting valve, drain the gas in the pot.
3. Do not plug the test tube plug too tightly (when using a silicone plug), do not use a rubber stopper.
4. Do not open the sterilization pot too early, and wait until the pressure and temperature in the sterilization pot are reduced to the same level as the room temperature or when the difference is not large, then turn on the sterilization pot.
If there are still bubbles in the above situation, water can be used as a control test for the medium group. If there is air bubbles in the medium group, and the control group has no air bubbles, it can be determined as the cause of the medium itself.
7. Inverted plate <br> The sterilized and thawed medium is cooled to 50 ° C and poured into a sterile dry Petri dish. The temperature of the medium should not be too high, otherwise it will easily form too much condensed water on the inner lid of the culture dish; if the temperature is too low, the medium will easily solidify into a block and cannot be made into a flat plate.
When pouring the flat plate, it should be carried out near the flame of the alcohol lamp, so as to prevent the external bacteria from falling into the flat plate, take the petri dish in the left hand, take the bottom of the triangular bottle in the right hand, and pull the cotton plug of the triangular bottle with the little finger of the left hand and the palm of the hand. At the mouth of the flask, open the slit of the petri dish with the thumb and forefinger until the mouth of the bottle just protrudes. Pour into the medium to cover the bottom. No more than one-third of the height of the dish, quickly cover the lid. Place on the table and gently rotate the dish to make the medium evenly distributed and condense.
8. Slanting surface <br> The agar medium contained in the test tube is placed on the wooden stick (or glass rod) immediately after sterilization, and is placed at an appropriate slope. After cooling, the agar solidifies. Beveled. The length of the bevel does not exceed one-half of the length of the test tube.
Nine, quality inspection <br> After the medium is sterilized, check it carefully and find that it is cracked, immersed in water, abnormal color, and the cotton plug is contaminated by the medium. It must be discarded and cannot be reused. And determine its final pH. Sterility tests and effects checks are also required. The sterility test is to take 1 tube (bottle)~2 tubes (bottle) from the sterilized medium, and incubate at 37 °C for 1 d to 2 d to confirm the growth of no bacteria; the effect check is to inoculate the relevant medium with the standard strain. Check the growth, morphology and biochemistry of the bacteria, which is consistent with the known situation. In both cases, the prepared medium can be used.
BIOBW, as the abbreviation of Beijing Baiou Bowei Biotechnology Co., Ltd., has .com ( National Standard Material Resource Platform website ), .cn (O2O platform), .org (microbial cell website), .org.cn (official platform) four domain names. The suffix can effectively and clearly show the business of Baiou Bowei! Beijing Baiou Bowei Biotechnology Co., Ltd. is a high-tech and high-quality biotechnology engineering company with independent research strength, experimental team and R&D team. It mainly supplies chromatographic consumables, chromatography instruments, solid phase extraction devices, chemical reagents, sample bottles and bottles. Products and services such as lamps, standards, microbiological technology, strain collection services, and ATCC product agents!
1. Weighing <br> Weigh the medicine needed for the medium with an electronic balance with an accuracy of 1/100. First, according to the formula, the amount of the various ingredients required to prepare a certain amount of the medium is calculated, and then accurately weighed with the balance. When weighing, weigh the paper into a scorpion to hold the medicine. The weighing paper folding method is shown in the figure.
Second, dissolve <br> The medium is placed in a beaker, slowly add a small amount of water, and stir with a glass rod while adding. If the medium does not contain agar, the medium does not need to be heated; if it contains agar, it needs to be heated and boiled with Bunsen burner or induction cooker. After completely dissolving, fill the required water and stir evenly (as shown in Figure 3). . If the amount of the culture medium is large, it can be heated and melted in a stainless steel pot. It can be heated with warm water and stirred at any time to prevent coking. If there is coking, the prepared medium cannot be used and must be reconstituted.
When the medium is prepared, it is not possible to dissolve the copper or iron container. Because the copper or iron container may exceed the content of copper and iron in the medium, the experiment is affected. (The copper content in the medium is more than 0.3 mg/L, and the bacteria should not grow. The iron content exceeds 0.14 mg/L, which hinders the bacteria from producing toxins. For drugs that are prone to reaction and produce precipitation, they should be dissolved separately, and then added to the medium such as dipotassium hydrogen phosphate and magnesium sulfate.
Third, adjust the pH
Although the medium contains a buffer substance component, the pH of the medium can be kept as much as possible within the required range, but if the medium to be dispensed does not meet the requirements, necessary adjustments are made. If you have a calibrated pH meter, you can use a pH meter. If not, use a precision pH test paper, and if necessary, use 1 mol/L sodium hydroxide or 1 mol/L hydrochloric acid (finely adjust 0.1-NaOH or 0.1 mol/L hydrochloric acid) Modulate the desired pH.
The pH of the base is generally 7.4 to 7.6, and it is also acidic or basic. The medium to be autoclaved with sodium hydroxide should be adjusted to 0.1 to 0.2 units higher when the pH is adjusted. When adjusting with sodium hydroxide, the pH of the medium should be lowered after autoclaving. 0.1~0.2. If the medium contains calcium carbonate, the pH may not be adjusted.
4. Filtration <br> The prepared medium can be omitted if there is no special requirement. If there is precipitation or turbidity, the liquid medium that needs clarification can be filtered with oil paper. The solid medium can be filtered with a double layer of gauze sandwiched between a thin layer of absorbent cotton. If the filtration method can not meet the requirements of clarification, the egg white clarification method can be used, that is, the medium is heated and cooled to 50-60 ° C, and is placed in a triangular bottle (not more than one-half of the capacity), and one is added according to 1000 mL. Egg white of 2 eggs, shake vigorously for 3-5min, autoclave at 121 ° C, 20 min, remove the hot filter.
5. Packing <br> The prepared medium is packed in a triangular bottle, test tube and other containers according to different purposes, and the test tube is divided. If the test tube is large, an automatic liquid dispenser can be used. If the test tube is small, the funnel can be used. Dispense.
The amount of dispensing does not exceed two-thirds of the volume of the container. The triangular bottle should not exceed one-half of the volume; the agar slope should not exceed one-fifth of the length of the test tube. After sterilization, the inclined surface is one-third of the medium amount, and the bottom layer is three. The amount of the second is suitable; the semi-solid agar is one-third of the length of the test tube; the high-grade agar used for inoculation or preservation is divided into one quarter or one third of the length of the test tube. To inoculate anaerobic bacteria to achieve two-thirds; agar plates, the inner diameter of 90mm is 13mL ~ 15mL is appropriate, the inner diameter of 70mm plate is 8mL ~ 10mL, the agar plate produced if the surface moisture is more, The plate was inverted and placed in a 37 ° C incubator for 30 min, dried and reused. Each batch of medium should be separately dispensed with a small glass bottle (about 20 mL) and sterilized simultaneously with the batch medium to determine the final pH of the batch.
6. Sterilization <br> The packaged medium should be sterilized immediately. The types of sterilization methods are as follows:
1. High-pressure steam sterilization method Most of the heat-resistant medium can be used in this method. When the small amount is dispensed, use 121 ° C, 15 min; when the sterilization amount is large, use 121 ° C, 30 min to sterilize; the sugar-containing medium can only Sterilize at 113~115°C for 15min to avoid damage to sugar.
2. Boiling sterilization method This method can be used for a medium containing a substance that is not resistant to high temperature.
3. Filter sterilization and sterilization by filtration, and add the medium quantitatively by aseptic technique. Blood and antibiotics can be extracted by aseptic technique and added to the medium cooled to about 50 °C. This method can be used when the medium contains a substance that is not heat resistant.
When the LST medium is sterilized, there may be bubbles in the fermentation tube. In order to prevent bubbles in the fermentation tube, the following measures can be taken:
1. The small inverted tube is immersed in the medium (no air bubbles are left) and then added to the test tube containing LST.
2. Before the sterilizing pot closes the venting valve, drain the gas in the pot.
3. Do not plug the test tube plug too tightly (when using a silicone plug), do not use a rubber stopper.
4. Do not open the sterilization pot too early, and wait until the pressure and temperature in the sterilization pot are reduced to the same level as the room temperature or when the difference is not large, then turn on the sterilization pot.
If there are still bubbles in the above situation, water can be used as a control test for the medium group. If there is air bubbles in the medium group, and the control group has no air bubbles, it can be determined as the cause of the medium itself.
7. Inverted plate <br> The sterilized and thawed medium is cooled to 50 ° C and poured into a sterile dry Petri dish. The temperature of the medium should not be too high, otherwise it will easily form too much condensed water on the inner lid of the culture dish; if the temperature is too low, the medium will easily solidify into a block and cannot be made into a flat plate.
When pouring the flat plate, it should be carried out near the flame of the alcohol lamp, so as to prevent the external bacteria from falling into the flat plate, take the petri dish in the left hand, take the bottom of the triangular bottle in the right hand, and pull the cotton plug of the triangular bottle with the little finger of the left hand and the palm of the hand. At the mouth of the flask, open the slit of the petri dish with the thumb and forefinger until the mouth of the bottle just protrudes. Pour into the medium to cover the bottom. No more than one-third of the height of the dish, quickly cover the lid. Place on the table and gently rotate the dish to make the medium evenly distributed and condense.
8. Slanting surface <br> The agar medium contained in the test tube is placed on the wooden stick (or glass rod) immediately after sterilization, and is placed at an appropriate slope. After cooling, the agar solidifies. Beveled. The length of the bevel does not exceed one-half of the length of the test tube.
Nine, quality inspection <br> After the medium is sterilized, check it carefully and find that it is cracked, immersed in water, abnormal color, and the cotton plug is contaminated by the medium. It must be discarded and cannot be reused. And determine its final pH. Sterility tests and effects checks are also required. The sterility test is to take 1 tube (bottle)~2 tubes (bottle) from the sterilized medium, and incubate at 37 °C for 1 d to 2 d to confirm the growth of no bacteria; the effect check is to inoculate the relevant medium with the standard strain. Check the growth, morphology and biochemistry of the bacteria, which is consistent with the known situation. In both cases, the prepared medium can be used.
BIOBW, as the abbreviation of Beijing Baiou Bowei Biotechnology Co., Ltd., has .com ( National Standard Material Resource Platform website ), .cn (O2O platform), .org (microbial cell website), .org.cn (official platform) four domain names. The suffix can effectively and clearly show the business of Baiou Bowei! Beijing Baiou Bowei Biotechnology Co., Ltd. is a high-tech and high-quality biotechnology engineering company with independent research strength, experimental team and R&D team. It mainly supplies chromatographic consumables, chromatography instruments, solid phase extraction devices, chemical reagents, sample bottles and bottles. Products and services such as lamps, standards, microbiological technology, strain collection services, and ATCC product agents!
Nootropics,Alpha-GPC,Choline,CDP-Choline,Coluracetam
PYSON Co. ,Ltd. , https://www.pysonbio.com