Microbial sample processing method
First, the primary (initial) sample suspension <br> After the sample is weighed by a certain weight or after taking a certain volume, add 9 times the weight or volume of the specific dilution, and after the external force is fully homogenized, the suspension is obtained. Turbid liquid, solution or emulsion.
When some of the initial suspensions contain larger sample particles, they should be allowed to settle horizontally. This gives the primary (initial) suspension (10-1).
Second, the secondary ten-fold gradient dilution <br> Take a specific volume of the primary suspension, add 9 times the specific volume of the dilution and mix well, so the primary suspension is diluted ten times (10-2 ), and then proceeding sequentially (10-3, 10-4....10-n) to obtain a series of dilutions until a dilution that meets the target microorganism culture requirements is obtained. Such an operation is called a secondary ten-fold gradient dilution and the resulting series of dilutions are collectively referred to as secondary ten-fold gradient dilutions.
Third, the principle of dilution operation <br> The primary suspension is prepared to make the microorganisms in the sample as evenly distributed as possible, and then in the next secondary ten-fold gradient dilution, the number of microorganisms can be uniformly indexed according to the index Decrease, so that after cultivation, it is convenient for colony counting operations.
In order to strictly narrow the counting range of each dilution and improve the accuracy of the counting, a general evaluation of the microbial contamination level of the sample should be made first, and then at least two dilutions of the appropriate serial dilutions should be selected, and at least each dilution should be inoculated. Two medium plates were cultured. Both the primary sample suspension and the secondary ten-fold gradient dilution sample solution should be evenly oscillated to ensure that the microorganisms are evenly distributed in the solution before proceeding to the next step.
Oscillation should be performed on the vortex mixer (see figure). Use the right thumb and middle finger to clamp the test tube, the right index finger against the test tube cap to prevent the test tube cap from flying when vibrating, and the three fingers force the bottom of the test tube against the rubber vibrating head of the vortex mixer. There are usually two gears for the vortex mixer to operate: one is that the vibrating head continues to vibrate, and the other is that only the object will vibrate against the vibrating head, usually using the latter. If there is no vortex mixer, you can use the method of hand-vibration mixing.
IV. Diluent 1. General Principles for Formulating Diluents In order to make the results more reproducible, it is recommended that when formulating the diluent, whether it is weighed one by one with a single reagent for dehydration or a dehydrated commercial finished medium, the laboratory personnel The following principles must be strictly followed when preparing the diluent:
(1) The various reagents for preparing the diluent or the dry powder of the dehydrated finished medium must be guaranteed.
(2) The water used to prepare the diluent must be qualified distilled water. The water's various indicators (pH, water activity, conductivity, electrical resistivity, total number of colonies) must meet the requirements for the water used for the preparation of the medium.
(3) The components of the diluent must be sufficiently dissolved to form a uniform solution without precipitation. After the dilution is prepared, the pH should meet the survival requirements of the target microorganism, especially the pH of the dilution after autoclaving. The normal range of the pH error should be ±0.2 (at 25 °C).
2. Ordinary dilutions commonly used in ordinary dilutions have a concentration of 0.1%, pH 6.8 ~ 7 sterile protein water (according to protein 胨 1.0g, sodium chloride 8.5g, distilled water 1000 mL ratio mixed to completely dissolve), phosphate Buffer, 0.85% sodium chloride solution, 2% buffered protein, water, etc. Some additives may be added as the case may be. For example, an emulsifier or a surfactant may be added to a sample having a high fat content; a foam inhibitor, an antifoaming agent, or the like may be added to a sample which is easy to foam during a homogeneous operation.
When preparing the primary sample solution, the sample may change the nature of the diluent due to the large proportion of the sample in the entire solution system, especially when the sample is insoluble in water, and the sample is in the diluent. The solubility will be affected. This will affect the pH or water activity (Aw) of the solution, so when it is not possible to determine if the sample has such an effect on the dilution, the pH and water activity of the first dilution should be determined. A phosphate buffer can be added to the dilution to stabilize the pH change.
It is particularly important to note that for some high-solubility dried samples (milk powder, baby food), the water activity is very low when the primary sample solution is made, so distilled water should be selected as the diluent. For the different samples, choose the most suitable dilution, which actually needs to be obtained through a series of tests, the selected dilution should have a high recovery rate.
The dilution preparation of the sample should be completed within 30 minutes.
3. For the qualitative or quantitative detection of anaerobic microorganisms in foods for the dilution of anaerobic microorganisms, the oxidation must be minimized, and the dilution should have an antioxidant effect. When preparing a sample solution, oxygen should be avoided as much as possible. In the detection of anaerobic bacteria that are extremely sensitive to oxygen, in addition to the use of appropriate dilutions, there are special equipment to assist, such as the use of anaerobic workstations as a protective measure for the bacteria.
4. Neutralizing Dilutions Some samples contain antibacterial substances, disinfectants, and other components that kill or inhibit microorganisms. It is necessary to add certain organic or inorganic compounds to neutralize these components to eliminate their effects.
For alcohols and phenolic disinfectants, the diluent can be used as a common nutrient broth; for chlorine-containing disinfectants, iodine-containing disinfectants, and peroxide disinfectants, 0.1% sodium thiosulfate should be added to the diluent; Chlorhexidine, quaternary ammonium disinfectant, 3% (mass concentration) Tween-80 and 0.3% lecithin should be added to the diluent; for aldehyde disinfectant, 0.3% glycine should be added to the diluent; For various compound disinfectants containing surfactants, 3% (mass concentration) Tween-80 should be added to the diluent to neutralize the residual effect of the sample solution;
For samples such as garlic, garlic, garlic, and onion, D/E neutralizing broth can be used because the sample contains ingredients that inhibit bacterial growth.
5. Halophilic bacteria, osmotic dilutions to study halophilic bacteria (such as: salt samples), should use 15% sterile sodium chloride solution as a diluent. When the osmotic bacteria count, 20% sterile sucrose solution can be used as the diluent. The purpose of this is to protect the bacteria, because the ordinary diluent is too large for the bacteria, because the liquid osmotic pressure is too large, the bacteria It will expand and rupture due to the penetration of liquid, resulting in the death of bacteria and the inability to culture and count. This requires a sterile hypertonic solution to be used as a diluent to avoid bacterial death due to osmosis.
V. Dispensing and Sterilization of Diluent <br> Dispense the diluent into a suitable container for the preparation of the primary suspension. The volume of the container should be appropriate and not too small to prevent liquid spillage during autoclaving.
The dilution was dispensed into a test tube with a ten-fold gradient dilution. Generally, 9 mL of the dilution solution was placed in each tube. After sterilization, the volume change of the diluent in each tube should be within ±2%.
When counting certain microorganisms, some different media are used. Perhaps the volume of the diluent (or a part of the diluent) used for these microorganisms is much larger than 9 ml. In this case, the dilutions are loaded. The test tube or flask should be reasonably selected according to the actual liquid volume.
Place the flask or tube containing the diluent in a matching stopper. The general dilution conditions for the diluent are 121 ° C, 15 min,
Special attention should be paid to the pH change of the diluent before and after sterilization. After some dilutions or media are sterilized, the pH will decrease. Because the change of pH affects the biological activity of the target microorganism, it is also a part that is easily overlooked in manual operation. In addition to using precision pH test paper, it is better to equip with a pH electronic meter with an error of ±0.1. Within the pH unit.
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!
First, the primary (initial) sample suspension <br> After the sample is weighed by a certain weight or after taking a certain volume, add 9 times the weight or volume of the specific dilution, and after the external force is fully homogenized, the suspension is obtained. Turbid liquid, solution or emulsion.
When some of the initial suspensions contain larger sample particles, they should be allowed to settle horizontally. This gives the primary (initial) suspension (10-1).
Second, the secondary ten-fold gradient dilution <br> Take a specific volume of the primary suspension, add 9 times the specific volume of the dilution and mix well, so the primary suspension is diluted ten times (10-2 ), and then proceeding sequentially (10-3, 10-4....10-n) to obtain a series of dilutions until a dilution that meets the target microorganism culture requirements is obtained. Such an operation is called a secondary ten-fold gradient dilution and the resulting series of dilutions are collectively referred to as secondary ten-fold gradient dilutions.
Third, the principle of dilution operation <br> The primary suspension is prepared to make the microorganisms in the sample as evenly distributed as possible, and then in the next secondary ten-fold gradient dilution, the number of microorganisms can be uniformly indexed according to the index Decrease, so that after cultivation, it is convenient for colony counting operations.
In order to strictly narrow the counting range of each dilution and improve the accuracy of the counting, a general evaluation of the microbial contamination level of the sample should be made first, and then at least two dilutions of the appropriate serial dilutions should be selected, and at least each dilution should be inoculated. Two medium plates were cultured. Both the primary sample suspension and the secondary ten-fold gradient dilution sample solution should be evenly oscillated to ensure that the microorganisms are evenly distributed in the solution before proceeding to the next step.
Oscillation should be performed on the vortex mixer (see figure). Use the right thumb and middle finger to clamp the test tube, the right index finger against the test tube cap to prevent the test tube cap from flying when vibrating, and the three fingers force the bottom of the test tube against the rubber vibrating head of the vortex mixer. There are usually two gears for the vortex mixer to operate: one is that the vibrating head continues to vibrate, and the other is that only the object will vibrate against the vibrating head, usually using the latter. If there is no vortex mixer, you can use the method of hand-vibration mixing.
IV. Diluent 1. General Principles for Formulating Diluents In order to make the results more reproducible, it is recommended that when formulating the diluent, whether it is weighed one by one with a single reagent for dehydration or a dehydrated commercial finished medium, the laboratory personnel The following principles must be strictly followed when preparing the diluent:
(1) The various reagents for preparing the diluent or the dry powder of the dehydrated finished medium must be guaranteed.
(2) The water used to prepare the diluent must be qualified distilled water. The water's various indicators (pH, water activity, conductivity, electrical resistivity, total number of colonies) must meet the requirements for the water used for the preparation of the medium.
(3) The components of the diluent must be sufficiently dissolved to form a uniform solution without precipitation. After the dilution is prepared, the pH should meet the survival requirements of the target microorganism, especially the pH of the dilution after autoclaving. The normal range of the pH error should be ±0.2 (at 25 °C).
2. Ordinary dilutions commonly used in ordinary dilutions have a concentration of 0.1%, pH 6.8 ~ 7 sterile protein water (according to protein 胨 1.0g, sodium chloride 8.5g, distilled water 1000 mL ratio mixed to completely dissolve), phosphate Buffer, 0.85% sodium chloride solution, 2% buffered protein, water, etc. Some additives may be added as the case may be. For example, an emulsifier or a surfactant may be added to a sample having a high fat content; a foam inhibitor, an antifoaming agent, or the like may be added to a sample which is easy to foam during a homogeneous operation.
When preparing the primary sample solution, the sample may change the nature of the diluent due to the large proportion of the sample in the entire solution system, especially when the sample is insoluble in water, and the sample is in the diluent. The solubility will be affected. This will affect the pH or water activity (Aw) of the solution, so when it is not possible to determine if the sample has such an effect on the dilution, the pH and water activity of the first dilution should be determined. A phosphate buffer can be added to the dilution to stabilize the pH change.
It is particularly important to note that for some high-solubility dried samples (milk powder, baby food), the water activity is very low when the primary sample solution is made, so distilled water should be selected as the diluent. For the different samples, choose the most suitable dilution, which actually needs to be obtained through a series of tests, the selected dilution should have a high recovery rate.
The dilution preparation of the sample should be completed within 30 minutes.
3. For the qualitative or quantitative detection of anaerobic microorganisms in foods for the dilution of anaerobic microorganisms, the oxidation must be minimized, and the dilution should have an antioxidant effect. When preparing a sample solution, oxygen should be avoided as much as possible. In the detection of anaerobic bacteria that are extremely sensitive to oxygen, in addition to the use of appropriate dilutions, there are special equipment to assist, such as the use of anaerobic workstations as a protective measure for the bacteria.
4. Neutralizing Dilutions Some samples contain antibacterial substances, disinfectants, and other components that kill or inhibit microorganisms. It is necessary to add certain organic or inorganic compounds to neutralize these components to eliminate their effects.
For alcohols and phenolic disinfectants, the diluent can be used as a common nutrient broth; for chlorine-containing disinfectants, iodine-containing disinfectants, and peroxide disinfectants, 0.1% sodium thiosulfate should be added to the diluent; Chlorhexidine, quaternary ammonium disinfectant, 3% (mass concentration) Tween-80 and 0.3% lecithin should be added to the diluent; for aldehyde disinfectant, 0.3% glycine should be added to the diluent; For various compound disinfectants containing surfactants, 3% (mass concentration) Tween-80 should be added to the diluent to neutralize the residual effect of the sample solution;
For samples such as garlic, garlic, garlic, and onion, D/E neutralizing broth can be used because the sample contains ingredients that inhibit bacterial growth.
5. Halophilic bacteria, osmotic dilutions to study halophilic bacteria (such as: salt samples), should use 15% sterile sodium chloride solution as a diluent. When the osmotic bacteria count, 20% sterile sucrose solution can be used as the diluent. The purpose of this is to protect the bacteria, because the ordinary diluent is too large for the bacteria, because the liquid osmotic pressure is too large, the bacteria It will expand and rupture due to the penetration of liquid, resulting in the death of bacteria and the inability to culture and count. This requires a sterile hypertonic solution to be used as a diluent to avoid bacterial death due to osmosis.
V. Dispensing and Sterilization of Diluent <br> Dispense the diluent into a suitable container for the preparation of the primary suspension. The volume of the container should be appropriate and not too small to prevent liquid spillage during autoclaving.
The dilution was dispensed into a test tube with a ten-fold gradient dilution. Generally, 9 mL of the dilution solution was placed in each tube. After sterilization, the volume change of the diluent in each tube should be within ±2%.
When counting certain microorganisms, some different media are used. Perhaps the volume of the diluent (or a part of the diluent) used for these microorganisms is much larger than 9 ml. In this case, the dilutions are loaded. The test tube or flask should be reasonably selected according to the actual liquid volume.
Place the flask or tube containing the diluent in a matching stopper. The general dilution conditions for the diluent are 121 ° C, 15 min,
Special attention should be paid to the pH change of the diluent before and after sterilization. After some dilutions or media are sterilized, the pH will decrease. Because the change of pH affects the biological activity of the target microorganism, it is also a part that is easily overlooked in manual operation. In addition to using precision pH test paper, it is better to equip with a pH electronic meter with an error of ±0.1. Within the pH unit.
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!
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