Application of biochemical incubator in multi-tube fermentation and membrane method

Incubator fermentation and application of a multi-tube membrane filter method

The biochemical incubator has a two-way temperature regulation system for cooling and heating, and the temperature controllable function is a scientific research institution, college, production unit or department laboratory of biology, genetic engineering, medicine, health and epidemic prevention, environmental protection, agriculture, forestry and animal husbandry. The important test equipment is widely used in low temperature constant temperature test, culture test, environmental test and so on.

1. Multi-tube fermentation

In the initial fermentation experiment, the fermentation tube was filled with a single or triple lactose peptone culture solution, and a small glass tube was inverted inside. Each sample was inoculated with three different water samples. The same inoculum water sample should have five tubes. The water samples were inoculated separately into the fermentation tube containing the lactose peptone culture solution, and cultured at 37 ° C ± 0.5 ° C for 24 h ± 2h. Lactose can play a role in selection because many bacteria cannot ferment lactose, while coliforms can ferment lactose and produce acid to produce gas. Bromocresol purple is added to the culture solution as an acidity indicator. After the bacteria produce acid, the culture solution turns from purple to yellow. The fermentation tube producing acid-producing gas indicates that the test is positive. If the gas is not obvious in the inverted tube, the fermentation tube can be tapped, and small bubbles rise to be positive.

The fermentation experiment slightly oscillates the fermentation tube with positive results of the initial fermentation test, and the culture (2-3 rings) is transferred to the EC culture solution with a 3 mm inoculating loop, and cultured at 44.5 ° C ± 0.5 ° C for 24 h ± 2 h (increased culture temperature) Can cause growth of coliforms that are not conducive to the natural environment). Immediately after the culture, the fermentation tube produced gas was confirmed to be positive for fecal coliforms.

2. Filter method

The filter membrane is a microporous membrane with a pore size of 0.45 μm. The water sample is injected into the sterilized filter with the filter membrane. After suction filtration, the bacteria are trapped on the membrane, and then the membrane is attached to the M-FC medium and cultured at 44.5 ° C ± 0.5 ° C. 24 h ± 2 h. The fecal coliform colonies were blue or blue-green on M-FC medium, and other non-fecal coliform colonies were gray, yellowish or colorless. Normally, non-fecal coliform colonies are rarely seen on M-FC medium due to temperature and selective action of the rose salt reagent.

Two. Considerations for the two methods

1. Requirements for culture temperature In addition to living in the intestine, bacteria in the total coliforms are often present in water and soil in the natural environment, but the suitable temperature for coliform cultivation in the natural environment is At about 25 ° C, if cultured at 37 ° C, it can still grow, but if the culture temperature is raised to 44.5 ° C, it will no longer grow, and the coliform bacteria directly from the feces, used to grow around 37 ° C, such as The growth temperature can be continued until the culture temperature is raised to 44.5 °C. Therefore, it is possible to distinguish the coliforms in the natural environment from the coliforms in the feces by increasing the culture temperature, and both methods are based on this. Place the inoculated sample or medium containing the filter into the culture dish and sink to the bottom of the water bath.

2. Chlorinated water sample treated with chlorine treated disinfected water, the water contains a certain amount of residual chlorine, so that the coliform group is damaged or inhibited. When collecting water samples, the sulfur should be added to the sampling bottle in advance. Sodium sulphate and sodium thiosulfate can be dechlorinated to resuscitate and repair damaged bacteria, thus avoiding the phenomenon of low or even false negative counts. In addition, residual chlorine is particularly effective in inhibiting the bacteria cultured by the membrane method. Therefore, the water sample should be diluted with a large amount of sterile water during filtration to reduce residual chlorine.

3. Preservation of sample The container for sampling should be covered before use. Wrap the bottle cap and bottle neck with kraft paper. Dry the oven at 160°C-170°C for 2h, or use high pressure steam sterilizer 121. Sterilize for 15 min at °C. The correct preservation of the water sample after sampling is also important. If improperly stored, the coliform bacteria in the sample will die or regenerate under certain conditions, which will affect the accuracy of the test results. Immediately after receiving the water sample, the test chamber should be tested. If it cannot be detected for any reason, immediately place the sample in the refrigerator (2 °C - 10 °C) and test within 2 hours.

4. Preparation and preservation of culture materials

(1). After the culture medium used in the multi-tube fermentation method is dispensed into each fermentation tube, the fermentation tube should be placed in a high-pressure steam sterilizer as soon as possible, and sterilized at 115 ° C for 20 minutes (note: this temperature should also be controlled well) , to achieve the role of sterilization, but also to prevent the decomposition of culture materials, and then stored in the refrigerator or in the dark.

(2). The M-FC medium used in the membrane method mostly uses the purchased product. The purity and quality of the aniline blue in the medium often affect the color of the colony. Therefore, the aseptically packaged medium is inoculated before use. Typical fecal coliforms were used to identify the color of the comparative colonies to identify their stability.

5. Results statistics

(1). The result of the multi-tube fermentation method is to calculate the MPN value of the fecal coliform bacteria per liter of water according to the number of positive results of the fermentation tubes of different inoculum sizes, and the corresponding MPN index from the MPN table. MPN is the abbreviation of “Most Probable Number”, which refers to the maximum possible number. It is a method for estimating the microbial density and hygienic quality in water according to statistical theory, so it is very accurate to accurately judge the positive number of different inoculum fermentation tubes. Important, otherwise it will lead to large deviations.

(2). The result of the filter method is to count the colonies which are blue or blue-green on the filter to calculate the number of fecal coliforms per liter of water sample. Therefore, in order to facilitate counting and reduce errors, the ideal water sample volume is 20-60 fecal coliform colonies grown on a filter.

3. Discussion on the application in practice

1. Applicability of the test method to the sample

(1). The multi-tube fermentation method is theoretically applicable to various water samples, since it is only necessary to determine the culture medium in which the sample is cultured (single or triple lactose) according to the sampling amount of the water sample. Due to the limitation of the volume of the fermentation tube, it is more suitable for detecting source water, surface water and pollution source wastewater (sampling amount is small), and if the amount of water inoculated is not the three inoculum in the MPN table, it is necessary to use the formula Conversion.

(2). The membrane method mainly uses a suction filtration device to trap the bacteria on the filter membrane, and then attaches the membrane to the solid medium for cultivation. Therefore, it can be used to detect a large volume of water sample, but is limited by the pore size of the membrane. When detecting water samples with high turbidity (waste source wastewater) and non-E. coli bacteria (heheshui), the colony count statistics have a certain influence. In addition, if there is a high toxic substance in the water sample, it will also be Accumulation is formed on the filter membrane to inhibit bacterial culture.

2. Detection time and cumbersome operation process

(1). The multi-tube fermentation method needs to pass the initial fermentation test and the double fermentation test to obtain the corresponding MPN index from the MPN table according to the number of positive results of the fermentation tube with different inoculation amount, and finally obtain each The MPN value of the fecal coliform bacteria in the ascending water. It can be seen that the culture time of the multi-tube fermentation method requires at least 2 days, and the number of inoculum and the number of fermentation tubes per inoculation amount are strictly required (the MPN meter uses 9 tubes and 15 tubes of fermentation tubes, and the inoculum amount is 10ml, 1ml, 0.1ml 3 volume query), otherwise the results are difficult to count.

(2). Membrane method At present, the NPS (filter + medium + culture dish) is mainly used for bacterial retention and culture. The operation is simple. It can be used only by wetting the medium pad with a small amount of sterile water. The culture time is 1 In the day or so, the results can be obtained faster than the multi-tube fermentation method, and also have high reproducibility and precision. In addition, due to the use of single-piece aseptic packaging, the sterilization time is saved, and secondary pollution during operation is avoided, and the filter membrane with long colonies can be stored as a test record after ultraviolet sterilization and drying, and is more in line with the measurement certification. specification.

3. Inspection cost considerations

(1). The fermentation tube, small glass inverted tube and plastic cover used in the multi-tube fermentation method can be reused after washing, autoclaving and ultraviolet disinfection, and the liquid medium can be prepared by itself or using a commercially available integrated medium. The cost of testing is not high. Due to its cumbersome method, there is a standard method for the detection of fecal coliforms in water and wastewater certified by US EPA. This detection technology widely used in countries such as the United States, Japan and Canada is also available in China. Use (Beijing Environmental Testing Center, Beijing Drainage Group Testing Center, etc.), this method is to use the IDEXX colilert reagent to detect fecal coliforms in water, although it is based on the principle of multi-tube fermentation, but it greatly simplifies its operation. . Of course, along with the simplification of the operation, the cost of testing is also greatly improved. The equipment is basically imported. The initial investment is between RMB 80,000 and RMB 100,000, and since the sampling bottles, reagents and sealed counting discs are all once For sexual use, the cost of testing a single sample will also be around RMB 200.

(2). The filter device mainly used in the filter device (polished stainless steel 3 manifold filter, 500ml funnel, oil-free diaphragm vacuum positive pressure dual-purpose pump and manual sterile water dispenser) is also basically imported, currently a lot The units use the products produced by the German Sartorius company, the initial investment is about 20,000 to 30,000 yuan (including 100 sets of aseptically packaged NPS, 12 yuan / each set). Since each sample needs to be cultured in three different dilution ratios, three sets of NPS are used, so the cost of testing a single sample is about 50 yuan.

Recommendation

It can be seen from the comparison of the two aspects that the membrane method has the advantages of time saving, material saving, low equipment requirements and more water samples can be collected, and with the establishment of emergency detection mechanism, when we need us The advantages of all kinds of sudden environmental pollution incidents (whether the hospital wastewater is treated with chlorine, whether the rivers and lakes are polluted by domestic wastewater) and the positive results are obtained more quickly.

Therefore, in the actual testing work, we can use the membrane method as the main detection method, and take measures to overcome the limitations of the water turbidity, other strains and toxic substances, and quickly reflect the water quality, for the supervision and management department. Provide scientific data; multi-tube fermentation method as an auxiliary method, through which to identify the suspicious species cultured by the filter method, so that the test results are more accurate, only in this way, the bacteriological detection in water can be carried out smoothly and efficiently.