? Bacteria in water samples include many "genus" and many "species". The hygiene evaluation standard of my country's "Hygienic Standards for Drinking Water" 2006 [1] is based on the total number of bacterial colonies and total coliform bacteria. group, heat-resistant coliforms and Escherichia coli as microbial indicators.
Total coliforms can come from the intestines of humans and warm-blooded animals and the natural environment, including Escherichia, Citrobacter, Klebsiella and Enterobacter. A large group of Gram-negative non-spore-forming bacteria that can ferment lactose, produce acid and gas, and are aerobic and facultatively anaerobic after 24 hours of cultivation at 37°C. Their growth characteristics on eosin-methylene blue plates are: (1) deep purple-black with metallic luster; (2) purple-black with no or slightly metallic luster; (3) lavender-red colonies.
Thermotolerant coliforms, also known as fecal coliforms, mainly include Escherichia and heat-resistant Klebsiella [6]. They use the method of increasing temperature to convert the natural large intestine into Bacteria are distinguished from coliforms in feces. They are a group of coliforms that can grow at 44.5°C. Only coliforms derived from animal and human feces can grow, while coliforms from nature cannot. Heat-tolerant growth[8-9].
Escherichia coli (Escherichiacoli), formerly often called Escherichia coli, is widely present in the intestines of humans and warm-blooded animals and is the dominant aerobic bacteria in the intestines of humans and animals. flora, intestinal bacteria necessary to maintain some physiological functions of the host. Although most Escherichia coli are non-pathogenic bacteria, when the host's immunity is reduced or the bacteria invade the external tissues or organs of the intestine, they can cause Extraintestinal infection. Certain serotypes can produce toxins and are pathogenic Escherichia coli, which can cause diarrhea in humans. Therefore, Escherichia coli in drinking water has become one of the important pathogenic bacteria of intestinal diseases. [2]
The logical relationship between their subordinate scopes is as follows
? The 2006 version of the "Hygienic Standards for Drinking Water" adds testing for Escherichia coli in drinking water.
In order to improve laboratory testing capabilities, our laboratory has recently carried out quality control activities for the detection of Escherichia coli in drinking water. The quality control activities have achieved the following goals:
1. Use the heat-resistant coliforms isolated at work, the standard strain Enterobacter aerogenes, and the standard strain Escherichia coli to conduct tests, observe the fluorescence of different coliforms in the EC-MUG culture medium, and obtain positive specimens Fluorescent visual effects.
2. The test tubes used were soaked in acid, treated with distilled water and directly cleaned with tap water to find factors affecting the fluorescence effect of Escherichia coli.
3. Conduct a comparative test using Beijing Luqiao EC-MUG culture medium reagent and Qingdao Haibo EC-MUG culture medium reagent to evaluate whether the reagents routinely used in the laboratory are excellent.
1. Materials and instruments
1.1. EC-MUG culture medium, purchased from Beijing Luqiao Co., Ltd. and Qingdao Haibo
1.2. Eosin-Melan culture medium , purchased from Beijing Land Bridge Co., Ltd.
1.3 Nutrient broth, purchased from Beijing Luqiao Co., Ltd.
1.4 incubator, 36℃ ±1℃; 44.5±0.5℃; wavelength 366nm UV lamp
2 Methods and results
2.1 Method principle
Escherichia coli can specifically produce β-glucuronidase (β-glucuronidase). This enzyme can decompose the EC-MUG fluorescent substrate on the culture medium to release a fluorescent product, making the culture medium at 366 Characteristic fluorescence is produced under nm ultraviolet light, and this technology is used to detect Escherichia coli.
2.2 Operation steps and results
The picture above shows the production of Enterobacter aerogenes and Escherichia coli in EC-MUG culture medium at 44.5°C under the background of fluorescent lamps. If the liquid of Enterobacter aerogenes is clear, it means that Enterobacter aerogenes cannot grow and reproduce at 44.5℃; if the liquid of Escherichia coli is turbid, it means that Escherichia coli can grow and reproduce at 44.5℃.
The above-cultured EC-MUG culture medium was observed using a 366nm ultraviolet lamp under the background of fluorescent lamp observation. Enterobacter aerogenes is still clear and has no blue light; Escherichia coli liquid at different dilutions has obvious blue light, and there is no obvious difference between the blue lights. Therefore, when observing the fermentation effect of Escherichia coli, you can use a 366nm ultraviolet lamp to observe under ordinary light, and the results will be significant.
The picture above shows the experimental effect of the aforementioned cultured EC-MUG culture medium in a dark room. Escherichia coli produces fluorescence after fermentation, and there is no significant visual difference between bacterial liquids of different dilutions; it is very obviously different from the negative effect of Enterobacter aerogenes. The Enterobacter aerogenes culture tube has weak fluorescence, which may be due to the fact that the EC-MUG medium itself has fluorescence!
This picture is a comparison of the effects of sterilized distilled water and EC-MUG culture medium. Test tubes 1 and 2 are acid-cleaned test tubes, and test tubes 5 and 6 are not acid-cleaned.
Observed in a dark room, Enterobacter aerogenes in EC-MUG culture tubes has obvious fluorescence compared to distilled water, taking pictures with a mobile phone. In actual observation, this fluorescence is relatively weak, but fluorescence is still produced. . Therefore, the fluorescence that ordinary test tubes and distilled water may produce is negligible, but it is an experimental fact that the reagent itself produces a certain amount of fluorescence.
Therefore, in order to reduce the lack of personal observation experience, it is recommended that during the experiment, markers should be used for comparison and observation, so that the intuitive effect of fluorescent yin and yang can be seen in the mind.
The heat-resistant coliforms in the picture above are working strains detected in daily work. The laboratory confirmed that they can produce coliforms with a rust-like metallic luster on eosin-melan plates. Belongs to heat-resistant coliforms. At present, the bacteria can grow turbidly in EC-MUG culture medium after culture at 44.5°C.
Observed with 366nm ultraviolet wave of three-purpose ultraviolet lamp under the background of fluorescent lamp, each test tube has a slight blue light, but the actual visual observation is not very strong. The photos taken with the mobile phone show a slight fluorescence effect, and the color of Enterobacter aerogenes seems to be darker. It can be judged that all 10 test tubes are negative for Escherichia coli.
The picture above shows the EC-MUG fluorescence effect of Enterobacter aerogenes and heat-resistant coliforms of working strains in a dark room. Mobile phone photos show strong fluorescence, but the actual visual effect is weak. If there is no positive control for comparison, it is easy to mistakenly think that the result is positive for Escherichia coli. However, when observed in a dark room, there is no difference between the heat-resistant coliforms and Enterobacter aerogenes, and the result can also be judged as negative.
Comparison of the fluorescence effects of Enterobacter aerogenes and laboratory thermotolerant coliforms after high-temperature culture in EC-MUG. Enterobacter aerogenes cannot grow at high temperatures, the liquid is clear, and it cannot decompose fluorescent substrates. The heat-resistant coliforms of this laboratory's working strain can grow at high temperatures, so the liquid is turbid. However, the working strain of this laboratory does not decompose the MUG fluorescent substrate on the culture medium to release a fluorescent product, which can be compared with Enterobacter aerogenes (commercial standard strains), it was determined to be fluorescence negative, so it is possible that the heat-resistant coliform working strain in our laboratory is a heat-resistant Klebsiella genus. (Heat-resistant coliforms, also known as fecal coliforms, are composed of Escherichia and heat-resistant Klebsiella. It is not a bacteriological classification name, but a term in the field of health bacteria.)
? The following pictures show the observation effect of EC-MUG culture using two different reagents using the same treated test tubes:
The fluorescence effect is observed in a dark room. The contrast between yin and yang of Beijing Luqiao products is obvious.
Under fluorescent lamps, use ultraviolet lamp to observe the fluorescence renderings. The contrast between yin and yang of Beijing Luqiao products is obvious.
Observing the fluorescence renderings in a dark room, Qingdao Haibo products have obvious contrast between yin and yang.
Under a fluorescent lamp, use an ultraviolet lamp to observe the fluorescence effect diagram. The contrast between yin and yang of Qingdao Haibo products is obvious.
The picture above is a photo of the experimental culture results under normal light.
Through comparison of two reagents from Beijing Luqiao and Qingdao Haibo, it was found that the two reagents produced the same fluorescence effect when cultivating Escherichia coli in test tubes treated in different ways with different dilution factors.
3. Discussion
3.1. According to relevant literature and experimental results, the test tube itself will produce a trace amount of fluorescence [3]. The test tubes used in this experiment were divided into two parts. One part was soaked in acid for more than seven days and then cleaned with distilled water; the other part was cleaned directly with tap water. Both types of test tubes were sterilized at a high temperature of 165°C for more than 2 hours. The two types of test tubes were evenly filled with distilled water control, Enterobacter aerogenes (standard strain), heat-resistant coliforms (working strain), and Escherichia coli strain (standard strain). There is no difference in experimental results between test tubes treated with the same strain in different methods, indicating that the fluorescence that may be produced by general test tubes has no impact on the experiment.
3.2. The EC-MUG medium reagent itself will overflow certain fluorescent substances after cultivation. If there is no positive control for comparison, it may be mistaken for a true positive, resulting in a false positive result. Therefore, junior experimenters or daily work should bring standard strains for simultaneous experiments and observe the experimental results.
3.3. The positive results of the EC-MUG culture medium produced by the two reagent manufacturers have significant fluorescence effects. In actual operation, the test results of Escherichia coli in daily drinking water are different from the negative control. If there is no abnormality, the result can be reported as no Escherichia coli detected.
3.4. GB/T5750.12-2006 detects Escherichia coli using multi-tube fermentation method, filter membrane method, and Escherichia coli enzyme substrate method. The first recommended method is the multi-tube fermentation method, which is based on the detection of total coliform lactopeptone fermentation and selective cultivation of EC-MUG, which reduces the workload and work expenses from batch work. In daily drinking water monitoring, the first method is simpler and easier to implement than the second filtration method; compared with the third method, the enzyme substrate method, it is more economical and affordable. Therefore, the first multi-tube fermentation method is still a method worthy of promotion in routine testing work.
3.5. There is no significant difference in the fluorescence effects of Escherichia coli liquids with different dilutions in EC-MUG culture medium. Therefore, after total coliform fermentation, in the next experimental operation, pipettes should be used in one tube, and cross-contamination in different fermentation tubes should not occur to prevent high positive values.
References
[1] GB5749-2006, Hygiene Standard for Drinking Water [S]. Beijing: China Standard Publishing House, 2007
[2], zzzzzz. Water, mmm mmm mmmJ]. Henan Journal of Preventive Medicine 2009 20(3):185-216.
[8] GGkkk. Daily Drink[J]. Preventive Medicine Forum, 2008, 14(12) : 1163-1164.
[9]hhhhhhhhh et al. Agricultural survey in southern China [J]. Public Health and Preventive Medicine, 2014, 25(4):11-13.
[gggg[J]. Environmental Science and Technology, 2010, 23(1): 67-70
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