Biochemical incubator at the application of multi-tube fermentation method

1.           Multiple tube fermentation methodFermentation fermentation tube that has the single-fold or three times the lactose peptone, and included a small glass tube upside down. Each sample of water with three different sample volume inoculated, inoculated with the same amount of water should be 5, the water samples were inoculated with peptone lactose fermentation tubes, in 37 ℃ ± 0.5 ℃ incubated 24h ± 2h. Can play the role of lactose selection, because many bacteria can ferment lactose, and coliform bacteria can ferment lactose and produce acid and gas. In the culture medium with bromocresol purple as pH indicator bacteria produce acid, the culture medium is from purple to yellow. Acid gas in the fermentation tubes that test positive, such as gas production in the inverted tube was not obvious, can tap the fermentation tube, small bubbles rising to the positive.

Complex fermentation minor oscillations early positive results of the fermentation fermentation test tubes, inoculated with a 3mm loop, culture （2-3 Central） transferred to EC medium, in 44.5 ℃ ± 0.5 ℃ incubated 24h ± 2h （higher incubation temperature can result from the natural environment is not conducive to the growth of coliform bacteria）. Observed immediay after c*tion, fermentation gas pipe is confirmed to be positive for fecal coliform.

2.           Membrane filtration membrane is a microporous membrane, pore size 0.45μm. Will have been sterilized water placed into the membrane filter, after filtration, bacteria Jibei trapped in the membrane The membrane is then affixed to the M-FC medium, in 44.5 ℃ ± 0.5 ℃ cultured 24 h ± 2h. Fecal coliform bacteria in the M-FC medium blue or blue-green, other non-fecal coliform bacteria into the gray, light yellow or colorless. Under normal circumstances, due to temperature and salt rose selective reagents role in the M-FC medium rare to see non-fecal coliform colonies.

二. Note the two methods.

1. Total coliform bacteria culture temperature requirements of bacteria in the gut than to live in foreign, in the natural environment of the water and soil often exist, but these coliform bacteria live in the natural environment training the most appropriate temperature for 25 ℃ and 37 ℃ for if you can still grow, but if the incubation temperature further increased to 44.5 ℃, while no growth, but directly from the excreta of coliform bacteria, are used to grow around 37 ℃, If the culture temperature to 44.5 ℃ still growing. Therefore, the method can be used to improve the natural environment, culture temperature in the coliform and fecal coliform bacteria distinguish between the two methods are based on this rationale. Good samples or inoculated with medium containing membrane into biochemical incubator, the inside temperature must reach the required temperature （44.5 ± 0.5 ℃）, the be placed. Such as the use of constant temperature water bath, its surface is higher than inoculum face, put the dish membrane water bath to sink to the bottom..

2. Treatment of chlorinated water samples disinfected by chlorine treatment of water, the water contains a certain amount of residual chlorine, coliform bacteria are damaged or subject to inhibition in water samples collected in the sample bottle should be added ahead of thiosulfate sodium thiosulfate can be dechlorinated to damage the bacteria to recover and repair, to avoid counting the low or even false negative results of the phenomenon. In addition, residual chlorine on the filter method of its inhibition of the bacterial culture is particularly so when the filtration of water samples should be a lot of water dilution to reduce the residual chlorine.

3. Sample containers used for the preservation of samples to be covered prior to use caps, the caps, and bottlenecks in brown paper wrapped office, home oven 160 ℃ -170 ℃ dry heat sterilization 2h, or high-pressure steam sterilizer 121 ℃ off bacteria 15min. Right after the water sample preservation is also important, such as improperly stored samples of coliform bacteria can die or re-growth under certain conditions, these will affect the accuracy of test results. Received water sample testing room immediay after detection, such as for some reason can not detect when the samples should be immediay placed in the refrigerator （2 ℃ -10 ℃） and was detected within 2 hours.

4. Preparation and preservation of material culture.

 ⑴. Multiple fermentation medium used in the sub-tube installed in the fermentation, after fermentation tubes should put in as soon as possible in the high-pressure steam sterilizer, sterilizing at 115 ℃ 20min （Note: The temperature should be controlled well, both to the role of sterilization, but also to prevent the loss of training material is broken down）, and then stored in the refrigerator or dark alternative. ⑵. Filter method used by the M-FC medium for greater use of outsourcing of finished products, aniline blue medium purity and quality tend to influence the color of the colony, therefore, aseptic packaging, complete culture medium before use, the best typical fecal coliform first inoculation, compared to observe the color of colonies, to identify its stability.

5. Results and Statistics.

 ⑴. Multiple fermentation results are based on different inoculum fermentation tube the number of positive results that emerged from the MPN table to look up the appropriate MPN index, calculated per liter of water, the MPN value of fecal coliform bacteria. MPN is the "Most Probable Number" in the abbreviation of the largest possible number, which is based on statistical theory, estimates of bacterial density in water and sanitation, quality approach, so accuray determine amount of inoculum fermentation is the number of positive tubes important, otherwise will lead to larger deviations.

⑵. Membrane filtration membrane on the results of a count showed a blue or blue-green colonies, to calculate per liter of water in the fecal coliform. Therefore, to facilitate counting, reduce errors, the ideal volume of water is a growth of 20-60 months on the membrane fecal coliform bacteria.

三. In the practical application of

1. Detection of applicability of the samples .

⑴. Multiple fermentation because only the sample volume under the water to determine how the samples were cultured in the medium （one-fold or three times the lactose） were cultured, so in theory applicable in various water samples, but due to limited volume of the fermentation tube, the more suitable test source water, pollution of surface water and wastewater （sample volume is not）, and if the water sample volume inoculated MPN table is not the three inoculation When we need a conversion formula.

⑵. Filter method is the use of filtration devices mainly bacteria trapped in the membrane, the membrane is then affixed to the solid medium, it can be used to detect larger water samples, but limited by the membrane pore size in the detection of high turbidity （water pollution）, non E. coli bacteria density type （river water） in water samples, on the colony counting statistics have a certain influence, in addition, toxic substances in water samples by high also in the filter accumulation of membrane formation, inhibition of bacterial culture.

3.       Detection time and operation of the tedious process of degree.

⑴.       Multi-tube fermentation method to go through primary fermentation test and test complex fermentation process before the two inoculation under different fermentation control the number of positive results that emerged from the MPN table to look up the corresponding MPN index to the conclusion that fecal coliform bacteria in water per liter MPN value. Can be seen, multiple fermentation culture time at least 2 days or more, and on the inoculum size, inoculation of fermentation tubes for each number, there are stringent requirements （MPN table to use more 9, 15 fermentation tubes, inoculation 10ml, 1ml, 0.1ml3 kind of volume of inquiries）, the result difficult to count.

⑵.     Filter method sets the current principal use of NPS （membrane + media + dish） for bacterial retention and training, simple operation, use only sterile water to wet the medium with a small amount of cushion can be used, incubation time 1 days, can be faster than the multi-tube fermentation method to obtain the results, also has a high degree of reproducibility and precision. In addition, aseptic packaging as a single chip, saving the sterilization time, but also to avoid the second pollution during the operation, and long a colony of the membrane film can be UV sterilized, dried as a test record of permanent preservation, more measurement certification standard.

三. Detection of cost considerations.

   ⑴. Multi-tube fermentation method used in fermentation tubes, small glass tubes and plastic back cover can be cleaned, autoclaved, and after the repeated use of ultraviolet disinfection, the liquid medium can be self-prepared or commercially available have been used with good integrated media, the cost of testing is not high. Because of its method is relatively complex, currently there is a through the US EPA-certified water and wastewater standards for fecal coliform detection methods to gradually replace it, that in the United States, Japan and Canada, widely used in countries such as the detection technology in China has some use （Beijing Environmental Monitoring Center, Beijing Drainage Group Testing Center, etc.） This method is to use the IDEXX colilert reagents to detect the fecal coliform in water, although it is based on the principle of multi-tube fermentation method, but to shift its operations greatly simplified. Of course, along with simplified operation, inspection costs to rise substantially, essentially all imported equipment, front-probably from 80,000 to 100000 yuan between, and because the sampling bottles, reagents, and plate counts were sealed off the use of a single sample testing costs will reach about RMB 200.

⑶.       Filter method mainly used in filtration devices （polished stainless steel manifold filter 3, 500ml funnel, oil-free diaphragm vacuum pumps and manual positive pressure dual-use sterile water plus receiver） is also basically came from abroad, in many cases units are the German company Sartorius products, its initial investment in about 20 thousand to 30 thousand yuan or so （including 100 sets of aseptic packaging of the NPS, 12 yuan / each）. Since each sample be diluted with 3 different filter than the training, to use three sets of NPS, so the cost of a single test sample is about 50 yuan.

四. Proposal.

 In several ways from the comparison between the two can be seen, membrane filter with time-saving, materials, equipment requirements, and can be low in samples collected more advantages, but also with the establishment of the contingency detection mechanism, when you need us pollution of various unexpected events （hospital waste water treatment is chlorination, rivers are polluted by domestic wastewater） to respond quickly, faster access to positive results when the advantages become more prominent. Therefore, the actual testing work, we can filter method as the primary detection method, and to take steps to overcome its vulnerability to water turbidity, bacteria and toxic substances interfere with other limitations, rapid response water quality, for the supervision and management to provide scientific data; multi-tube fermentation method as an auxiliary method to on the membrane through its suspicious bacteria were cultured and identified, so that more accurate test results, the only way to smooth the water bacteriological testing and efficient conduct.