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1 Translated English of Chinese Standard: GB/T Buy True-PDF Auto-delivery. NATIONAL STANDARD OF THE PEOPLE S REPUBLIC OF CHINA GB ICS C 51 Partially replacing GB/T Standard examination methods for drinking water - Microbiological parameters 生活饮用水标准检验方法微生物指标 Issued on: December 29, 2006 Implemented on: July 01, 2007 Issued by: Ministry of Health of the People's Republic of China; Standardization Administration of the People's Republic of China. Buy True-PDF Auto-delivery. Page 1 of 55

2 Table of Contents Foreword Total bacterial count Total coliforms Thermotolerant coliform bacteria Escherichia coli Giardia Cryptosporidium Buy True-PDF Auto-delivery. Page 2 of 55

3 Foreword GB/T 5750 "Standard Test Method for Drinking Water" is divided into the following parts: - General principles; - Collection and preservation of water samples; - Water analysis quality control; - Organoleptic and physical parameters; - Nonmetal parameters; - Metal parameters; - Aggregate organic parameters; - Organic parameters; - Pesticides parameters; - Disinfection by-products parameters; - Disinfectants parameter; - Microbiological parameters; - Radiological parameters. This standard replaces total number of bacteria and total coliforms in the part II of GB/T Standard examination methods for drinking water. As compared with GB/T , the main changes are as follows: - ADJUST the structure in accordance with GB/T Directives for standardization - Part 1: Rules for the structure and drafting of standards ; - ADD 7 testing methods for 4 indicators including thermotolerant coliform bacteria, Escherichia coli, Giardia, and Cryptosporidium in drinking water.; This standard is proposed by and shall be under the jurisdiction of the Ministry of Health of the People's Republic of China. The responsible drafting organizations of this standard: China Center for Disease Control and Prevention Safety Institute for Environment and Healthrelated Product. Buy True-PDF Auto-delivery. Page 3 of 55

4 Standard examination methods for drinking water - Microbiological parameters 1 Total bacterial count 1.1 Plate counting method Scope This standard specifies the use of plate counting method for the determination of total bacterial counts in drinking water and its source water. This method applies to the determination of total bacterial counts in drinking water and its source water Terms and definitions The following terms and definitions apply to this standard Standard plate-count bacteria The total number of bacteria contained in 1 ml of water sample obtained after 48 h incubation at 37 C under aerobic conditions on nutrient agar Culture media and reagents Nutrient agar Ingredients: A Peptone 10 g B Beef paste 3 g C Sodium chloride 5 g D Agar 10 g ~ 20 g E Distilled water 1000 ml Preparation method: After mixing above components, HEAT to dissolve it, ADJUST the ph to 7.4 ~ 7.6, CONTAIN it into glass containers (if Buy True-PDF Auto-delivery. Page 5 of 55

5 USE the sterile pipette to take undiluted water samples and two ~ three 1 ml of water samples of the appropriate dilution degree, respectively INJECT it into the sterilized plate. The following operation is the same as the drinking water testing procedure Colony counting and reporting methods When doing plate colony counting, it can use naked eye to directly observe it, if necessary, it shall make inspection with a magnifying glass to prevent omission. After recording the number of colonies in each plate, it shall calculate the average count of colonies in the same dilution, which is used for the calculation of next step. When obtaining the average of the dilutions, if one of the plates has more platelet-like colonies, it should not be used, but the plates without platelet colonies shall be used to calculate the average count of colonies of this dilution. If the platelet colonies are less than half of the plate, whilst the distribution of colony count on this other half is very even, the colonies of this half of plate is counted and multiplied by 2 to get the count of colonies of the entire plate. Then the average count of colonies of this dilution is calculated Selection and reporting methods for different dilutions First SELECT the average count of colonies between 30 ~ 300 to make calculation, if the average count of colonies of only one dilution meet this range, then MULTIPLY the count of colonies by the dilution factor and REPORT it (SEE example 1 in Table 1) If there are two dilutions, the count of colonies grown is between 30 ~ 300, it is determined depending on the ratio of the two, if the ratio is less than 2, it shall report the average of the two (such as example 2in Table 1). If the ratio is more than 2, it shall report the total count of colonies with a small dilution (example 3 in Table 1). If it is equal to 2, it shall also report the count of colonies of smaller dilution (SEE example 4 in Table 1) If the average count of colonies of all dilutions is greater than 300, then it shall multiply the average count of colonies of the highest dilution by the dilution factor and report it (see Example 5 in Table 1) If the average count of colonies of all dilutions is smaller than 30, then it shall multiply the average count of colonies of the lowest dilution by the dilution factor and report it (see Example 6 in Table 1) If the average count of colonies of all dilutions is not between 30 ~ 300, then it shall multiply the average count of colonies closest to 30 or 300 by the dilution factor and report it (see Example 7 in Table 1) If the plates of all dilutions are free from colony growth, it shall be reported as undetected. Buy True-PDF Auto-delivery. Page 7 of 55

6 Small inverted tube Slides Testing procedure Lactose fermentation test TAKE 10 ml of water sample, INOCULATE it into 10 ml of double lactose peptone culture solution, TAKE 1 ml of water sample, INOCULATE it into 10 ml of single lactose peptone culture solution, TAKE another 1 ml of water sample, INJECT it into 9 ml of sterilized normal saline, MIX it uniformly, PIPETTE 1 ml (that is, 0.1 ml of water sample) into 10 ml single lactose peptone culture solution, INOCULATE 5 tubes for each dilution. If the tap water that has been processed must be tested regularly or once a day, it can directly inoculate five sets of 10 ml water sample double culture medium, each set is inoculated with 10 ml of water sample When testing the source water, if the pollution is serious, it shall increase the dilution. It can inoculate 1, 0.1, 0.01 ml or even 0.1, 0.01, ml. Each dilution is inoculated to 5 tubes, each water sample is totally inoculated to 15 tubes. When inoculating the water sample less than 1 ml, it must perform 10 times incremental dilution, take 1 ml for inoculation, and change one 1 ml sterile graduated pipette for each increment of dilution PLACE the inoculated tube in the incubator at 36 C ± 1 C for 24 h ± 2 h. If all lactose peptone culture tubes are not producing gas acid, then they can be reported as negative for the total coliforms. If they produce acid or gas, FOLLOW the steps below Separation culture TRANSPLANT the fermentation tubes producing acid and gas onto the eosin methylene blue agar plate, CULTURE it in an incubator at 36 C ± 1 C for 18 ~ 24 h. OBSERVE the colony morphology, SELECT the colonies with the following characteristics for Gram staining, microscopic examination and confirmation test. Dark purple black colonies with metallic luster; Purple black colonies without or with slight metallic luster; Pale purple colonies with darker color center Confirmation test After the above stained microscopic examination, it is gram-negative bacillus, Buy True-PDF Auto-delivery. Page 12 of 55

7 min per each sterilization. After the first two boiling, it is necessary to replace the water to rinse it for 2 ~ 3 times, to remove the residual solvent Filter sterilization: USE the ignited alcohol swab to perform flame sterilization, or USE steam sterilizer for autoclaving at kpa (121 C, 15 lb) for 20 min Filtered water sample USE the sterile tweezers to take the edge of the sterilized membrane filter, LET the rough surface face upwards, ATTACH it to the sterilized filter bed, FIX the filter, POUR 100 ml of water sample (if the water sample contains more bacteria, it can reduce the filtered water sample volume or otherwise dilute the water sample) into the filter, OPEN the filter valve, PERFORM suction filtration at Pa (minus 0.5 atmosphere) Culture After filtration of the water sample, MAKE suction for about another 5 s, CLOSE the filter valve, TAKE off the filter, USE sterile tweezers to take the edge of the membrane filter, TRANSFER it onto the magenta sodium sulfite medium, LET the bacteria catching face of the membrane filter face upwards, LET the membrane filter be in complete and close attachment with the culture medium, there shall be no air bubbles between them, INVERT the plate, PLACE it into the 37 C incubator to culture it for 24 h ± 2 h Results observations and reports SELECT colonies that meet the following characteristics for Gram staining and microscopic examination: Purple red colonies with metallic luster; Deep red colonies without or with slight metallic luster; Pale red colonies with darker center The Gram-negative bacillus is further inoculated with lactose peptone culture solution, cultured at 37 C for 24 hours. If there is acid production and gas production, the total coliforms are determined to be positive CALCULATE the count of total coliforms growing on the membrane filter in accordance with the formula (1), REPORT the total coliform colonies (CFU/100 ml) per 100 ml of water. Total coliform colonies (CFU/100 ml) = (Number of total coliform colonies counted 100) / Filtered water sample volume (ml)...(1) Buy True-PDF Auto-delivery. Page 19 of 55

8 are positive If the water which is not subject to chlorination is detected, and only the thermotolerant coliform bacteria are to be detected, or when investigating the contamination of the thermotolerant coliform bacteria of the source water, it may use a direct multi-tube thermotolerant coliform bacteria method, that is, during the step I lactose fermentation test, INOCULATE the lactose peptone culture solution in accordance with the total coliform to a 44.5 C ± 0.5 C water bath for culture, the following steps are same as Results report Based on the number of positive tubes confirmed to be thermotolerant coliform bacteria, CHECK the most probable number (MPN) search form, REPORT the most probable number (MPN) value of the thermotolerant coliform bacteria per 100 ml of water sample. 3.2 Membrane filter method Scope This standard specifies the use of membrane filter method for the determination of the thermotolerant coliform bacteria in drinking water and low turbidity source water. This method applies to the determination of thermotolerant coliform bacteria in drinking water and low turbidity source water Terms and definitions The following terms and definitions apply to this standard Membrane filter technique for thermotolerant coliform bacteria The thermotolerant coliform membrane filtration method refers to using the membrane filter with a bore size 0.45 μm to filter the water sample, where the bacteria is retained on the membrane, and the membrane filter being affixed to the selective medium added with lactose, and cultured at 44.5 C for 24 h, to detect the thermotolerant coliform bacteria in water through the forming characteristic colonies Culture media and reagents MFC culture medium Ingredients Buy True-PDF Auto-delivery. Page 27 of 55

9 Preparations are the same as in Filtered water sample is same as in Culture: After the filtration of the water sample, CONTINUE suction for approximately another 5 s, CLOSE the filter valve, TAKE off the filter, USE sterile tweezers to clamp the edge of the membrane filter, TRANSFER it on the MFC culture medium, LET the bacteria collecting face of the membrane face upwards, LET the membrane be in close contact with the culture medium, there shall be free from air bubbles between them, INVERT the dish, PLACE it in a 44.5 C water-isolated incubator for 24 h ± 2 h. If using a constant temperature water bath, it shall use a plastic dish, COVER the dish tightly, or USE waterproof tape to seal each dish, STACK the culture dishes into a plastic bag, IMMERSE it into a constant temperature water bath at 44.5 C, CULTURE it for 24 h ± 2 h. The thermotolerant coliform colonies are blue on this culture medium, and the non-thermotolerant coliform colonies are gray to creamy The suspected colonies are transplanted onto the EC culture medium, for culture at 44.5 C for 24 h ± 4 h, if gas is produced, it is confirmed as the thermotolerant coliform bacteria Results report The number of thermotolerant coliform colonies confirmed is counted, and the number of thermotolerant coliform bacteria in the water is expressed in a colony-forming unit (CFU) of thermotolerant coliform bacteria in 100 ml of water, as shown Where (2). Thermotolerant coliform bacteria (CFU/100 ml) = (The number of thermotolerant coliform colonies counted 100) / Filtered water sample volume (ml)...(2) 4 Escherichia coli 4.1 Multi-tube fermentation method Scope This standard specifies the use of multi-tube fermentation method for the determination of Escherichia coli in drinking water and its source water. This method is applicable to the determination of Escherichia coli in drinking water and its source water Terms and definitions The following terms and definitions apply to this standard. Buy True-PDF Auto-delivery. Page 29 of 55

10 Inoculation The membrane filter with typical colony growth through total coliforms membrane filter method is subject to Escherichia coli testing. The membrane filters are transferred to NA-MUG plates under aseptic conditions, the bacteria collecting side faces upwards for culture Culture The inoculated NA-MUG plates are incubated at 36 C ± 1 C for 4 h Results observations and reports The cultured NA-MUG plates are irradiated in a dark place by a UV lamp having a power of 6 W at a wavelength of 366 nm. If blue fluorescence is generated at the colony edge or on the back of the colony, it indicates that the water sample contains Escherichia coli. The number of colonies producing blue fluorescence is recorded and reported, the format of the report is identical to that of the total coliform membrane filter method. 4.3 Enzyme substrate method Scope This standard specifies the use of enzyme substrate method for the determination of Escherichia coli in drinking water and its source water. This method applies to the detection of Escherichia coli in drinking water and its source water. This method can determine whether the water sample contains Escherichia coli and the most probable number (MPN) of Escherichia coli in 24 h. This method can simultaneously detect total coliforms. For the method, SEE clause Terms and definitions The following terms and definitions apply to this standard Enzyme substrate technique for Escherichia coli The production of β-d-galactosidase on selective culture medium decomposes the chromogen substrate to releases the chromogen, which makes the culture medium change color, and the production of β-glucuronidase decomposes the Buy True-PDF Auto-delivery. Page 33 of 55

11 results are expressed in MPN/100 ml. If all test tubes do not produce fluorescence, it can be reported that Escherichia coli is not detected hole quantitative plate method The quantitation plate in which the water sample has been incubated for 24 h to turn yellow are irradiated by a UV light at a wavelength of 366 nm in the dark. If blue fluorescence is generated, it indicates that the cavity of this quantitative plate contains Escherichia coli COUNT the number of cavities with fluorescent response. CHECK Table 5 to find the most probable number of Escherichia coli it represents. The results are expressed in MPN/100 ml. If all test tubes do not produce fluorescence, it can be reported that Escherichia coli is not detected. 5 Giardia 5.1 Immunomagnetic separation fluorescent antibody method Scope This standard specifies the use of immunomagnetic separation fluorescence antibody method for the determination of Giardia cysts and Cryptosporidium oocysts in drinking water and its source water. This method is applicable to the determination of Giardia cysts and Cryptosporidium oocysts in drinking water and source water Terms and definitions The following terms and definitions apply to this standard Giardia A protozoal parasite that may be found in water or other media. There are two species, whose hosts are: G. intestinalis (human) and G. muris (rat) Cryptosporidium A protozoan parasite that may be found in water or other medium, there are 6 species, and their possible hosts are: C. parvum (mammals, including humans); C. boileyi and C. meleagridis (birds); C. muris (rat); C. serpeatis (reptile) and C. nasorum (fish). Buy True-PDF Auto-delivery. Page 35 of 55

12 C Suitable pressure pump (diversion pump, peristaltic pump); D Pump tube; E Clips; F Water meter; G Flow control valve (1 L/min to 4 L/min) Elutriation/concentration/purification equipment Envirochek method A Filter clip: horizontal oscillating device with arm, the arm has vertical mounting filter clips, maximum frequency is 600 r/min; B 175 ml conical centrifuge tube; C Centrifuge: 175 ml graduated conical centrifuge tubes, and centrifuges with an acceleration of 1500 g; D Vortex mixer; E Plastic pipette bulb; F 10 ml pipette; G 50 ml pipette; H 100 ml graduated measuring cylinder; I One-side planar test tube, 125 mm 16 mm, with tube plug, one side is 60 mm 10 mm plane; J Magnetic particle concentrator (MPC-M) for one-side planar test tube; K Conical 5 ml microcentrifuge tube with stopper; L Pasteur pipette Filta-Max method A Manual or automatic Filta-Max elutriation main equipment and matching equipment (concentrate tube and base, washing tube and stainless-steel siphon tube); B Manual vacuum pump; C Magnetic stirrer and stir bar; Buy True-PDF Auto-delivery. Page 37 of 55

13 Non-ionic surfactant Twee-20 Ultrapure water 0.1 ml 900 ml DISSOLVE 1.44 g of disodium hydrogen phosphate, 0.24 g of dihydrogen phosphate, 0.2 g of potassium chloride, and 8 g of sodium chloride into 900 ml of ultrapure water. STIR it for 20 min until it is completely dissolved, ADD 0.1 ml of non-ionic surfactant Tween-20, CONTINUE stirring for another 10 min, then USE ultrapure water to dilute it to 1000 ml. C Filta-Max Xpress fast method for buffer elutriation (PETT buffer solution): Sodium pyrophosphate tetra-basic decahydrate EDTA tri-sodium salt Tris-HCl (1 mol/l) Tween g 0.3 g 10 ml 0.1 ml ADD 0.2 g of sodium pyrophosphate tetra-basic decahydrate and 0.3 g of EDTA tri-sodium salt to 900 ml of ultrapure water, STIR it for 10 min until it is completely mixed. Then ADD 10 ml of 1.0 mol/l Tris-HCl and STIR it for 5 min to make it mixed. Then ADD 0.1 ml of Tween-80 and STIR it for 10 min to mix it (Tween-80 has a high viscosity, so be careful when taking it). Finally, USE ultrapure water to dilute it to 1000 ml, ADJUST the ph to 7.4 ± mol/l hydrochloric acid solution mol/l sodium hydroxide solution Pure methanol DAPI storage solution: In a flask containing 1 mg of 4' 6-diamino-2- phenylindole (DAPI), INJECT 500 μl of pure methanol (2 mg/l). PRESERVE it in darkness at 4 C for 15 days DAPI staining solution: USE 50 ml of PBS to dilute 10 μl of DAPI parent solution, PREPARE it every day, STORE it in a dark place at 4 C g/l sodium hypochlorite solution Alkaline detergents Pure Giardia lamblia cyst: The concentration is 100 cysts/ml, it can be stored at 4 C for 2 months Pure Cryptosporidium parvum Oocysts: The concentration is 100 oocysts/ml, it can be stored at 4 C for 2 months. Buy True-PDF Auto-delivery. Page 41 of 55

14 i TURN on the pump, EMPTY the filter bag as soon as possible. After filtration, it shall place the filter bag in a dark place at 4 C for preservation, generally not more than 72 h. C Elutriation a REMOVE the ethylene plug from the inlet of the filter bag, USE the measuring cylinder to add approximately 110 ml of elutriation buffer solution to the outer chamber of each filter bag. b INSERT the filter bag into the clamp with the arm horizontal shaker. The outlet valve of the filter bag is at the 12 o'clock position. c TURN on the oscillator switch, SET the speed to 80% of the maximum speed, then OSCILLATE the sample for 10 minutes. d POUR the elutriation solution from the filter bag into a 175 ml conical centrifuge tube, USE 110 ml of elutriation buffer solution to refill the external cavity of the filter bag. e INSERT the filter into the clamp of the oscillator. This time, the position of the outlet valve is 90 degrees rotated along its axis from its original position. At 80% power, OSCILLATE it for another 10 minutes. f REPEAT the step d, carefully REMOVE the vinyl cap, POUR the elutriation solution from the filter bag into a 175 ml conical centrifuge tube. D Concentration a CENTRIFUGE the 175 ml centrifuge tube which contains the 1500 g of elutriation sample for 15 min. SLOW down naturally so as not to disturb the sediment. b Carefully USE pipette to remove the supernatant, so that the supernatant is just above the sediment (do not disturb the sediment). c If the volume of the compacted sediment is less than or equal to 0.5 ml, ADD reagent water to the centrifuge tube to make its total volume reach to 10 ml. PLACE the test tube in a rotary stirrer for 10 s ~ 15 s, to resuspend the sediment. d If the volume of the compacted sediment is greater than 0.5 ml, USE the formula (3) to determine the total volume required in the centrifuge tube: Total required volume (ml) = volume of sediment 10 ml/0.5 ml... (3) In order to adjust the resuspended sediment to a compacted sediment volume Buy True-PDF Auto-delivery. Page 43 of 55

15 plug to allow the effluent to concentrate to 30 ml ~ 40 ml. Gently POUR the concentrate into a 50 ml centrifuge tube. 3) Second elutriation. ADD 600 ml of PBST buffer solution to the concentration tube again, then CONNECT it to the elutriation tube. REPEAT the first elutriation process for only 10 times. 4) Second elutriation solution concentration. ADD first concentrate to the second elutriation solution. REPEAT the concentration process as described above. 5) TRANSFER the 3 μm membrane filter to the provided bag, ADD 5 ml of PBST buffer solution, USE hands to gently rub the membrane filter from outside the bag. CLEAN it for 2 times in this way. MIX the cleaning solution with the concentrate. b Automatic elutriation steps 1) First elutriation. PLACE the 3 μm filter into the concentrator, ASSEMBLE the concentration tube and the elutriation tube. TURN on the power of the automatic elutriator. REMOVE the filter module from the bracket, INSTALL it on the top of the piston of the elutriator. USE a quick connector to connect the throat of the elutriator to the elutriation tube. PRESS down the F1 key on the control panel, REMOVE the bolts from the filter module. Then CONNECT the stainless-steel siphon again. INJECT 600 ml of PBST buffer solution into the concentration tube, then CONNECT it to the quick connector. PRESS down the F1 key to start the first infiltration, then PRESS the F3 key for the first elutriation. REMOVE the concentration tube, PRESS the F4 key to remove any residual liquid from the filter. 2) First elutriation solution concentration. CONNECT the concentration tube to the magnetic stir bar, PLACE it on a magnetic stir plate, STIR it at 60 r/mm ~ 120 r/min. CONNECT the vacuum pump to the concentrator to create a vacuum at a pressure of 13.3 kpa ~ 40.0 kpa. OPEN the live plug to allow the effluent concentrate to 30 ml ~ 40 ml. Gently POUR the concentrate into a 50 ml centrifuge tube. 3) Second elutriation. ADD 600 ml of PBST buffer solution to the concentration tube again, then CONNECT it to the elutriation tube. PRESS down the F3 key to start the elutriation. REMOVE the concentration tube, PRESS the F4 key to remove any residual liquid from the filter. 4) Second elutriation solution concentration. ADD first concentrate to the second elutriation solution. REPEAT the concentration process as described above. Buy True-PDF Auto-delivery. Page 45 of 55

16 K PLACE the microcentrifuge tube in another magnetic particle concentrator (MPC-M). The MPC-M has a magnetic strip at the microcentrifuge tube. L USE hands to gently shake the test tube at 180. SHAKE about 180 degrees per second for about 1 minute. At the end of this step, beads and oocysts will form a brown dot on the back of the test tube. M Immediately SUCK out the supernatant from the test tubes and caps left on the MPC-M. If more than one sample is processed at the same time, three 180- degree shaking or rolling motions are performed before the supernatant of each tube is sucked out. BE careful not to disturb the attachment to the wall adjacent to the magnet. DO not shake the test tube. When performing these steps, DO not remove the tube from the MPC-M Separation of magnetic beads from sporocyst/oocyst complex A REMOVE the magnetic strip from the MPC-M. B ADD 50 μl of 0.1 mol/l hydrochloric acid (HCl) to the above microcentrifuge tube, VORTEX it for 10 s. C PLACE the test tube on the MPC-M, LET it rest vertically for 10 min at room temperature. D Forcefully VORTEX it for 5 s ~ 10 s. E ENSURE that all samples are at the bottom of the test tube, then PLACE the microcentrifuge tube on the Dynal MPC-M. F PLACE the magnetic strip on the MPC-M again, USE hands to gently shake the test tube which is head-to-end connected with an angle about 90 to the ground, so that the cap top and substrate of the test tube are alternatively inclined up and down. MAINTAIN the inclination frequency approximately once per second for 30 s. G PREPARE a well-shaped slide, ADD 5 μl of a 1 mol/l sodium hydroxide (NaOH) solution to the sample well. H DO not remove the microcentrifuge tube from the MPC-M. TRANSFER all samples from microcentrifuge tubes on MPC-M to sample wells which contain sodium hydroxide. DO not disturb the beads on the back wall of the test tube. I REPEAT steps A-F, then TRANSFER the sample to the same well-shaped slide Dyeing PLACE the well-shaped slides with sample in a 42 C incubator, Buy True-PDF Auto-delivery. Page 49 of 55

17 EVAPORATE it dry ADD one drop (50 μl) of pure methanol to each well which contains the dry sample, LET it dry naturally for 3 min ~ 5 min USE the test tube to prepare the required volume (50 μl per well) of anti-cryptosporidium antibody and anti-giardia monoclonal antibody fluorescein isothiocyanate (FITC) working dilution (1/1: Cellabs/PBS) in a test tube ADD 50 μl of the above fluorescein isothiocyanate (FITC) monoclonal antibody working dilution into the well which contains sample. PLACE the slides in a wet chamber to incubate it at 37 C for about 30 minutes After 30 minutes, TAKE out the slide, USE a clean Pasteur pipette which has a vacuum source at the top end to gently pipette off the excess fluorescein-labeled monoclonal antibody from each side of the well ADD 70 μl of PBS to each well. After 1 min ~ 2 min of quiescence, SUCK out excess PBS ADD 50 μl of DAPI solution (prepared at the time of use, that is, add 10 μl of 2 mg/ml DAPI which is dissolved in pure methanol to 50 ml of PBS) to each well, LET it stand at room temperature for about 2 min SUCK out excess DAPI solution ADD 70 μl of PBS to each well. After 1 min ~ 2 min of quiescence, SUCK out excess PBS ADD 70 μl of reagent water to each well. After standing for 1 min, SUCK out excess reagent water After allowing the slides to dry in the dark, ADD a drop of mounting medium which prevents the fluorescence from weakening to the center of each well COVER the well slide with a coverslip, STORE it in a dry cassette for future reference Microscope TURN on the microscope and mercury lamp. After preheating for 10 min, CHECK it under a 200x fluorescence microscope, MAKE further confirmation under a 400x fluorescence microscope. COUNT the number for all wells. The sporocyst of Giardia is oval in shape. They have a length of 8 μm ~ 14 μm and a width of 7 μm ~ 10 μm. The sporocyst wall emits green apple Buy True-PDF Auto-delivery. Page 50 of 55