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1、INTERNATIONAL STANDARD IS0 10705-I First edition 1995-08-01 Water quality - Detection and enumeration of bacteriophages - Part 1: Enumeration of F-specific RNA bacteriophages Qua/it 9 mm) and kanamycin (100 pg; 9 mm). 6.3.3 Glycerol, 870 g/litre. 6.4 Microbiological reference cultures Salmonella typ
2、himurium strain WG49, phage type 3 Nal (F 42 /ac:Tn5), NCTC 12484. Bacteriophage MS2, NCTC 12487 or ATCC 15597-Bl . Escherichia co/i K-l 2 Hfr from appropriate culture col- lection, e.g. NCTC 12486 or ATCC 23631. NOTE 1 The NCTC strains are available from the National Collection of Type Cultures, 61
3、 Colindale Avenue, London NW9 6HT, England. The ATCC strains are available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, U.S.A. 7 Apparatus and glassware Usual microbiological laboratory equipment, including 7.1 Hot-air oven for dry-heat sterilization and an a
4、utoclave. Apart from apparatus supplied sterile, glassware and other equipment shall be sterilized ac- cording to the instructions given in IS0 8199. 7.2 Incubator or water bath, thermostatically controlled at 37 “C + 1 “C. 7.3 Incubator or water bath, thermostatically controlled at 37 “C f 1 “C and
5、 equipped with a rotary platform at 100 min- + 10 min- . 7.4 Water bath, thermostatically controlled at 45 “C f 1 “C. 7.5 Water bath or equivalent device, for melting agar media. 7.6 pH-meter. 7.7 Counting apparatus, with indirect, oblique light. 7.8 Deep freezer, thermostatically controlled at - 20
6、 “C * 5 “C. 2 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/22/2007 20:45:15 MDTNo reproduction or networking permitted without license from IHS -,-,- Q IS0 IS0 10705-1:1995(E) 7.9 Dee
7、p freezer, - 7O”C;tff-?;) Log-phaseculture , INOCULUMCULTURE Store on melting ice, use within 2 h (10.1) Figure 2 - Scheme for culturing, maintenance and quality control of host strain WG49 4 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NAS
8、A Technical Standards 1/9972545001 Not for Resale, 04/22/2007 20:45:15 MDTNo reproduction or networking permitted without license from IHS -,-,- 0 IS0 IS0 10705-1:1995(E) 9.1.2 Preparation of working cultures 9.3 Quality control of host strain WG49 Thaw one vial of stock culture (9.1 .I) at room tem
9、- perature and inoculate on a plate of McConkey agar (A.71, or another lactose-containing medium, in such a way that single colonies will be obtained. Incubate at37”C1”Cfor18hf2h.Add50mlofTYGBto (A.11 a 300 ml conical flask (7.16) and warm to room temperature. Select three to five lactose-positive c
10、ol- onies from the McConkey agar and inoculate material from each of these colonies in the flask with TYGB. Incubate for 5 h f 1 h at 37 “C f 1 “C while shaking at 100 min- + 10 min- . Add 10 ml of glycerol (A.61 and mix well. Distribute into plastics vials (7.19) in I,2 ml aiiquots and store at - 7
11、0 “C + 10 “C for a maximum of 2 years. Control the quality of the work- ing culture according to 9.3. NOTES Use a culture as prepared in 9.2. At times t = 0 h and t = 3 h, also inoculate two plates of McConkey agar (A.71, or another lactose-containing medium with the same dilution series, and incuba
12、te at 37 “C + 1 “C for 24 h f 2 h. From plates yielding between 30 and 300 colonies, count the number of lactose-positive and lactose-negative colonies and calculate the percentage of lactose-negative colonies. At times t = 0 h and t = 3 h, spread 0,l ml of the IO- dilution on a plate of McConkey ag
13、ar or alterna- tive, place one disk with nalidixic acid (Nal) and one disk with kanamycin (Km) on the plates and incubate for 24 h + 2 h at 37 “C + 1 “C. Measure inhibition zones around the antibiotic disks. 3 If a great number of tests is anticipated, several conical flasks can be inoculated in par
14、allel. 4 If quality control fails, prepare new inocula from the stock culture. After repeated failures, or if the stock culture is depleted, obtain a new lyophilized ampoule of the refer- ence culture. Do not subculture repeatedly in the laboratory. The host strain is acceptable if the following cri
15、teria are met: plate count on TYGA (9.2) at 0 h: 0,5 to 3 x IO cfp/ml; plate count on TYGA (9.2) at 3 h: 7 to 40 x IO cfp/ml; 9.2 Calibration of turbidity measurements lactose-negative colonies (plasmid segregation) 80 %. 10.2 Method for samples with high bacterial background flora Add nalidixic aci
16、d to ssTYGA (A.3) until a final con- centration of 100 pg/ml is obtained. NOTE 10 Nalidixic acid is stable when heated. It can ei- ther be added from a filter-sterilized solution (A.4) (0,2 ml/50 ml) after melting of sslYGA or can be added to T/GA before autoclaving. 10 Procedure 10.3 Confirmatory t
17、est 10.1 Standard procedure Take one vial of working culture from the freezer and thaw it at room temperature. Add 50 ml of TYGB (A.1) to a nephelometric conical flask (7.17), or plain conical flask (7.16). Adjust the spectrometer reading to 0 as described in 9.2 and prewarm to room tem- perature. I
18、noculate 0,5 ml of working culture. Incu- bate at 37 “C + 1 “C while shaking at 100 min- * 10 min- . Measure turbidity every 30 min. At a turbidity corresponding to a cell density of approxi- mately IO8 cfu/ml (based on data obtained in 9.2), take the inoculum culture from the incubator and quickly
19、cool on melting ice. Use within 2 h. NOTE 7 It is essential that the culture is quickly cooled to prevent loss of F-pili by the cells, which will negatively influence recovery. Melt bottles of ssTYGA (A.3), cool to 44 “C to 50 “C, aseptically add calcium-glucose solution (A.1 ) (0,5 ml/50 ml) and di
20、stribute 2,5 ml aliquots into cul- ture tubes with caps, placed in a water bath at 45 “C f 1 “C. To each tube, add 1 ml of sample (or dilution or concentrate). Examine each volume or di- lution step at least in duplicate. Add 1 ml of inoculum culture, mix carefully and pour the contents over the sur
21、face of a 9 cm TYGA plate (A.2). Distribute evenly, allow to solidify on a perfectly horizontal, cool surface and incubate the plates upside-down at 37 “C + 1 “C for 18 h + 2 h. NOTES 8 Do not stack more than 6 (preferably 4) plates. 9 The addition of ice-cold sample and host culture to the top-agar
22、 may lead to a sharp drop in temperature and solidification of the medium. Assure a sufficient time inter- val between these two steps to allow reheating. However, make sure that inoculated tubes remain in the water bath for not more than 10 min. Count the number of plaques appearing on each plate w
23、ithin 4 h, using indirect oblique light. In parallel with the series of plates described under 10.1, prepare a similar series with RNase-solution (A.5) added to the tubes of ssTYGA until a final con- centration of 40 pg/ml is obtained (i.e. 100 1 of RNase solution to 2.5 ml of ssTYGA in a tube). NOT
24、ES 11 Confirmatory tests should at least be carried out a) when examining new sampling points; b) regularly at fixed sampling points when NRNase/N (see clause 11) is usually less than 10 %; c) always at fixed sampling points when NRNase/N is usually 10 %; d) if large, circular, clear plaques with sm
25、ooth edges (probably somatic Salmonella phages) are regularly seen. 12 In rare cases, RNA-phages may not be inhibited by RNase at 40 pg/ml and it may be necessary to increase the concentration of RNase to 400 pg/ml. 10.4 Samples with low phage counts Proceed according to 10.1 but with the following
26、modifications: - IO ml of ssTYGA, 1 ml of host culture and 5 ml of sample in duplicate per dilution step; - pour over 50 ml of TYGA in a 14 cm Petri dish. NOTE 13 This procedure will be able to detect up to 1 pfu/50 ml or 100 ml, if 10 or 20 plates are inoculated in parallel. Due to the high consump
27、tion of culture media, it may be advisable to use concentration methods which will also be necessary for even lower counts. 10.5 Quality assurance With each series of samples, examine a procedural blank using sterile diluent as the sample and a stan- 6 Copyright International Organization for Standa
28、rdization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/22/2007 20:45:15 MDTNo reproduction or networking permitted without license from IHS -,-,- f3 IS0 IS0 10705-1:1995(E) dard preparation of MS2 (see 9.3). Plot the results on a control ch
29、art. Optionally, also use a naturally polluted standard sample, taken from sewage or surface water, diluted to approximately 100 pfp/ml in peptone saline solution and glycerol (5 g/l) and stored at - 20 “C + 5 “C or - 70 “C + 5 “C. Discard the standard samples if the concentration of RNA-phages decr
30、eases. NOTE 14 In the absence of easily available standardized reference materials, any programme for the exchange of standard samples between laboratories should be encour- aged. If sensitivity to phages is lost (this is unusual but it may happen very suddenly and completely), prepare a new set of
31、inocula according to 9.1.2. 11 Expression of results Select plates with 30 to 300 plaques. From the num- ber of plaques counted, and taking into account the results of previous confirmatory tests, calculate the number concentration of (plaque-forming particles of) F-specific RNA bacteriophages in 1
32、ml of the sample as follows: CPfP = n N - NRNase x F where C PfP is the confirmed number concentration of F-specific RNA bacteriophages per milli- litre; N is the total number of plaques counted on WG49 plates according to 10.1, 10.2 or 10.4; N RNase is the total number of plaques counted on WG49 pl
33、ates with RNase according to 10.3; n is the number of replicates; F is the dilution (or concentration) factor (l/5 in the case of 10.4). 12 Test report The test report shall contain the following information: a) b) cl d) e) a reference to this part of IS0 10705; all details necessary for complete id
34、entification of the sample; if a confirmatory test was used and the ratio of NRNase to N. as a percentage; the results expressed in accordance with clause 11; any other information relevant to the method. 7 Copyright International Organization for Standardization Provided by IHS under license with I
35、SO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/22/2007 20:45:15 MDTNo reproduction or networking permitted without license from IHS -,-,- IS0 10705-1:1995(E) Annex A (normative) Culture media, reagents and diluents A.1 Tryptone-yeast extract-glucose broth (TYGB) Basal medium Tr
36、ypticase peptone Yeast extract NaCl Distilled water log lg 8g 1 000 ml Dissolve the ingredients in hot water. Adjust the pH so that after sterilization it will be 7,2 f 0,l at 25 “C. Distribute the medium into bottles in volumes of 200 ml, and sterilize in the autoclave at 121 “C + 1 “C for 15 min.
37、Store in the dark at 4 “C + 2 “C for not longer than 6 months. Calcium-glucose solution CaCl,.2H,O Glucose Distilled water 3g log 100 ml Dissolve the ingredients in the water while heating gently. Cool to room temperature and filter-sterilize through a 0,22 pm membrane filter. Store in the dark at 4
38、 “C + 2 “C for not longer than 6 months. Complete medium Basal medium 200 ml Calcium-glucose solution 2 ml Aseptically add calcium-glucose solution to basal me- dium and mix well. If it is not for immediate use, store in the dark at 4 “C f 2 “C for not longer than 6 months. A.2 Tryptone-yeast extrac
39、t-glucose agar (NGN Basal medium Trypticase peptone 10 g Yeast extract Ig NaCl 8g Agar 12 g to 20 g 1) Distilled water 1 000 ml 1) Depending on the gel strength of the agar. Dissolve the ingredients in boiling water. Adjust the pH so that after sterilization it will be 7,2 f 0,l at 25 “C. Distribute
40、 the medium into bottles in volumes of 200 ml, and sterilize in the autoclave at 121 “C f 1 “C for 15 min. Store in the dark at 4 “C f 2 “C for not longer than 6 months. Complete medium Basal medium Calcium-glucose solution (A.11 200 ml 2 ml Melt the basal medium and cool to between 45 “C and 50 “C.
41、 Aseptically add calcium-glucose solution, mix well and pour into Petri dishes as follows: - 20 ml in dishes of diameter 9 cm; - 50 ml in dishes of diameter 14 cm. Allow to solidify and store in the dark at 4 “C f 2 “C for not longer than 6 months, if it is well protected against desiccation. 8 Copy
42、right International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/22/2007 20:45:15 MDTNo reproduction or networking permitted without license from IHS -,-,- Q IS0 IS0 10705-1:1995(E) A.3 Semi-solid tryptone-y
43、east extract-glucose agar (ssTYGA) Prepare basal medium according to A.2 but use half of the mass of agar (6 g to 10 g), depending on gel strength; the gel strength of ssTYGA is critical to ob- tain good results and if possible different concen- trations should be tested. Distribute into bottles in
44、50 ml volumes. A.4 Nalidixic acid solution Nalidixic acid NaOH (1 mol/l) Distilled water 250 mg 2 ml a ml Dissolve nalidixic acid in NaOH solution, add distilled water and mix well. Filter-sterilize through an 0,22 pm membrane filter. Store at 4 “C + 2 “C for not longer than 8 h or at - 20 “C + 2 “C
45、 for not longer than 6 months. A.5 RNase solution RNase Distilled water 100 mg 100 ml Dissolve RNase in water while heating for 10 min at 100 “C. Distribute into plastics cups in 0,5 ml volumes and store at - 20 “C for not longer than 1 year. Thaw at room temperature before use. A.6 Glycerol (steril
46、e) I Glycerol (870 g/l) 100 ml I Distribute into bottles in 20 ml volumes and sterilize in the autoclave at 121 “C + 1 “C for 15 min. Store in the dark for not longer than 1 year. A.7 McConkey agar Peptone 20,o g Lactose JO,0 9 Bile salts 5,o g Neutral red 75 mg Agar 12gto20g Distilled water 1 000 m
47、l Dissolve the ingredients in boiling water. Adjust the pH so that after sterilization it will be 7,4 f 0,l at 25 “C. Distribute the medium into bottles in volumes of 200 ml, and sterilize in the autoclave at 121 “C f 1 “C for 15 min. Cool to between 45 “C and 50 “C and pour 20 ml into Petri dishes
48、of diameter 9 cm. Allow to solidify and store in the dark at 4 “C + 2 “C for not longer than 6 months. A.8 Peptone saline solution Peptone Sodium chloride Distilled water I,0 9 83 9 1 000 ml Dissolve the constituents in about 950 ml of the wa- ter by boiling. Adjust the pH with sodium hydroxide solu
49、tion or hydrochloric acid (1 mol/l), so that after sterilization it will be 7,0 + 0,l. Make up to 1 000 ml with the water, dispense in convenient volumes and autoclave at 121 “C + 1 “C for 15 min. Store in the dark for not longer than 6 months. 9 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Techni