ISO-10705-1-1995.pdf
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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
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