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1、JIS JAPANESE I NDIJSTR IAL STANDARD Translated and Published by Japanese Standards Association JIS K 0350-30-10 :zoo2 Testing methods for detection and enumeration of heterotrophic bacteria in industrial water and wastewater ICs 13.060.70 Reference number : JIS K 0350-30-10 : 2002 (E) PROTECTED BY C
2、OPYRIGHT Copyright Japanese Standards Association Provided by IHS under license with JSALicensee=IHS Employees/1111111001, User=Wing, Bernie Not for Resale, 03/12/2007 08:27:12 MDTNo reproduction or networking permitted without license from IHS -,-,- K 0350-30-10 : 2002 Foreword T h i s translation
3、has been made based on the original Japanese Industrial Standard established by the Minister of Economy, Trade and Industry through deliberations at the Japanese Industrial Standards Committee according to the proposal of establishing a Japanese Industrial Standard from the Japan Industrial Water As
4、sociation (JIWA)/the Japanese Standards Association (JSA), with a draft of Industrial Standard based on the provision of Article 12 Clause 1 of the Industrial Standardization Law. JIS K 0350 consists of the following four parts. JIS K 0350-10-10 Testing methods for standard plate counts of hetero- t
5、rophic bacteria in industrial water and wastewater JIS K 0350-20-10 Testing methods for detection and enumeration of coliform organisms in industrial water and waste- water JIS K 0350-30-10 Testing methods for detection and enumeration of heterotrophic bacteria in industrial water and waste- water J
6、IS K 0350-40-10 Testing method for enumeration of total bacteria in industrial water and wastewater Date of Establishment: 2002-03-20 Date of Public Notice in Official Gazette: 2002-03-20 Investigated by: Japanese Industrial Standards Committee Standards Board Technical Committee on Environment and
7、Recycling Policy JIS K 0350-30-10 : 2002, First English edition published in 2002-12 Translated and published by: Japanese Standards Association 4-1-24, Akasaka, Minato-ku, Tokyo, 107-8440 JAPAN In the event of any doubts arising as to the contents, the original JIS is to be the final authority. O J
8、SA2002 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from the publisher. Printed in Japan PROTECTED BY COPYRIGHT Cop
9、yright Japanese Standards Association Provided by IHS under license with JSALicensee=IHS Employees/1111111001, User=Wing, Bernie Not for Resale, 03/12/2007 08:27:12 MDTNo reproduction or networking permitted without license from IHS -,-,- JAPANESE INDUSTRIAL STANDARD JIS K 0350-30-10 : 2002 Testing
10、methods for detection and enumeration of heterotrophic bacteria in industrial water and wastewater 1 Scope This Japanese Industrial Standard specifies testing methods for detec- tion and enumeration of heterotrophic bacteria in industrial water and wastewater. 2 Normative references The standards gi
11、ven in Attached Table 1 contain pro- visions which, through reference in this Standard, constitute provisions of this Standard. The most recent editions of the standards (including amendments) shall be applied. 3 Definitions For the purposes of this Standard, the definitions shall be in ac- cordance
12、 with JIS K 0101, JIS K 0102, JIS K 0550 and JIS K 0211, and the fol- lowing definition shall apply. 3 . 1 heterotrophic bacteria Heterotrophic bacteria here refer to all bacteria which form colonies in the culture medium when culturing at 25 k 1 “C for 7 days using the culture medium of which nutri
13、tious substance content is comparatively low. 4 Matters in common Matters in common shall be as follows: 4 . 1 accordance with JIS K 0050. General rules The general matters common to chemical analysis shall be in 4 . 2 fied in JIS K 0557. Water Water used in this Standard shall be A2 water or A3 wat
14、er(1) speci- Note (1) The metal-made still shall not be used. Reagents The reagent specified in JIS shall be used. When there is no reagent specified in JIS, the reagent giving no hindrance to the test shall be used. For the concentration of the solution of reagents, in general, the mass concen- tra
15、tion shall be expressed in the unit of g/L or mg/L and for the molality, in the unit of mom or m mol/L. In addition, on treating compounds, the mass as anhydride shall be used. The concentration indicated in parentheses following the name of the solution of reagents refers to the rough value of conc
16、entration except in the case of a reference solution. For example, sodium hydroxide solution (3. morn) refers to sodium hydroxide solution of approximately 1 mol/L. The concentration of liquid reagents shall be expressed in the mixture ratio re- agents (a + b) with water (or other liquid reagents).
17、This expression indicates that reagents of a ml and water (or other liquid reagents) of b ml are mixed. In addition, when liquid reagents (for example, hydrochloric acid) specified in Table 3 of JIS K 0050 are used without dilution, only the names of reagents shall be stated. PROTECTED BY COPYRIGHT
18、Copyright Japanese Standards Association Provided by IHS under license with JSALicensee=IHS Employees/1111111001, User=Wing, Bernie Not for Resale, 03/12/2007 08:27:12 MDTNo reproduction or networking permitted without license from IHS -,-,- 2 K 0350-30-10 : 2002 e) f) Water to be used for the prepa
19、ration of reagents shall be that of 4.2. The names of reagents shall be coordinated as much as possible with those men- tioned in the nomenclature of compounds specified by the Chemical Society of Japan and in JIS reagents, which are based on the nomenclature system for inorganic compounds and for o
20、rganic compounds prescribed by International Union of Pure and Applied Chemistry (IUPAC). Care shall be sufficiently taken in accordance with the related laws and regu- lations for treating reagents and wastewater and the like. g) 4.4 Sterilizing procedures of implement and the like Sterilizing proc
21、edures of implement and the like shall be carried out as follows: a) Dry heat sterilization This is used for sterilization of glass-made and metal- made implements. Sterilize them at approximately 170 “C for approximately 1 h. High-pressure steam sterilization This is used for sterilization of cultu
22、re media, dilution water, a sample container, culture media that have been used, etc. Sterilize them at 121 “C for 15 min to 20 min. Flame sterilization This is used for the sterilization of a test tube and the mouth part of a flask. Sterilize them by keeping the mouth part aslant in flame and turni
23、ng for a while, after and before cultivating operation. b) c) 4.5 lows: Disinfection operations Disinfection operations shall be carried out as fol- a) Before and after test operation, disinfect fingers and a testing bench. Use cresol soap solution (10 g/L), cationic soap solution (i g/L to 10 g/L),
24、 or alcohol for dis- infection (which is specified in the Japanese Pharmacopoeia) or ethanol (80 vol%) ethanol (95) specified in JIS K 81021, for disinfection of fingers. For the disinfection of a testing bench, either spray cationic soap solution (10 g/L), alcohol for disinfection or ethanol (80 vo
25、l%), etc, or wipe with the cloth moistened with these solutions. Employed implements such as a pipette, a sample container, a water sampler, and so on, shall be immersed in disinfection solution such as cresol soap solu- tion (30 g/L to 50 g/L) and the like for 1 day (or shall be sterilized by high-
26、pres- sure steam), and washed with water sufficiently until disinfection solution is completely removed. Sterilize the test tube, petri dish or the like, which has been used for culture test, by high-pressure steam method in 4.4 b) together with culture media, then throw away the culture media, and
27、wash sufficiently with water. Note (2) Discard used plastic petri dish or the like after adequate sterilizing. b) c) 4.6 Glass ware implements For glass ware implements, those which are speci- fied in JIS R 3503 and JIS R 3505 shall generally be used. However, when special implements are required, o
28、ne example of them for each item shall be illustrated or explained. Moreover, when heating operations are involved, the borosilicate glass-1 specified in JIS R 3503 shall be used. PROTECTED BY COPYRIGHT Copyright Japanese Standards Association Provided by IHS under license with JSALicensee=IHS Emplo
29、yees/1111111001, User=Wing, Bernie Not for Resale, 03/12/2007 08:27:12 MDTNo reproduction or networking permitted without license from IHS -,-,- 3 K 0350-30-10 : 2002 5 Sample 5 . 1 sampler. Sampling Sampling shall be carried out using a sample container or water 5.1.1 Implements Implements shall be
30、 as follows: a) Water sampler A Heyroth water sampler(3). The water sampler, put in a portable box, shall be sterilized by dry heat sterilization in 4.4 a). Fig. 1 shows an example. Note (3) For sampling from the depth at which sampling with a Heyroth water sampler is difficult, a Vandorn water samp
31、ler shall be used. In this case, transfer the sample to the sample container in b). . A : Glass-made sample B : Stopper C : Chain D : Chain for stopper opening E : Chain for metal fittings of bottle holder plate F : Bottle holder plate G : Portable box H : Cover of portable box I : Weight container
32、(100 mi) Fig. 1 An example of a Heyroth water sampler b) Sample container 100 ml glass bottle with a ground stopper. Carry out dry heat sterilization in 4.4 a) or high-pressure steam sterilization in 4.4 b) on the sample container(4) whose stopper part and neck part have been covered with aluminium
33、foil (or parchment paper), etc. Alternatively, a sterilized polyethyl- ene bottle for bacteria test may be used. Take care so that the sample con- tainer is not subjected to contamination until sampling begins. Note (4) When sampling the water which contains oxidizing material such as re- sidual chl
34、orine, place 20 mg to 30 mg of sodium thiosulfate pentahydrate (powdered) specified in JIS K 8637 into the sample container, and then sterilize it by high-pressure steam method in 4.4 b) or by Oxirane (eth- ylene oxide) method. The commercially available sterilized polyethylene bottle for bacte- ria
35、 test filled with sodium thiosulfate beforehand may be used. PROTECTED BY COPYRIGHT Copyright Japanese Standards Association Provided by IHS under license with JSALicensee=IHS Employees/1111111001, User=Wing, Bernie Not for Resale, 03/12/2007 08:27:12 MDTNo reproduction or networking permitted witho
36、ut license from IHS -,-,- 4 K 0350-30-10 : 2002 5 . 1 . 2 Operations Sampling shall be carried out as follows: Sampling of surface water When the surface water in lake and marsh, river, waterway, overflow, water tank, etc. can be directly sampled, take the sample into a sample container. On this occ
37、asion, care shall be taken so that the wa- ter touched with hands and fingers is not sampled. When the surface water can not be sampled directly, take it with a water sampler(3). Sampling water at definite depth Take the water at a definite depth using a water sampler(3). Sampling from water tap Whe
38、n the material of water tap is proof against the flame sterilization, sterilize a water tap by the flame sterilization in 4.4 c), in advance of opening the tap, and after discharging the water in the pipe suf- ficiently, take water into a sample container. When the flame sterilization is not practic
39、al, remove the dirt on the periph- ery and inside of water tap, and disinfect with ethanol (80 vol%), etc. in ad- vance. Open the tap and after discharging the water in the pipe sufficiently, take water into a sample container. Sampling from piping and apparatus Similarly to c), take water into a sa
40、mple container. Handling of sample Carry out the test immediately after sampling. When test is not carried out immediately, preserve it in a dark place at O “C to 5 “C (do not freeze) and carry out the test within 9 h. 6 Testing method For the test of heterotrophic bacteria, culture a certain amount
41、 of sample or diluted sample added to two or more petri dishes at 25 +_ 1 “C for 7 days using the agar culture media (PGY agar culture medium or R2A agar culture me- dium), and count the number of colonies formed on and inside the culture media. 6 . 1 Reagents and culture media Reagents and culture
42、media shall be as follows: 6 . 1 . 1 Water In accordance with 4.2. 6 . 1 . 2 Dilution water Physiological saline solution or phosphate buffer solution (pH 7.2) shall be used. Physiological saline solution Dissolve 8.5 g of sodium chloride specified in JIS K 8150 in water to make total 1 L. Transfer
43、the proper amount (approxi- mately half the container content) of this solution into a glass bottle or an Er- lenmeyer flask in 6 . 2 . 2 , stopper it, then carry out the high-pressure steam sterilization in 4.4 b) for approximately 15 min. Phosphate buffer solution (pH 7 . 2 ) Dissolve 34 g of pota
44、ssium dihydrogen- phosphate specified in JIS K 9007 in approximately 500 ml of water, drip so- dium hydroxide solution (1 mol/L) (Prepare using sodium hydroxide specified in JIS K 8576) to adjust the pH value to 7.2, and add carbonic acid free-water specified in JIS K 0557 to make total 1 L. Take 1.
45、25 mL of this solution, and add water to make total 1 L. Carry out the same sterilizing procedure as that in a) on this solution. PROTECTED BY COPYRIGHT Copyright Japanese Standards Association Provided by IHS under license with JSALicensee=IHS Employees/1111111001, User=Wing, Bernie Not for Resale,
46、 03/12/2007 08:27:12 MDTNo reproduction or networking permitted without license from IHS -,-,- 5 K 0350-30-10 2002 6 . 1 . 3 Agar culture media PGY agar culture medium or R2A agar culture me- dium shall be used. a) PGY agar culture medium Add 2 g of peptone (Use pancreatin hydrolyzate of casein), 1
47、g of yeast extract (powdered), 0.5 g of D(+)-glucose specified in JIS K 8824 and 15 g of agar (powdered) specified in JIS K 8263 in 1 L of water, and heat to dissolve them. Adjust the pH value after sterilization to 7.0 f 0.1 using sodium hydroxide solution (i molk) or hydrochloric acid (i mom) (Pre
48、- pare using hydrochloric acid specified in JIS K 8180). Transfer a proper amount to a large test tube (30 mm X 200 mm) or an Er- lenmeyer flaskP), and stopper it with a metal, plastics, or silicone sponge stop- per which is proof against the high-pressure steam sterilization, carry out the high-pre
49、ssure steam sterilization in 4 . 4 b) for approximately 15 min, and after sterilization, use it for testing, keeping it at approximately 50 OC. When pre- serving, put it in a cold and dark place with care to prevent dehydration. Do not use the culture medium after long term preservation. R2A agar culture medium After adding 0.5 g of pro