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資料2: Quantitative Analytical Methods of Bacteria Used for Test-bed Tests (MEPC52/2/7)
MARINE ENVIRONMENT PROTECTION
COMMITTEE
52nd session
Agenda item 2
MEPC 52/2/7
9 July 2004
Original: ENGLISH
HARMFUL AQUATIC ORGANISMS IN BALLAST WATER
Quantitative Analytical Methods of Bacteria Used for Test-bed Tests
Proposal for development of the Guidelines for Approval of Ballast Water Management Systems
Submitted by Japan
SUMMARY
Executive summary:
This document contains comments to the draft Guidelines for Approval of Ballast Water Management Systems, including proposal for the quantitative analytical methods of bacteria used for test-bed tests.
Action to be taken: Paragraph 10
Related documents: BWM/CONF/INF.5
Introduction
1 At MEPC 51 the Ballast Water Working Group decided to develop 13 guidelines for the uniform implementation of the International Convention for the Control and Management of Ships' Ballast Water and Sediments. One of these guidelines, Guidelines for Approval of Ballast Water Management Systems, needs documents on quantitative analytical methods of bacteria used for test-bed tests.
2 This document presents a first draft of the analytical methods. The draft, which can be found in the annex to this document, have to be further developed. Japan wishes to finalize the Guidelines for Approval of Ballast Water Management Systems by MEPC 52 and to start type approval process using standardized procedure by the Organization.
Outline of the methods
3 The regulation D-2 of the Convention defines the maximum allowable number of indicator pathogenic bacteria in discharging ballast water. Ballast Water Management Systems must be able to decrease these bacteria, not to exceed the D-2 standard at discharge. But it is not recommended to use the pathogenic bacteria in influent water for evaluation of prototype treatment systems of ballast water. Also it is not recommended to try to detect the specific pathogenic strains during the quantitative analysis, because the procedure contains time consuming steps and need a specific facility to protect human health and public sanitation. Therefore, Japan thinks it necessary and appropriate to make alternative tests composed of two different type of analysis as follows.
.1 First test: termination of heterotrophic bacteria of 104 CFU per milliliter or more in influent water to the level of 102 CFU per milliliter. This test aims to confirm the effectiveness of treatment against bacteria in general.
.2 Second test: confirmation of presence of proxy bacteria of the indicator pathogenic bacteria less than the permitted level described in the D-2. This test aims to observe the effectiveness of the treatment against the indicator pathogenic bacteria.
Heterotrophic bacteria used in the first step
4 Heterotrophic bacteria of 104 CFU per milliliter can be found in natural environment commonly in both marine and fresh waters, and therefore it is not necessary to add any cultured bacteria.
5 As heterotrophic bacteria often recover its number within a day after disappearance of termination stress under eutrophic condition, the termination efficacy shall be measured immediately before and after treatment. It is not necessary to analyze concentration of heterotrophic bacteria at the end of each experiment, because it varies depending on experiment condition.
Proxy bacteria
6 Proxy bacteria is a group of bacteria, which include the pathogenic bacteria. It is easier technically and saves time and costs to make quantitative analysis of proxy bacteria than that of specific pathogenic species.
7 Indicator pathogenic bacteria refered in the regulation D-2 of the Convention are Escherichia coli, Enterococcus and Vibrzo cholerae. Proxy bacteria for them are Coliform, Enterococcus group bacteria, and all clones of Vibrio cholerae (without specifying 01 and 0139), respectively.
8 Any of these proxy bacteria shall not be added to the influent water for test-bed tests. Observation shall focus on presence of naturally occurring proxy bacteria less than the permitted level described in regulation D-2 after treatment.
Proposal
9 Japan proposes the following methods for inclusion to the guidelines as an annex to evaluate treatment efficacy of indicator pathogenic bacteria:
.1 Sampling of test water,
.2 Quantitative analysis of heterotrophic bacteria and proxy bacteria for the indicator pathogenic bacteria.
Action requested of the Committee
10 The Committee is invited to note the proposals in this document, including the text in the annex, and take action as appropriate.
For reasons of economy, this document is printed in a limited number. Delegates are kindly asked to bring their copies to meetings and not to request additional copies.
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ANNEX
DRAFT
QUANTITATIVE ANALYTICAL METHODS OF BACTERIA USED FOR TEST-BED TESTS
1 INTRODUCTION
1.1 The indicator bacteria specified in regulation D-2, Ballast Water Performance Standard, have pathogenic properties and it is not recommended to use such bacteria in influent water for evaluation of prototype treatment systems of ballast water. Also it is not recommended to try to detect the specific pathogenic strains during the quantitative analysis, because the procedure contains time consuming steps and need a specific facility to protect human health and public sanitation. The following two different types of analysis are considered as alternative tests:
.1 First test: termination of heterotrophic bacteria of 104 CFU per milliliter or more in influent water to the level of 102 CFU per milliliter. This test aims to confirm the effectiveness of treatment against bacteria in general.
.2 Second test: confirmation of presence of proxy bacteria of the indicator pathogenic bacteria less than the permitted level described in the D-2. This test aims to observe the effectiveness of the treatment against the indicator pathogenic bacteria.
1.2 Indicator pathogenic bacteria refered in regulation D-2 are Escherichia coli, Enterococcus and Vibrio cholerae. Proxy bacteria for them are Coliform, Enterococcus group bacteria, and all clones of Vibrio cholerae (without specifying 01 and 0139), respectively.
1.3 This draft text describes a method to confirm treatment efficacy on heterotrophic bacteria and proxy bacteria for the indicator pathogenic bacteria used for a test-bed test which is a part of procedures for approval and certification of a single piece of equipment, or a series of equipment (Type Approval) of the Guidelines for Approval of Ballast Water Management Systems (G8).
2 SAMPLING METHODS
2.1 The apparatus for sampling shall be sterilized by burning sterilization, dry heat sterilization, heat sterilization, autoclaving, or filtration depending on their application.
2.2 The samples of cca. 500 ml shall be collected in a sterilized bottle of 500 ml capacity.
2.3 The samples shall be analyzed as soon as possible after collection to minimize changes in bacterial population. The recommended maximum elapsed time between collection and analysis of samples is 8 hours. When analysis cannot begin within 8 hours maintain sample at a temperature below 4℃ and analysis must be started within 24 hours.
3 QUANTITATIVE ANALYTICAL METHODS
3.1 All analyses use either a Pour Plate Method or Spread Plate Method and results on concentration of bacteria are shown using colony forming unit (CFU).
3.2 The standard quantitative analytical methods for heterotrophic bacteria and Coliform are provided in ISO 8199 (1988) and ISO 9308-2 (2000), respectively. These standard methods shall be followed in their analyses. As no standard method is available in ISO for Enterococcus group and Vibrio cholerae, methods desribed in the paragraph 3.3 are recomended.
3.3 At least five replicate agar plates shall be used for one analytical chance for each bacterium.
3.4 Medium, incubate condition and characters of colony
3.4.1 Heterotrophic bacteria
Medium: Plate Count Agar/Standard Methods Agar, PYG agar, R2A agar, Nutrient agar, Trypticase soy agar (Trypticase glucose extract agar), Eugon agar, Marine Agar.
Prepare one of these medium by using distilled water for analysis of fresh water bacteria and artificial seawater for marine bacteria.
Incubate condition: temperature may be determined arbitrarily within a range of 10 to 35℃ by fitting it to water temperature during test-bed tests. All culture must be incubated within ±0.5℃ of the set temperature.
Character of colony : various depending on bacteria species
3.4.2 Coliform
Medium: MacConckey II Agar with MUG, Violet Red Bile Agar with MUG or Nutrient Agar with MUG
Incubate condition: 35 to 37℃ for 4 hours
Character of colony: colonies exhibit blue fluorescence under UV irradiation
3.4.3 Enterococcus group
Medium: M-Enterococcus agar
Incubate condition: 36℃ for 48±3 hr
Character of colony: transparent colonies surrounded by a dark brown or black zone
3.4.4 Vibrio cholerae
Medium: TCBS plate medium (thiosulfuric acid, citric acid, bile acid, white sugar)
Incubate condition: 35℃ for 24-48 hr
Character of colony: yellow colonies
3.5 The following are two examples of methods commomly used for analysis of bacteria. Choose an appropriate method in accordance with the laboratory facilities. Methods can be modified within reason. If other methods are available, such as standard analytical methods of waste water, they can be used as long as scientific comparability is kept. However, same method shall be used for a series of tests to take comparable data.
3.5.1 Pour plate method
The sample water admixes with agar medium in a Petri dish or a test tube, and the number of bacterium colony grown in and on the agar medium is counted.
3.5.1.1 Prepare a 10-times dilutions series of water sample. Dilution strength must be decided depending on the expecting concentration of bacteria in the water sample.
3.5.1.2 Inoculate 1 ml of water sample of the series to the center of a empty sterilized Petri dishes.
3.5.1.3 Pour 15 to 20 ml liquefied medium maintained at 45 to 50℃ into each Petri dish, and mix melted medium thoroughly with sample or dilution.
3.5.1.4 After medium solidifies, invert plates and place in incubater.
3.5.1.5 Alternatively, dilute 9 to 19 ml of agar medium in a test tube that has been kept at a temperature between 45 and 50℃ with 1ml of each dilution of the series 3.5.1.1, pour the mixture onto a Petri dish.
3.5.1.6 After the medium solidifies, invert plates and place in incubater.
3.5.1.7 Count the number of colonies that have been formed in and on each plate after a specific incubation period. Select the final count plates so that the total number colonies on a plate is between 20 to 200 colonies. Calculate the number of bacteria per ml of sample from the number of colonies per plate, the volume of the collected sample, and the dilution ratio.
3.5.2 Spread-plate method
Place the sample water onto surface of the agar medium and calculate the number of grown colony of bacteria formed after cultivation.
3.5.2.1 Prepare a 10-times dilution series of water sample. Dilution strength must be decided depending on the expecting concentration of bacteria in the water sample.
3.5.2.2 Add 15 to 20 ml of agar medium, dissolved by heating, into a sterilized Petri dishes, allow it solid by cooling, and predry the surface appropriately by keeping them in an incubator for a few hours.
3.5.2.3 Drop 0.1ml of water sample or dilution prepared by the step 3.5.2.1 onto the centers of appropriately predried agar plates using a sterilized pipette and spread it evenly over surface area of the plate using a sterilized spreader.
3.5.2.4 Cultivate bacteria by setting the plate upside down.
3.5.2.5 Count colonies grown on the agar plate, following the method described in 3.5.1.7.
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