阅读说明：本技术 一种水质中微生物的测定方法 (Method for measuring microorganisms in water ) 是由 赵爱英 张妍楠 常青 王富强 姚学文 陈巧霞 席子涵 焦小霞 董芳芳 于 2021-03-18 设计创作，主要内容包括：本发明涉及微生物检测技术领域,具体涉及一种水质中微生物的测定方法,包括以下步骤；1)培养基的制备；2)接种：菌落总数的接种以无菌操作方法用灭菌吸管吸取1ml充分混匀的水样,注入灭菌平皿中,倾注约15ml融化并冷却到40-50℃的营养琼脂培养基,旋摇平皿,使水样与培养基充分混匀。同时做平行及空白对照；总大肠菌群的接种为取10ml水样接种到10ml双料乳糖蛋白胨培养液中；3)培养；4)记录及结果观察：菌落总数结果采用直接计数；总大肠菌群若初发酵有产酸产气现象则进行进一步分离培养,在EMB平板上有典型菌落特征则进行证实试验并对照MPN表查询,确定最终检测结果。(The invention relates to the technical field of microorganism detection, in particular to a method for determining microorganisms in water quality, which comprises the following steps; 1) preparing a culture medium; 2) inoculation: inoculating the total number of colonies, using a sterile pipette to absorb 1ml of fully mixed water sample, injecting the water sample into a sterile plate, pouring about 15ml of nutrient agar culture medium which is melted and cooled to 40-50 ℃, and rotating the plate to fully mix the water sample and the culture medium. Parallel and blank comparison is carried out at the same time; the inoculation of total coliform group bacteria is to take 10ml of water sample to inoculate into 10ml of two-material lactose peptone culture solution; 3) culturing; 4) recording and result observation: directly counting the total number of the colonies; and (3) further performing separation culture if the total coliform group has the phenomenon of acid and gas production in the initial fermentation, performing a confirmation test if the EMB plate has typical colony characteristics, and inquiring according to the MPN table to determine the final detection result.)

1. A method for measuring microorganisms in water, which is characterized by comprising the following steps:

1) preparation of a culture medium: the culture medium of the total number of the bacterial colonies is nutrient agar, and the culture medium of the coliform is mainly prepared by lactose peptone, an EMB flat plate and the like and is the same as the nutrient agar;

2) inoculation: inoculating the total number of colonies, using a sterile pipette to absorb 1ml of fully mixed water sample, injecting the water sample into a sterile plate, pouring about 15ml of nutrient agar culture medium which is melted and cooled to 40-50 ℃, and rotating the plate to fully mix the water sample and the culture medium. Parallel and blank comparison is carried out at the same time; the inoculation of coliform group bacteria is to take 10ml of water sample to inoculate into 10ml of two-material lactose peptone culture solution, take 1ml of water sample to inoculate into 10ml of single-material lactose peptone culture solution, take another 1ml of water sample to inject into 9ml of sterilized normal saline, absorb 1ml (namely 0.1ml of water sample) to inject into 10ml of single-material lactose peptone culture solution after mixing evenly, each dilution inoculates 5 tubes;

3) culturing: putting the sample in an incubator at 36 +/-1 ℃, culturing the total number of colonies for 48h, and culturing coliform groups for 24h and 2 h;

4) recording and result observation: directly counting the total number of the colonies; and (3) further performing separation culture if the coliform group has the phenomenon of acid and gas production in the initial fermentation, performing a confirmation test if the characteristic of the typical colony exists on an EMB (Electron multiplying B) plate, and inquiring according to an MPN (Multi-Point network) table to determine a final detection result.

Technical Field

The invention relates to the technical field of microorganism detection, in particular to a method for determining microorganisms in water.

Background

In the water quality detection process, the main detection indexes of microorganisms comprise total bacterial colony number, total coliform group, heat-resistant coliform group, escherichia coli group and the like, and the detection results of the indexes can reflect whether water quality is polluted or sanitary and safe, so that the basic guarantee of human drinking water safety is provided.

However, in the process of sampling water quality microorganism detection items in remote (remote) areas, on the premise that sampling modes, sampling equipment and sampling of water quality samples are strictly operated according to relevant national standard requirements, whether factors such as transportation distances and storage modes influence the detection results of microorganisms or not is a problem.

Disclosure of Invention

In view of this, the present application provides a method for determining microorganisms in water, which aims at determining whether factors such as a transportation distance and a storage mode have an influence on a detection result of the microorganisms under the premise that a sampling mode, sampling equipment and a sampling bag for containing a water quality sample are strictly operated according to relevant national standard requirements in a sampling process of a current detection method.

In order to solve the technical problems, the invention provides a technical scheme that 1, the method for measuring the microorganisms in the water comprises the following steps:

1) preparation of a culture medium: the culture medium of the total number of the bacterial colonies is nutrient agar, and the culture medium of the coliform is mainly prepared by lactose peptone, an EMB flat plate and the like and is the same as the nutrient agar;

2) inoculation: inoculating the total number of colonies, using a sterile pipette to absorb 1ml of fully mixed water sample, injecting the water sample into a sterile plate, pouring about 15ml of nutrient agar culture medium which is melted and cooled to 40-50 ℃, and rotating the plate to fully mix the water sample and the culture medium. Parallel and blank comparison is carried out at the same time; the inoculation of coliform group bacteria is to take 10ml of water sample to inoculate into 10ml of two-material lactose peptone culture solution, take 1ml of water sample to inoculate into 10ml of single-material lactose peptone culture solution, take another 1ml of water sample to inject into 9ml of sterilized normal saline, absorb 1ml (namely 0.1ml of water sample) to inject into 10ml of single-material lactose peptone culture solution after mixing evenly, each dilution inoculates 5 tubes;

3) culturing: putting the sample in an incubator at 36 +/-1 ℃, culturing the total number of colonies for 48h, and culturing coliform groups for 24h and 2 h;

4) recording and result observation: directly counting the total number of the colonies; and (3) further performing separation culture if the coliform group has the phenomenon of acid and gas production in the initial fermentation, performing a confirmation test if the characteristic of the typical colony exists on an EMB (Electron multiplying B) plate, and inquiring according to an MPN (Multi-Point network) table to determine a final detection result.

Compared with the prior art, the detailed description of the application is as follows: the invention solves the problem that whether the water quality of the drinking water has certain stability when the microorganism indexes reach the laboratory for detection under different transportation distances and storage conditions, provides certain data support and theoretical basis, and has theoretical guiding significance when detecting and operating related items of microorganisms in the field and remote areas. The invention optimizes the influence conditions of different water qualities on different microorganism index results under different storage temperature and time conditions, has more reliable test results, and has guiding significance for the detection of microorganism related items in remote areas and remote areas in the field.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.

Example 1

A method for measuring microorganisms in water, which is characterized by comprising the following steps:

1) preparation of a culture medium: the culture medium of the total number of the bacterial colonies is nutrient agar, the culture medium of the coliform group is mainly prepared from lactose peptone, an EMB flat plate and the like, the preparation process is the same as that of the nutrient agar, and the high-pressure sterilization treatment is carried out according to the instructions provided by a supplier on the premise of well checking and accepting the culture medium; the culture medium of coliform bacteria is mainly lactose peptone, EMB plate and other preparation processes as nutrient agar;

2) inoculation: inoculating the total number of the colonies, sucking 1ml of fully mixed water sample by a sterile pipette by an aseptic operation method, injecting the water sample into a sterile plate, pouring about 15ml of nutrient agar culture medium which is melted and cooled to 40 ℃, and shaking the plate to fully mix the water sample and the culture medium. Parallel and blank comparison is carried out at the same time; the inoculation of coliform group bacteria is to take 10ml of water sample to inoculate into 10ml of two-material lactose peptone culture solution, take 1ml of water sample to inoculate into 10ml of single-material lactose peptone culture solution, take another 1ml of water sample to inject into 9ml of sterilized normal saline, absorb 1ml (namely 0.1ml of water sample) to inject into 10ml of single-material lactose peptone culture solution after mixing evenly, each dilution inoculates 5 tubes;

3) culturing: putting the sample in a 35 ℃ incubator, culturing the total number of colonies for 48 hours, and culturing the coliform group for 22 hours;

4) recording and result observation: directly counting the total number of the colonies; and (3) further performing separation culture if the coliform group has the phenomenon of acid and gas production in the initial fermentation, performing a confirmation test if the characteristic of the typical colony exists on an EMB (Electron multiplying B) plate, and inquiring according to an MPN (Multi-Point network) table to determine a final detection result.

Example 2

A method for measuring microorganisms in water, which is characterized by comprising the following steps:

1) preparation of a culture medium: the culture medium of the total number of the bacterial colonies is nutrient agar, the culture medium of the coliform group is mainly prepared from lactose peptone, an EMB flat plate and the like, the preparation process is the same as that of the nutrient agar, and the high-pressure sterilization treatment is carried out according to the instructions provided by a supplier on the premise of well checking and accepting the culture medium; the culture medium of coliform bacteria is mainly lactose peptone, EMB plate and other preparation processes as nutrient agar;

2) inoculation: inoculating the total number of the colonies, sucking 1ml of fully mixed water sample by a sterile pipette by an aseptic operation method, injecting the water sample into a sterile plate, pouring about 15ml of nutrient agar culture medium which is melted and cooled to 45 ℃, and shaking the plate to fully mix the water sample and the culture medium. Parallel and blank comparison is carried out at the same time; the inoculation of coliform group bacteria is to take 10ml of water sample to inoculate into 10ml of two-material lactose peptone culture solution, take 1ml of water sample to inoculate into 10ml of single-material lactose peptone culture solution, take another 1ml of water sample to inject into 9ml of sterilized normal saline, absorb 1ml (namely 0.1ml of water sample) to inject into 10ml of single-material lactose peptone culture solution after mixing evenly, each dilution inoculates 5 tubes;

3) culturing: putting the sample in an incubator at 36 ℃, culturing the total number of colonies for 48 hours, and culturing coliform groups for 24 hours;

4) recording and result observation: directly counting the total number of the colonies; and (3) further performing separation culture if the coliform group has the phenomenon of acid and gas production in the initial fermentation, performing a confirmation test if the characteristic of the typical colony exists on an EMB (Electron multiplying B) plate, and inquiring according to an MPN (Multi-Point network) table to determine a final detection result.

Taking the example 2 as an example, the method specifically operates as follows:

selecting different water qualities in remote areas as samples, numbering the samples as No. 1-50 respectively, and carrying out correlation on the numbers according to the requirements of relevant standards under different transportation temperature conditions and different time conditions to influence the detection results of different microorganism indexes.

Study object

The types of microorganisms: and (3) inoculating total bacterial colonies, coliform groups, heat-resistant coliform groups (faecal coliform groups) and Escherichia coli groups in the water sample of the remote limited area.

Water quality: surface water, ground water and drinking water.

In remote areas: the finger is far away from the laboratory, and the sample can not be delivered to the laboratory for inspection according to the microorganism inoculation time limit after sampling. (Water sample delivered to laboratory in 24 h)

Main equipment

Refrigerator, biochemical incubator, clean bench, water bath, vortex mixer and other relevant experimental equipment.

Consumable material

Disposable sterilized gloves, masks, caps, 75% medical alcohol, absorbent cotton, marking pens, notebooks, waste collection bags, etc.

Method basis

Standard test method for drinking water microbiological indicators for life (GB/T5750.12-2006).

Procedure for the preparation of the

Firstly, sampling: sampling all water samples according to a sterile operation program, and taking two water samples from the same water sample

Second, experimental procedures and results

1. Preparation of a culture medium: the culture medium of the total number of the bacterial colonies is nutrient agar, and the high-pressure sterilization treatment is carried out according to the instructions provided by a supplier on the premise of well checking and accepting the culture medium; the culture medium of coliform bacteria is mainly lactose peptone, EMB plate and other nutrient agar.

2. Inoculation: inoculating the total number of bacterial colonies, using a sterile pipette to absorb 1ml of fully mixed water sample, injecting the water sample into a sterile plate, pouring about 15ml of nutrient agar culture medium which is melted and cooled to about 45 ℃, and rotating the plate to fully mix the water sample and the culture medium. Parallel and blank comparison is carried out at the same time; the inoculation of coliform bacteria is to take 10ml of water sample to inoculate into 10ml of two-material lactose peptone culture solution, take 1ml of water sample to inoculate into 10ml of single-material lactose peptone culture solution, take another 1ml of water sample to inject into 9ml of sterilized normal saline, absorb 1ml (namely 0.1ml of water sample) to inject into 10ml of single-material lactose peptone culture solution after mixing evenly, each dilution inoculates 5 tubes.

3. Culturing: the samples are placed in an incubator at 36 +/-1 ℃, the total number of colonies is cultured for 48 hours, and the coliform group is cultured for 24 hours or 2 hours.

4. Recording and result observation: counting the total number of colonies directly, performing isolation culture if the coliform group is fermented for the first time and produces acid and gas, performing a confirmation test on the EMB plate with typical colonies, and inquiring an MPN table to perform a result report

Results of the experiment

On the premise of ensuring that environmental factors influencing the detection result are not changed, inoculating the result of the total number of the cultured bacterial colonies in a time-limited manner at different storage temperatures and different conveying times (Table I); results for coliform (table two); results for heat-resistant coliform group bacteria (table three); escherichia coli (Table four). Wherein 50 water samples with the serial numbers of 01-50 are randomly extracted from the first table, and 10 water samples with the serial numbers of 01-10 are extracted from the second table to the third table. (temperature 20 ℃ C. humidity: 48%)

TABLE-Total number of colonies (unit: cfu/ml)

According to the method, a judgment value of 100cfu/mL is used as a measurement index according to the total number of the bacterial colonies of the drinking water, no matter whether a water quality detection judgment standard result is qualified or unqualified, under the condition that the refrigeration temperature is 4 ℃, the results of the total number of the bacterial colonies of 50 groups of different sampling points which arrive at a laboratory in 8 hours and arrive at the laboratory in 24 hours are compared, and the numerical difference is small.

But under the condition of normal-temperature storage of different water qualities, the total number of colonies reaches the detection result of the laboratory within 4 hours of storage, and if the total number of the colonies is smaller than the judgment standard value result and the detection value is smaller than 50cfu/mL, the total number of the colonies is qualified, the total number of the colonies reaches the detection result of the laboratory within 8 hours, and the total number of the colonies reaches 2-3 times of the total number of the colonies reaches the detection result of the laboratory within 4 hours; if the detection value reaching the laboratory within 4 hours is smaller than the judgment standard value result and the detection value is larger than 50cfu/mL and smaller than 100cfu/mL, the sample reaches the laboratory within 8 hours, the detection result of the sample is basically larger than the limit value of the judgment standard 100cfu/mL, the result is unqualified, and compared with the detection result reaching the laboratory within 4 hours, the sample shows very large change of 0-4 times difference, however, the detection result reaching the laboratory within 16 hours is basically consistent with the detection result reaching the laboratory within 8 hours, but the detection value is 0-2 times of the detection result reaching the laboratory within 8 hours, and the difference of the change times is not obvious. As can be seen from Table one, the numbers are 1-50.

Metel II Total coliform group (MPN/100ml)

(Note that sample numbers 1-10 in Table two correspond to sample numbers 1-10 in Table one)

As can be seen from the results of the total coliform group test of 10 groups of water quality, if the test result delivered to the laboratory within 4 hours is less than 2MPN/100mL under the condition of normal temperature storage, no matter the test result arrives at the laboratory within 8 hours and 16 hours or under the condition of 4 ℃ refrigeration, the test result arrives at the laboratory within 8 hours and 24 hours and is not changed, even if the total coliform group is detected, under the condition of 4 ℃ refrigeration, the test result of 8 hours storage and 24 hours storage is shown in group 3 and group 6, and the test result is basically not changed when the total coliform group is stored for 4 hours.

But the water is preserved under the normal temperature condition, when the water quality of the group 3 and the group 6 is preserved for 4 hours, and the detection results are respectively less than 50 and more than 50, the detection results of the preservation for 8 hours and the preservation for 16 hours are 1-1.5 times and 2-3 times of the preservation for 4 hours.

Pleurotus ostreatus (faecal coliform)

Since the heat-resistant Escherichia coli group and Escherichia coli group were further detected when the total Escherichia coli group was detected, the heat-resistant Escherichia coli group was detected in 3 groups and 6 groups. As can be seen from the table, if the test is carried out after being stored for 4 hours under the condition of normal temperature storage, the test result is not changed because the storage time is 8 hours and 16 hours, or the test result is not changed when the test is carried out under the condition of refrigeration at 4 ℃ for 8 hours and 24 hours.

Escherichia coli (Ex. coli) of Epimedium

(Note: sample No. 01, 02, 04, 05, 07, 08, 09, 10 did not detect the total coliform group, and heat-resistant coliform group and Escherichia coli were not detected again according to the relevant regulations in GB/T5750.12-2006.)

The results of Escherichia coli detection in the groups are consistent with those of Escherichia coli groups because the total Escherichia coli groups are detected in the groups 3 and 6, and the detection results are not changed due to the storage time of 8h and 16 when the groups are stored for 4h under the normal-temperature storage condition or the storage time of 8h and 24h under the 4 ℃ refrigeration condition.

Example 3

A method for measuring microorganisms in water, which is characterized by comprising the following steps:

1) preparation of a culture medium: the culture medium of the total number of the bacterial colonies is nutrient agar, the culture medium of the coliform group is mainly prepared from lactose peptone, an EMB flat plate and the like, the preparation process is the same as that of the nutrient agar, and the high-pressure sterilization treatment is carried out according to the instructions provided by a supplier on the premise of well checking and accepting the culture medium; the culture medium of coliform bacteria is mainly lactose peptone, EMB plate and other preparation processes as nutrient agar;

2) inoculation: inoculating the total number of colonies, using a sterile pipette to absorb 1ml of fully mixed water sample, injecting the water sample into a sterile plate, pouring about 15ml of nutrient agar culture medium which is melted and cooled to 50 ℃, and rotating the plate to fully mix the water sample and the culture medium. Parallel and blank comparison is carried out at the same time; the inoculation of coliform group bacteria is to take 10ml of water sample to inoculate into 10ml of two-material lactose peptone culture solution, take 1ml of water sample to inoculate into 10ml of single-material lactose peptone culture solution, take another 1ml of water sample to inject into 9ml of sterilized normal saline, absorb 1ml (namely 0.1ml of water sample) to inject into 10ml of single-material lactose peptone culture solution after mixing evenly, each dilution inoculates 5 tubes;

3) culturing: putting the sample into a 37 ℃ incubator, culturing the total number of colonies for 48 hours, and culturing coliform groups for 24 hours;

4) recording and result observation: directly counting the total number of the colonies; and (3) further performing separation culture if the coliform group has the phenomenon of acid and gas production in the initial fermentation, performing a confirmation test if the characteristic of the typical colony exists on an EMB (Electron multiplying B) plate, and inquiring according to an MPN (Multi-Point network) table to determine a final detection result.

The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.