阅读说明：本技术 一种薄膜过滤法检测注射用达托霉素中间产品中微生物计数的方法 (Method for detecting microbial count in daptomycin intermediate product for injection by using membrane filtration method ) 是由 唐咏群 曹宇 于 2020-06-03 设计创作，主要内容包括：本发明涉及一种薄膜过滤法检测注射用达托霉素中间产品中微生物计数的方法,以含吐温80的pH7.2磷酸盐缓冲液作为冲洗液,在添加有0.05mol/L～0.2mol/L乙二胺四乙酸二钠无菌溶液的胰酪大豆胨琼脂培养基中培养需氧菌,在沙氏葡萄糖琼脂培养基中培养霉菌及酵母菌,采用薄膜过滤法分别检测供试品溶液中需氧菌总数和霉菌及酵母菌的总数,所述供试品溶液为已添加各种辅料制成的注射用达托霉素中间产品。采用本发明的检测方法可以消除注射用达托霉素中间产品的抑菌性,确保检验结果的准确性,用于注射用达托霉素中间产品的微生物计数检查。(The invention relates to a method for detecting microbial count in daptomycin intermediate products for injection by a membrane filtration method, which comprises the steps of taking phosphate buffer solution with pH7.2 containing Tween 80 as flushing fluid, culturing aerobic bacteria in a trypticase soytone agar culture medium added with 0.05 mol/L-0.2 mol/L of ethylene diamine tetraacetic acid sterile solution, culturing mould and saccharomycetes in a Sabouraud's glucose agar culture medium, and respectively detecting the total number of the aerobic bacteria and the total number of the mould and the saccharomycetes in a test solution by adopting the membrane filtration method, wherein the test solution is the daptomycin intermediate products for injection, which are prepared by adding various auxiliary materials. The detection method can eliminate the bacteriostasis of the daptomycin intermediate product for injection, ensure the accuracy of the detection result and is used for the microbial counting inspection of the daptomycin intermediate product for injection.)

1. A method for detecting microbial count in daptomycin intermediate products for injection by a membrane filtration method is characterized in that phosphate buffer solution with pH7.2 and containing Tween 80 is used as flushing fluid, aerobic bacteria are cultured in a trypticase soytone agar culture medium added with 0.05 mol/L-0.2 mol/L of disodium ethylene diamine tetraacetate sterile solution, mould and saccharomycetes are cultured in a Sabouraud's glucose agar culture medium, the total number of the aerobic bacteria and the total number of the mould and the saccharomycetes in a test solution are respectively detected by the membrane filtration method, and the test solution is the daptomycin intermediate products for injection prepared by adding various auxiliary materials.

2. Method according to claim 1, characterized in that it comprises the following steps:

(1) and (3) moistening the filter membrane: wetting the filter membrane with phosphate buffer solution containing Tween 80 and pH7.2;

(2) determination of the total number of aerobic bacteria: after a test solution is filtered by a wetted filter membrane, washing the filter membrane by a phosphate buffer solution with the pH value of 7.2 and containing Tween 80, enabling the bacterial surface of the washed filter membrane to face upwards, pasting the filter membrane on a tryptone soytone agar culture medium containing 0.05 mol/L-0.2 mol/L disodium ethylene diamine tetraacetate sterile solution, culturing for 3-5 days at the temperature of 30-35 ℃, and detecting;

(3) and (3) total number of moulds and yeasts is determined: filtering the test solution with a wetted filter membrane, washing the filter membrane with a phosphate buffer solution containing Tween 80 and having a pH value of 7.2, enabling the bacterial surface of the washed filter membrane to face upwards, sticking the filter membrane to a Sabouraud's dextrose agar culture medium, culturing for 5-7 days at 20-25 ℃, and detecting.

3. The method according to claim 2, wherein the washing solution is a phosphate buffer solution of ph7.2 containing tween 80 at a volume concentration of 0.5% to 2%.

4. The method according to claim 3, wherein the washing solution is a phosphate buffer solution of pH7.2 containing Tween 80 at a volume concentration of 1%.

5. The method as claimed in claim 2, wherein the concentration of the sterile solution of disodium edetate is 0.05mol/L to 0.1 mol/L.

6. The method as claimed in claim 5, wherein the concentration of the sterile solution of disodium edetate is 0.05 mol/L.

7. The method according to claim 2, wherein the volume of the sterile solution of disodium edetate added to the tryptose soy agar medium is 0.1 to 0.2 percent of the total volume of the tryptose soy agar medium.

8. The method according to claim 7, wherein the volume of the sterile solution of disodium edetate added to the tryptose soy agar medium is 0.13-0.14% of the total volume of the tryptose soy agar medium; preferably 0.137%.

9. The method according to claim 4, wherein the phosphate buffer pH7.2 containing Tween 80 at a volume concentration of 1% is prepared by the following method: (1) weighing 34g of potassium dihydrogen phosphate and 7g of sodium hydroxide in a 1000mL triangular flask, adding purified water to dissolve the potassium dihydrogen phosphate and the sodium hydroxide to 1000mL, and preparing a phosphate stock buffer solution with the pH value of 7.2; (2) uniformly mixing purified water and a pH7.2 phosphate stock buffer solution according to the volume ratio of 800:1, and sterilizing to prepare a pH7.2 phosphate buffer solution; (3) and (3) uniformly mixing the phosphate buffer solution with the pH value of 7.2 and the Tween 80 according to the volume ratio of 99:1, and sterilizing to obtain the phosphate buffer solution with the pH value of 7.2 and the volume concentration of 1% Tween 80.

10. The method according to any one of claims 1 to 9, further comprising a negative control comprising the steps of:

(1) and (3) moistening the filter membrane: wetting the filter membrane with a phosphate buffer solution of pH7.2 containing Tween 80 at a volume concentration of 1%;

(2) determination of the total number of aerobic bacteria: washing the filter membrane by phosphate buffer solution with the volume concentration of 1% Tween 80 and the pH value of 7.2, enabling the bacterial surface of the washed filter membrane to face upwards, pasting the filter membrane on a tryptone soybean peptone agar culture medium containing 0.05mol/L ethylene diamine tetraacetic acid disodium sterile solution, culturing for 3-5 days at the temperature of 30-35 ℃, and detecting;

(3) and (3) total number of moulds and yeasts is determined: washing the filter membrane with a phosphate buffer solution with the volume concentration of 1% Tween 80 and the pH value of 7.2, enabling the bacterial surface of the washed filter membrane to face upwards, pasting the filter membrane on a Sabouraud's dextrose agar culture medium, culturing for 5-7 days at the temperature of 20-25 ℃, and detecting.

Technical Field

The invention belongs to the technical field of drug analysis, and particularly relates to a method for detecting microbial count in a daptomycin intermediate product for injection by a membrane filtration method.

Background

The microorganism counting inspection method is a method for inspecting the degree of microorganism pollution of non-specified sterilization preparations, raw materials and auxiliary materials thereof. The examination items include total aerobic count (TAMC), total mold and yeast count (TYMC) and control bacteria examination. The microbial counting inspection of the intermediate product of the sterile preparation is to control the aspects of raw and auxiliary materials, inner packaging materials, a feeding process, a liquid medicine preparation tank, a liquid medicine pipeline, production tools, a filtering system, filling environment guarantee and the like, so the microbial counting level of the intermediate product of the sterile preparation also needs to be monitored, and the main detection items are total aerobic bacteria (TAMC), total mould and yeast (TYMC).

At present, there are three main methods for microorganism counting and checking, which are respectively: plate method, membrane filtration method and MPN method. The plate method is simple to operate and has poor antibacterial activity for eliminating the product. The membrane filtration method has relatively complex operation and strong bacteriostatic ability for eliminating the product. The MPN method is not very complicated in operation, but is inferior to the membrane filtration method and the plate counting method in precision and accuracy.

Daptomycin for injection is indicated for blood stream infection (bacteremia) with right-side infective endocarditis caused by staphylococcus aureus (including methicillin-sensitive and methicillin-resistant). The components of the daptomycin intermediate product for injection are mainly shown in the following table:

formulation of	Function(s)	Amount of prescription	1mL of the composition
				Daptomycin	Active substance	500 mg/piece	81.5040mg/mL
Sodium hydroxide	pH regulator	Q.S.	Q.S.to pH
				Water for injection	Solvent(s)	Q.S.	Q.S.

When the test article has bacteriostatic activity, the following method is adopted for treatment so as to eliminate the bacteriostatic activity of the test article solution, and then the examination is carried out according to the law. The usual method is as follows. Increasing the volume of a diluent or a culture medium; adding proper neutralizing agent or deactivating agent; thirdly, adopting a thin film filtration method; and fourthly, the combined use of the methods. If no proper method for eliminating the bacteriostatic activity of the test sample exists, the recovery failure of the specific test bacteria indicates that the test sample has stronger antibacterial activity to the test bacteria, and simultaneously indicates that the test sample is not easily polluted by the microorganisms.

Disclosure of Invention

The invention aims to provide a method for detecting microbial count in daptomycin intermediate products for injection by a membrane filtration method on the basis of the prior art.

The technical scheme of the invention is as follows:

a method for detecting microbial counts in daptomycin intermediate products for injection by a membrane filtration method comprises the steps of taking a phosphate buffer solution with pH7.2 containing Tween 80 as a flushing fluid, culturing aerobic bacteria in a trypticase soytone agar medium (TSA) added with 0.05 mol/L-0.2 mol/L of ethylene diamine tetraacetic acid (EDTA-2Na) sterile solution, culturing mould and yeast in a Sabouraud's dextrose agar medium (SDA), and respectively detecting the total number of the aerobic bacteria and the total number of the mould and the yeast in a test solution by the membrane filtration method, wherein the test solution is a daptomycin intermediate product for injection prepared by adding various auxiliary materials, namely a daptomycin injection solution for injection meeting the requirements of pharmacopoeia.

The invention takes daptomycin intermediate product for injection as test solution, adopts a membrane filtration method, selects proper neutralizer to add into a culture medium for microorganism counting inspection, and concretely comprises the following steps:

(1) and (3) moistening the filter membrane: wetting the filter membrane with phosphate buffer solution containing Tween 80 and pH7.2;

(2) determination of the total number of aerobic bacteria: after a test solution is filtered by a wetted filter membrane, washing the filter membrane by a phosphate buffer solution with the pH value of 7.2 and containing Tween 80, enabling the bacterial surface of the washed filter membrane to face upwards, pasting the filter membrane on a tryptone soytone agar medium (TSA) containing 0.05 mol/L-0.2 mol/L disodium ethylene diamine tetraacetate sterile solution, culturing for 3-5 days at the temperature of 30-35 ℃, and detecting;

(3) and (3) total number of moulds and yeasts is determined: filtering the test solution with a wetted filter membrane, washing the filter membrane with a phosphate buffer solution containing Tween 80 and having a pH value of 7.2, enabling the bacterial surface of the washed filter membrane to face upwards, sticking the filter membrane on a Sabouraud's Dextrose Agar (SDA) culture medium, culturing for 5-7 days at 20-25 ℃, and detecting.

Daptomycin is a calcium ion-dependent antibiotic that has little antibacterial activity in the absence of calcium ions. The pH7.0 sterile NaCl-peptone buffer and trypticase Soy agar medium (TSA) used contained trace amounts of calcium ions. In the invention, in order to eliminate the bacteriostatic activity of a test solution during the determination of the total number of aerobic bacteria, 0.05 mol/L-0.2 mol/L disodium ethylene diamine tetraacetate (EDTA-2Na) is added into a trypticase soytone agar medium (TSA) to chelate calcium ions in the medium.

In the present invention, the kind of the washing solution is important and needs to be strictly controlled, and for example, the interference effect is not easily eliminated by using the commonly used pH7.0 sterile sodium chloride-peptone buffer solution and 0.1% peptone water solution, which affects the recovery rate of the detection microorganism count test. The method takes the phosphate buffer solution with the pH value of 7.2 and containing the Tween 80 as the flushing fluid, and can eliminate the interference effect, so that the recovery rate of the microbial count in the daptomycin intermediate product for injection meets the regulation.

In a preferred embodiment, the washing solution is a phosphate buffer solution with pH7.2 containing Tween 80 at a volume concentration of 0.5-2%. In a more preferred embodiment, the washing solution is a phosphate buffer solution of pH7.2 containing Tween 80 at a volume concentration of 1% without affecting the effect of the present invention.

In one embodiment, the phosphate buffer of pH7.2 containing Tween 80 at a volume concentration of 1% is prepared as follows: (1) weighing 34g of potassium dihydrogen phosphate and 7g of sodium hydroxide in a 1000mL triangular flask, adding purified water to dissolve the potassium dihydrogen phosphate and the sodium hydroxide to 1000mL, and preparing a phosphate stock buffer solution with the pH value of 7.2; (2) uniformly mixing purified water and a pH7.2 phosphate stock buffer solution according to the volume ratio of 800:1, and sterilizing to prepare a pH7.2 phosphate buffer solution; (3) and (3) uniformly mixing the phosphate buffer solution with the pH value of 7.2 and the Tween 80 according to the volume ratio of 99:1, and sterilizing to obtain the phosphate buffer solution with the pH value of 7.2 and the volume concentration of 1% Tween 80.

For the purposes of the present invention, the selection of the rinse solution and the concentration of EDTA-2Na added to trypticase Soytone agar medium (TSA) during the preparation both affect the recovery of microbial counts in the present assay for daptomycin intermediate for injection when determining the total number of aerobic bacteria. When the concentration of EDTA-2Na is lower, the bacteriostasis in the sample cannot be eliminated, so that the recovery rate of the test bacteria does not meet the specification; when the concentration of EDTA-2Na is higher, the survival condition of the microorganisms in the sample is destroyed, and the recovery rate of the test bacteria is also out of regulation.

According to the invention, when the total number of aerobic bacteria is measured, 0.05 mol/L-0.2 mol/L of ethylene diamine tetraacetic acid (EDTA-2Na) sterile solution is added into a tryptone soya peptone agar medium (TSA) used, so that the bacteriostasis in a sample can be eliminated, and the recovery rate of test bacteria is not in accordance with the specification. In a preferred scheme, 0.05 mol/L-0.1 mol/L of disodium ethylene diamine tetraacetate (EDTA-2Na) sterile solution is added into trypticase soytone agar medium (TSA), and the recovery rate of the microbial count in the daptomycin intermediate product for injection meets the requirement and is more stable. In a more preferred embodiment, a 0.05mol/L sterile solution of disodium ethylenediaminetetraacetate (EDTA-2Na) is added to trypticase Soy agar medium (TSA) without affecting the effect of the present invention.

In a preferred embodiment, the volume of the sterile solution of disodium ethylenediaminetetraacetic acid (EDTA-2Na) added to the tryptone Soy agar medium (TSA) used in the determination of the total number of aerobic bacteria is 0.1-0.2% of the total volume of the tryptone Soy agar medium.

In a more preferred embodiment, the volume of the sterile solution of disodium ethylenediaminetetraacetic acid (EDTA-2Na) added to the tryptic Soy agar medium (TSA) is 0.13-0.14% of the total volume of the tryptic Soy agar medium. For example, it may be specifically 0.13%, 0.132%, 0.134%, 0.137%, 0.138%, 0.139%, or 0.14%.

When the method of the invention is used for detecting the number of microorganisms in the daptomycin intermediate product for injection, the method also can comprise a negative control, and the method comprises the following steps:

(1) and (3) moistening the filter membrane: wetting the filter membrane with a phosphate buffer solution of pH7.2 containing Tween 80 at a volume concentration of 1%;

(2) determination of the total number of aerobic bacteria: washing the filter membrane by phosphate buffer solution with the volume concentration of 1% Tween 80 and the pH value of 7.2, enabling the bacterial surface of the washed filter membrane to face upwards, pasting the filter membrane on Tryptone Soy Agar (TSA) culture medium containing 0.1mol/L of Ethylene Diamine Tetraacetic Acid (EDTA) sterile solution, culturing for 3-5 days at the temperature of 30-35 ℃, and detecting;

(3) and (3) total number of moulds and yeasts is determined: washing the filter membrane with a phosphate buffer solution with the volume concentration of 1% Tween 80 and the pH value of 7.2, enabling the bacterial surface of the washed filter membrane to face upwards, pasting the filter membrane on a Sabouraud's Dextrose Agar (SDA), culturing for 5-7 days at the temperature of 20-25 ℃, and detecting.

By adopting the technical scheme of the invention, the advantages are as follows:

and (2) carrying out microbial counting inspection on the daptomycin intermediate product for injection by adopting a membrane filtration method, filtering a to-be-detected sample solution through a wetted filter membrane, flushing the filter membrane by a phosphate buffer solution with the pH value of 7.2 and containing Tween 80, and adding 0.05 mol/L-0.2 mol/L EDTA-2Na sterile solution into the TSA for aerobic bacteria counting to further remove the bacteriostatic action of the daptomycin intermediate product for injection on bacteria. The detection method can eliminate the bacteriostasis of the daptomycin intermediate product for injection, ensure the accuracy of the detection result and is used for the microbial counting inspection of the daptomycin intermediate product for injection.

Detailed Description

The detection method of the present invention is further illustrated by the following examples, which are not intended to limit the present invention in any way.

Preparation of the test:

1. reagent

Trypticase soy peptone agar medium TSA;

sabouraud glucose agar medium SDA;

phosphate buffer, pH 7.2;

pH7.2 phosphate buffer containing 1% Tween 80 by volume;

0.05mol/L EDTA-2Na sterile solution;

PVDF filter membrane;

test solution: the daptomycin intermediate product for injection is prepared by adding various auxiliary materials.

2. Bacterial strain

Staphylococcus aureus bacteria;

pseudomonas aeruginosa;

b, bacillus subtilis;

candida albicans;

aspergillus niger.

3. Instrument for measuring the position of a moving object

A biological safety cabinet;

a biochemical incubator (20-25 ℃);

a biochemical incubator (30-35 ℃);

a microbial limited filtration system;

a micropipette.

4. Buffer preparation

ph7.2 phosphate stock buffer preparation: 34g of potassium dihydrogen phosphate and 7g of sodium hydroxide were weighed into a 1000mL Erlenmeyer flask, and dissolved to 1000mL by adding purified water. Subpackaging, sterilizing and storing at 2-8 ℃.

preparation of phosphate buffer at pH 7.2: mixing purified water and stock buffer solution at a volume ratio of 800:1, and sterilizing to obtain Phosphate Buffer Solution (PBS) with pH of 7.2.

Ph7.2 phosphate buffer containing tween 80 at a volume concentration of 1% was prepared: mixing purified water and stock buffer solution at a volume ratio of 800:1 to obtain Phosphate Buffer Solution (PBS) with pH7.2, mixing Phosphate Buffer Solution (PBS) with pH7.2 and tween 80 at a volume ratio of 99:1, and sterilizing to obtain phosphate buffer solution with pH7.2 containing tween 80 at a volume concentration of 1%.

Preparation of tryptone Soy agar Medium containing EDTA-2 Na: taking a bottle of melted trypticase soy agar culture medium, adding 0.137mL of sterile 0.05 mol/L-0.1 mol/L EDTA-2Na sterile solution into each 100mL of culture medium, uniformly mixing, and pouring into a plate.

5. Procedure of the test

A: test group

A sterile filter with a pore size of not more than 0.45 μm is first wetted with about 50mL of pH7.2 phosphate buffer containing 1% Tween 80 by volume. Adding 100mL of the test solution into a filter cup, washing the filter cup by using 5X 100mL of phosphate buffer solution with pH7.2 and the volume concentration of 1% Tween 80, and adding 0.1mL of staphylococcus aureus with the volume concentration of not more than 100cfu into the last washing solution; finally, the filter membrane was removed aseptically and the bacteria side up was attached to a prepared dish of TSA medium containing EDTA-2 Na. Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger were operated in accordance with the protocol. One plate was prepared for each strain.

A sterile filter with a pore size of not more than 0.45 μm is first wetted with about 50mL of pH7.2 phosphate buffer containing 1% Tween 80 by volume. 100mL of the above test solution was added to the filter, and then the filter was washed with 5X 100mL of a pH7.2 phosphate buffer containing 1% Tween 80 by volume, and 0.1mL of Candida albicans, not more than 100cfu, was added to the last wash. And finally, taking out the filter membrane in a sterile mode, and attaching the bacteria face upwards to a prepared SDA culture medium plate. Aspergillus niger is operated according to the method. One plate was prepared for each strain.

B: bacteria liquid control group

Sterile filters with pore sizes no larger than 0.45 μm were taken and first wetted with about 50mL of phosphate buffer pH 7.2. 100mL of pH7.2 phosphate buffer was added to the filter, then the filters were rinsed with 5X 100mL of pH7.2 phosphate buffer, and 0.1mL of Staphylococcus aureus no greater than 100cfu was added to the last rinse. The filters were then removed aseptically and the bacteria were placed face up on prepared TSA media plates. Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger were operated in accordance with the protocol. One plate was prepared for each strain.

Sterile filters with pore sizes no larger than 0.45 μm were taken and first wetted with about 50mL of phosphate buffer pH 7.2. 100mL of pH7.2 phosphate buffer was added to the filter, the filters were then rinsed with 5X 100mL of pH7.2 phosphate buffer, and 0.1mL of Candida albicans, not greater than 100cfu, was added to the last rinse. And finally, taking out the filter membrane in a sterile mode, and attaching the bacteria face upwards to a prepared SDA culture medium plate. Aspergillus niger is operated according to the method. One plate was prepared for each strain.

C: control group of test article

A sterile filter with a pore size of not more than 0.45 μm is first wetted with about 50mL of pH7.2 phosphate buffer containing 1% Tween 80 by volume. 100mL of the above test solution was added to each of 2 filter cups, followed by filtration, and each filter was washed with 5X 100mL of a pH7.2 phosphate buffer containing 1% Tween 80 by volume. The filters were removed aseptically and the bacteria were attached face up to prepared TSA and SDA plates containing EDTA-2Na, respectively. One plate was prepared for each medium.

D: diluent control

A sterile filter with a pore size of not more than 0.45 μm is first wetted with about 50mL of pH7.2 phosphate buffer containing 1% Tween 80 by volume. 100mL of pH7.2 phosphate buffer containing 1% Tween 80 by volume was added to the filter, then the filters were washed with 5X 100mL of pH7.2 phosphate buffer containing 1% Tween 80 by volume, and 0.1mL of Staphylococcus aureus not greater than 100cfu was added to the last wash. The filters were then removed aseptically and the bacteria were plated face up on prepared TSA media plates containing EDTA-2 Na. Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger were operated in accordance with the protocol. One plate was prepared for each strain.

A sterile filter with a pore size of not more than 0.45 μm is first wetted with about 50mL of pH7.2 phosphate buffer containing 1% Tween 80 by volume. 100mL of pH7.2 phosphate buffer containing 1% Tween 80 by volume was added to the filter, then the filters were washed with 5X 100mL of pH7.2 phosphate buffer containing 1% Tween 80 by volume, and 0.1mL of Candida albicans of not more than 100cfu was added to the last wash. And finally, taking out the filter membrane in a sterile mode, and attaching the bacteria face upwards to a prepared SDA culture medium plate. Aspergillus niger is operated according to the method. One plate was prepared for each strain.

E: negative control

A sterile filter with a pore size of not more than 0.45 μm is first wetted with about 50mL of pH7.2 phosphate buffer containing 1% Tween 80 by volume. 100mL of pH7.2 phosphate buffer containing 1% by volume of Tween 80 was added to each of 2 filter cups, followed by filtration, and each filter was washed with 5X 100mL of pH7.2 phosphate buffer containing 1% by volume of Tween 80. The filters were removed aseptically and the bacteria were attached face up to prepared TSA and SDA plates containing EDTA-2Na, respectively. One plate was prepared for each medium. Negative controls should have no colony growth.

6. Culturing

TSA medium is used for aerobic culture, and SDA medium is used for mold and yeast culture.

The TSA culture medium in the test group, the bacterial liquid control group and the diluent control group is cultured for no more than 3 days at 30-35 ℃, and the SDA culture medium is cultured for no more than 5 days at 20-25 ℃.

The TSA culture medium in the test sample control group and the negative control group is cultured for 3-5 days at 30-35 ℃, and the SDA culture medium is cultured for 5-7 days at 20-25 ℃.

7. Microorganism enumeration method suitability confirmation acceptance criteria

In 3 independent parallel tests, the bacteria recovery rate of a diluent control group (the bacterial colony number value of the diluent control group accounts for the percentage of the bacterial colony number of the bacterial liquid control group) is between 50% and 200%, which indicates that the used diluent does not influence the microbial count of a test sample;

in 3 independent parallel tests, the bacteria recovery rate of a test group (the percentage of the colony number of the test group minus the colony number of a test sample control group to the colony number of a bacteria liquid control group) is between 50% and 200%;

when the recovery rates of the diluent control group and the test group are 50% to 200%, the number of aerobic bacteria, the number of mold and the number of yeast in the test sample can be checked by the method for counting the test sample solution.

When the method applicability is confirmed, if the recovery of one or more test bacteria does not meet the requirement by adopting the method, the method and the test conditions which are closest to the requirement are selected for checking the test solution.