YY/T 0918-2014Test method for determining bacterial retention of membrane/filter assembly utilized for infusion liquid filtration (English PDF)

3.1 log reduction value; LRV

The 10-base logarithm of the ratio of the number of challenge microorganisms to the number of filtrate microorganisms.

6.1 Stainless steel pressure vessel

6.2 Air conditioner

6.7 Liquid flow meter

6.8 Hose clamp

6.10 Biological safety cabinet

6.11 Ultra-clean workbench

The instrument assembly is shown in Figure 1.

8.2.5 Tryptic soy broth

Prepare according to manufacturer's instructions.

8.3 Analytical reagents and materials

8.3.1 M plate count agar

Prepare according to manufacturer's instructions.

8.3.2 Peptone water (1 g/L)

Dissolve the peptone into water; distribute it into screw-top bottles in appropriate volumes to prepare a 10-fold dilution; sterilize at 121 °C for 15 minutes.

8.4 Brevundimonas diminuta

Brevundimonas diminuta (ATCC 19146).

8.5 Analytical membrane

Diameter 47 mm, pore size 0.45 µm.

9.1 General

The following two methods have been widely used for the preparation of Brevundimonas diminuta suspensions. These methods are not exclusive of other equally effective methods. However, it is important that all Brevundimonas diminuta challenge suspensions used are monodisperse and comply with the provisions of Chapter 10.

9.2 Isolation and preservation of strains

Culture according to the instructions for Brevundimonas diminuta and check purity by the streak plate. Check whether the colony morphology is consistent and identify single colonies of Brevundimonas diminuta according to Chapter 10.

9.2.1 Stock cultures

Prepare the stock culture from the single colony isolated in 9.2. Inoculate tryptic soy agar slant and culture at 30 °C ± 2 °C for 24 h. Use sterile liquid paraffin to cover the slant and store at 4 °C. Check viability and purity weekly. Alternatively, tryptic soy semi-solid agar stab can be used instead of slant culture.

9.2.2 Long-term storage of cultures

Freeze-dry or store in liquid nitrogen.

9.3 Preparation of challenge stocks in saline lactose broth

9.3.1 Inoculate stock culture (9.2.1) into10 mL of sterile tryptic soy broth and culture it at 30 °C ± 2 °C for 24 hours.

9.3.2 Transfer 2 mL of the well-mixed broth culture solution into 1 L of sterile saline lactose broth; vortex to mix; incubate at 30 °C ± 2 °C for 24 hours.

Note: The bacterial suspension in saline lactose broth can be stored at 4 °C before use, for no more than 8 hours.

9.3.3 Determine the concentration of viable bacteria in the challenge bacterial suspension according to Chapter 11 (general concentration 107 CFU/mL ~ 108 CFU/mL).

9.3.4 Identify Brevundimonas diminuta according to Chapter 10

9.4 Preparation of Brevundimonas diminuta frozen bacteria paste

9.4.1 Inoculate stock culture (9.2.1) into 10 mL of sterile growth medium A (8.2.1) and culture it at 30 °C ± 2 °C for 24 hours.

9.4.2 Transfer 10 mL of the bacterial suspension obtained in 9.4.1 into 500 mL of sterile growth medium A, and incubate at 30 °C ± 2 °C for 24 hours.

9.4.3 Transfer 200 mL of the bacterial suspension obtained in 9.4.2 into 10 L of sterile growth medium A; prepare 10 L of seed culture solution; culture at 30 °C ± 2 °C for 24 hours.

9.4.5 When the culture reaches the stationary growth phase, collect the bacterial cells by continuous centrifugation.

9.4.6 Resuspend the bacterial cells in 2 ~ 3 times the volume of cold sterile buffer for storage.

9.4.7 Centrifuge the bacterial cell suspension and resuspend the bacterial cells in an equal volume of buffer for storage. Determine the bacterial concentration (viable bacterial concentration is generally 1 x 10^12 CFU/mL).

9.4.8 Transfer all the bacterial paste (such as 50 mL) into a sterile plastic centrifuge tube, use dry ice acetone or liquid nitrogen to freeze it; store it at -60 °C.

9.4.4 Inoculate the above 10 L of seed culture solution into 500 L of growth medium

A Incubate aerobically at 30 °C ± 2 °C. Monitor growth by spectrophotometric analysis at 500 nm and draw a growth curve.

9.5 Preparation of challenge stock solution by frozen strain paste

9.5.1 Use sterile forceps to clamp the test tube; immerse it in 80% (volume fraction) alcohol for disinfection; ignite the alcohol until most of the alcohol is burned out.

9.5.2 Aseptically remove the test tube cap; under normal temperature conditions, put the test tube into a conical flask containing a sterile magnetic stirring rod and stroke-physiological saline solution - containing 0.001 mol/L ~ 0.002 mol/L magnesium chloride (MgCl2) - of 20 times the volume of bacterial paste (for example, transfer 50 mL of frozen bacterial paste into 1 L of sterile solution).

Note: Magnesium chloride (MgCl2) needs to be dissolved in the solvent before adding the frozen bacteria paste.

9.5.3 Place the flask on the magnetic stirrer and mix until the contents in the test tube are evenly suspended (40 minutes).

9.5.4 Determine the viable bacterial concentration according to Chapter 11 (bacterial suspension concentration is generally 1 x 10^10 CFU/mL ~ 2 x 10^10 CFU/mL).

9.5.5 Identify Brevundimonas diminuta according to Chapter 10

10.1 Colony morphology

10.1.1 The colonies of Brevundimonas diminuta are beige, slightly raised, and completely transparent.

10.1.2 After culturing at 30 °C (optimal growth temperature) for 24 hours, it grows to the size of a needle tip, and the diameter will be 1 mm ~ 2 mm after cultivating for 36 h ~ 48 h.

10.2 Microscopic observation

10.2.2 Perform flagella staining (optional). Brevundimonas diminuta is characterized by polar single flagella.

10.2.1 Perform Gram staining

10.2.1.1 Use a compound optical microscope equipped with a calibrated eyepiece micrometer and an oil immersion lens of good resolution to examine the prepared strains. Observe the size and distribution of microorganisms in several fields of view.

10.2.1.2 The strains prepared by staining shall be Gram-negative, short rod-shaped bacteria with a size of (0.3 µm ~ 0.4 µm) × (0.6 µm ~ 1.0 µm), which mainly exists in the form of single cells.

10.3 Biochemical properties

lactose broth or sterile physiological saline, so that the test filter membrane per square centimeter can withstand the challenge of at least 107 microorganisms; mix thoroughly.

13.2 Bacterial challenge test and positive control test

13.2.1 Add the required volume of bacterial suspension prepared in Chapter 11 (at least 500 mL for each branch) into the pressure vessel.

13.2.2 Close valve C and adjustable valves A, B, C and D, and open valves A and B.

13.2.3 Pressurize the pressure vessel to 200 kPa.

13.2.4 Slowly open the adjustable valve D to allow the challenge bacteria suspension to fill the test sample filter assembly, the positive control filter assembly and their downstream analysis filter assemblies. Exhaust air from each filter assembly into a suitable disinfectant. When each filter assembly is filled with liquid, close its exhaust valve.

13.2.6 Open adjustable valve D, and use adjustable valves A and B to adjust the flow rate to an effective filtration area of 2 mL/min ~ 4 mL/min per square centimeter of the test filter membrane and the positive control filter membrane.

13.2.7 When all liquid filtration is completed, close valves A and B and adjustable valves A and B.

13.2.8 Close the air regulator and release the pressure in the container.

13.2.9 Remove each branch analysis filter assembly; evacuate the downstream for 15 seconds; remove all liquid; transfer the membrane from the filter assembly to the M plate count agar under sterile conditions; incubate at 30 °C ± 2 °C; record the number of colonies at the 72nd hour and on the 7th day.

13.2.10 Identify whether each colony is Brevundimonas diminuta or contaminating bacteria (Part 10).

13.2.5 Open adjustable valves A and B

13.3 Integrity test

After the challenge test is completed, if necessary, perform an integrity test on the test sample filter component according to YY/T 0929.1.

14.1 Identification of filters

Report the membrane/filter manufacturer's name, nominal pore size, effective filtration area and other relevant data.

14.2 Operating conditions

Report pressure, differential pressure, temperature, flow and other relevant parameters.

14.3 Challenge bacteria count

Report the bacterial concentration determined in step 11.7, and calculate and report the total bacterial count in the challenge suspension.

14.4 Number of bacteria in filtrate

14.4.1 Control

Report the number of colonies observed on the positive and negative control analysis filters.

14.4.2 Test sample

Report the number of colonies observed on the analysis filter membrane for the test sample. Report each colony as Brevundimonas diminuta or contaminating bacteria.

14.5 Filter integrity

If applicable, report the parameters and results of the integrity test in 12.1 and 13.3; indicate whether the filter can pass the integrity test; explain the basis for the judgment.

14.6 Filtration performance

Calculate and report the log reduction value.

14.7 Results evaluation

The test is invalid when bacterial colonies appear on the negative control analysis filter or non-test microorganisms appear on the test analysis filter.

When the test sample analysis shows that no bacterial colonies appear on the filter membrane and the positive control is positive, judge that the sterilization requirements are met. […]