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APPENDIX 10 MICROBIOLOGICAL TESTS

10.1 STERILITY TEST​

      The test is designed to reveal the presence, if any, of contamination with viable micro-organisms in pharmaceutical or medical articles intended for parenteral administration or for other sterile applications, which, according to the Pharmacopoeia, are required to be sterile. A satisfactory negative result, however, only indicates that no contaminating micro-organism has been found in the sample examined in the conditions of the test. Nevertheless, because the sample to be tested is randomly selected from the particular batch it represents, it is thereafter assumed that the whole batch passes the sterility test, which is at present the only method available to the various authorities who have to examine a product for sterility.

      Alternative procedures or procedural details may be employed to demonstrate that an article is sterile, provided the results obtained are at least of equivalent reliability. Where a difference appears, or in the event of a dispute, when evidence of microbial contamination is obtained by the procedure given in this Pharmacopoeia, the result so obtained is conclusive of failure of the article to meet the requirements of the test.

Test Conditions

      Adventitious microbial growth that is transmitted to an article or to inoculated test culture media from the environment during the course of a sterility test invalidates the results of the test. Hence, it is necessary to demonstrate that the proper precautions have been taken to exclude extraneous micro-organisms throughout the test period.

      The test should be carried out under aseptic conditions in an area as free from contamination as possible by the use of disinfecting agents, ultraviolet lamps and air filters. Ultraviolet lamps and disinfecting aerosols should not be used during actual testing operation. The test manipulations should be carried out in a clean room (class 10,000) under a laminar flow hood, with operators dressed in sterilized, static-free clothing, including head- and foot-wears. The air pressure in the testing room should be more than that of the exterior area. The performance of the laminar flow hood should be monitored by particulate count, settle plates, or slit-sampling devices, and the performance of the filters and ultraviolet lamps checked routinely. Regular, simultaneous control test with known sterile preparations are also advisable.

Culture Media

      The culture media used for sterility tests for bacteria and fungi should be capable of supporting the growth of a wide variety of micro-organisms, with both aerobic and anaerobic growth characteristics, including the types found in the environment of the manufacturing operations. More than one culture medium will generally be needed to fulfil these criteria.

      The following culture media have been found suitable for the test for Sterility. Fluid thioglycolate medium is intended primarily for the culture of anaerobic bacteria but will also sustain the growth of aerobic bacteria. Soybean-casein digest medium is intended primarily for the culture of aerobic bacteria but will also sustain the growth of fungi.

      A. Preparation

      Culture media for the tests may be prepared as described below, or dehydrated mixtures yielding similar formulations may be used, provided that, when reconstituted as directed by the manufacturer or distributor, they have growth-promoting properties equal or superior to those obtained from the formulae given herein. Media are sterilized in an autoclave using a validated process.

      I. FLUID THIOGLYCOLATE MEDIUM (FLUID MERCAPTO ACETATE MEDIUM)

L-Cystine	0.5 g
Sodium chloride	2.5 g
Dextrose monohydrate	5.5 g
Agar, granulated (moisture content not in excess of 15 per cent)	0.75 g
Yeast extract (water-soluble)	5.0 g
Pancreatic digest of casein	15.0 g
Sodium thioglycolate (or thioglycolic acid 0.3 ml)	0.5 g
Resazurin sodium Solution (0.01 per cent w/v, freshly prepared)	1.0 ml
Water	1000 ml

                                                       

      Mix and heat until solution is effected. Adjust the pH of the solution with sodium hydroxide TS so that, after sterilization, it will have a pH of 7.1±0.2. Filter while hot through a filter paper, if necessary. Transfer the medium to suitable containers that provide a ratio surface to depth of medium such that not more than the upper half of the medium has undergone a colour change indicative of oxygen uptake at the end of the incubation period, and sterilize as directed above. If more than the upper one-third of the medium has a pink colour, the medium may be restored once by heating the containers until the pink colour disappears. When ready for use, not more than the upper one-third of the medium in a container should have a pink colour.

      Use Fluid Thioglycolate Medium by incubating it under aerobic conditions.

      II. ALTERNATIVE FLUID THIOGLYCOLATE MEDIUM (for devices having tubes with small lumina)

L-Cystine	0.5 g
Sodium chloride	2.5 g
Dextrose monohydrate	5.5 g
Yeast extract (water-soluble)	5.0 g
Pancreatic digest of casein	15.0 g
Sodium thioglycolate (or thioglycolic acid 0.3 ml)	0.5 g
Water	1000 ml

      Mix and heat until solution is effected. Adjust the pH of the solution with sodium hydroxide TS so that, after sterilization, it will have a pH of 7.1±0.2. Filter, if necessary. Place in suitable vessels, and sterilize by steam under pressure (see Steam Sterilization under “Sterilization and Sterility Assurance” (Appendix 12). The medium is freshly prepared or heated on a steam-bath and allowed to cool just prior to use. Do not reheat.

      Use Alternative Thioglycolate Medium in a manner that will assure anaerobic conditions for the duration of the incubation period.

      III. SOYBEAN-CASEIN DIGEST MEDIUM

Pancreatic digest of casein	17.0 g
Papaic digest of soybean meal	3.0 g
Sodium chloride	5.0 g
Dipotassium hydrogenphosphate	2.5 g
Dextrose monohydrate	2.5 g
Water	1000 ml

      Dissolve the solids with water, heating slightly to effect a solution. Cool the solution to room temperature, and adjust the pH with sodium hydroxide VS so that, after sterilization, it will have a pH of 7.3±0.2. Filter, if necessary, and dispense into suitable containers. Sterilize as directed above or by a validated filtration process. Incubate under aerobic conditions.

      B. Properties and suitability

      All the media to be used must comply with the following tests, carried out on each batch before or in parallel with the test on the article being examined.

      (1) STERILITY Incubate portions of the media intended mainly for the detection of bacteria at 30º to 35º and those intended mainly for the detection of fungi at 20º to 25º for not  less than 14 days or by incubating uninoculated containers as negative controls during a sterility test procedure. No growth of micro-organisms occurs.

      (2) GROWTH PROMOTION Inoculate duplicate test containers of each medium with 10 to 100 viable microorganisms listed in Table 1, and incubate according to the conditions specified for it. The test media are satisfactory if evidence of growth appears within 5 days. This test can be conducted simultaneously with the use of the media for sterility test purposes. However, the sterility test is considered invalid if the sterility of the media or this growth promotion test is not successful.

      (3) VALIDATION TESTS FOR BACTERIOSTASIS AND FUNGISTASIS This validation is performed when the test for sterility has to be carried our on a new product or whenever there is a change in the experimental conditions of the test. The validation may be performed simultaneously with the test for sterility of the product to be examined, but before the results of this test are being interpreted. The procedures are as follows:

      Membrane Filtration Method: Filter the test sample and rinse the membrane with minimum of three 100-ml portions of the appropriate rinsing fluid. Inoculate the final rinse with less than 100 colony-forming units, for each appropriate micro-organism specified in Table 1. Repeat the rinse procedure on another filter that has not been exposed to the specimen under test. This filter will serve as the positive control. Incubate these filters for not more than 7 days under the condition indicated in Table 1 and compare the growth.

      If the growth of each test organism in the test containers is visually comparable to the growth in the positive control, use the same amounts of article, number and volume of rinses, and medium when conducting the sterility test. If the growth of the test organisms in the test containers is not visually comparable to that in the positive control, the amount of article used is bacteriostatic or fungistatic. Repeat the test, using a larger number of rinses. Changes in the type of membrane filter used and in the use of neutralizing agents, if

Table 1 Test Micro-organisms for Growth Promotion and the Validation Tests

available, may reduce the antimicrobial effect of the article. If five rinses, each of about 500 ml, fail to neutralize the antimicrobial residue on the test filter membrane, proceed with sterility test.

      Direct Inoculation Method: Inoculate two containers of each sterility test medium with less than 100 colony-forming units, using the volume of medium (see Tablet 3) for each appropriate micro-organism specified in Table 1. Add the specified portion of the article under test to one of the inoculated containers of each medium. The other inoculated container is the positive control. Repeat the procedure for each appropriate micro-organism, and incubate the containers at the appropriate temperature for not more than 7 days.

      If the growth of the test organisms in the test container is not visually comparable to that of the inoculated control container, the article is bacteriostatic or fungistatic. The use of a sterile neutralizing agent, such as polysorbate 80, lecithin, azolectin, or β-lactamase, may be appropriate. If a neutralizing agent is not effective, establish suitable increased volumes of medium. Use the smallest volume of medium in which the growth of test micro-organisms in the presence of the article is not adversely affected. If the medium volume is increased to 2000 ml and antimicrobial activity is still present, proceed with the sterility test using the 2000 ml of medium. Volumes of medium more than 2000 ml may be needed for testing medical devices to permit complete immersion of the device.

Sampling of Test Samples

      Unless otherwise directed in the individual monographs, test the number of articles specified in Table 2. If the contents of each article are of sufficient quantity (see Tables 3 and 4), they may be divided so that equal appropriate portions are added to each of the specified media. If each article does not contain sufficient quantities for each medium, use twice the number of articles indicated in Table 2.

Opening the containers

      Cleanse the exterior surfaces of ampoules and closures of vials and bottles with a suitable decontaminating agent, and gain access to the contents in suitable aseptic manner. If the vial contents are packaged under vacuum, admit sterile air by means of a suitable sterile device, such as a needle attached to a syringe barrel filled with nonabsorbent cotton.

      For purified cotton, gauze, surgical dressings, and related Pharmacopoeial articles, open the package or container aseptically.

Quantity of Article

      When using the Membrane Filtration Method, unless otherwise specified elsewhere in this Appendix or in the individual monograph, use whenever possible the entire contents of each container, but not less than the quantities specified in Tables 3 and 4. When using the Direct Inoculation Method, use the quantities indicated in Tables 3 and 4.

Incubation

      Unless otherwise directed in the individual monograph, incubate the test mixture for 14 days with Fluid thioglycolate medium or Alternative thioglycolate medium, where so indicated, at 30º to 35º, and with Soybean-casein digest medium at 20º to 25º. For products terminally sterilized by a validated moist heat process, incubate the test sample for not less than 7 days, if the Membrane Filtration Method is used.

Table 2 Minimum Number of Articles to Be Tested in Relation to the Number of Articles in the Batch

Table 3 Quantities of Article for Liquid Products* ​

Table 4 Quantities of Article for Solid Products​

Test for Sterility of the Article

      The test may be carried out using either Method I, Membrane Filtration, or Method II, Direct Inoculation, as described below, taking into account any modifications described in the appropriate section. Method I is to be preferred whenever the nature of the article to be examined permits, that is, for filterable liquids and for those miscible with, or soluble in, aqueous or oily solvents that do not have an antimicrobial effect under the conditions of the test.

      Method I: Membrane Filtration The sterility testing of the articles should be performed when feasible by membrane filtration of the test substances. This procedure is applicable in the sterility testing of nonbacteriostatic or non-fungistatic liquids or soluble powders, and is particularly appropriate where the article is an oil, an ointment, or a cream that can be put into solution with non-bacteriostatic or non-fungistatic dilution fluids or solvents. The membrane filtration technique is suitable also in the sterility testing of liquids and soluble powders that possess inherent bacteriostatic or fungistatic properties. Certain devices also may be appropriately tested for sterility of the critical pathways by the membrane filtration technique.

      Strict aseptic precautions are needed in the manipulations of the tests; the frequent use of negative controls is highly recommended.

      Apparatus A suitable unit consists of a closed reservoir and a receptacle between which a properly supported membrane or membranes of appropriate porosity is (are) placed. A membrane generally suitable for sterility testing has a nominal porosity of not more than 0.45 μm, and a diameter of approximately 47 mm. Cellulose nitrate filters, for example, are used for aqueous, oily and weakly alcoholic solutions and cellulose acetate filters, for example, for strongly alcoholic solutions. The membranes having hydrophobic edges or low product binding characteristics that minimize inhibitory product residue may be needed for certain products, e.g. for antibiotics. The apparatus must be so designed that solution to be examined can be introduced and filtered under aseptic conditions and it must permit the removal of the membrane for transfer to the culture medium or be suitable for carrying out the incubation after adding the culture medium to the apparatus itself. The entire unit may be assembled and sterilized with the membrane(s) in place prior to use in the test, or the membranes may be sterilized separately by steam under pressure, or by any method that yields proper performance.

      Where the article to be tested is an oil, sterilize the membrane separately, and after thorough drying, assemble the unit, using aseptic precautions.

      Dissolving, dilution and rinsing fluids The following fluids are to be used for dissolving, diluting or rinsing articles under tests for sterility. They must be sterile and do not have antibacterial or antifungal properties.

FLUID A Dissolve 1 g of either peptic digest of animal tissue or dried meat peptone in water to make 1000 ml, filter or centrifuge to clarify, if necessary. Adjust to a pH of 7.1±0.2, dispense into containers, and sterilize in an autoclave using a validated process.

FLUID B Prepare Fluid B by adding 1 g of polysorbate 80 to each litre of Fluid A. Adjust to pH 7.1±0.2, dispense into flasks, and sterilize in an autoclave using a validated process.

FLUID C Dissolve 5 g of either peptic digest of animal tissue or dried meat peptone, 3 g of beef extract and 10 g of polysorbate 80 in water to make 1000 ml, filter or centrifuge to clarify, if necessary. Adjust to a pH of 6.9±0.2, dispense into flasks, and sterilize in an autoclave using a validated process.

FLUID D Adjust, if necessary, the pH of isopropyl myristate to be used as Fluid D to not less than 5.5, and sterilize by filtration through a 0.22-μm membrane filter. Fluid D must also be free from antimicrobial properties.

      Generally, Fluid A is used for dissolving watersoluble solids, for dilution before filtration the liquid article miscible with aqueous vehicles, and for washing the membrane(s) by filtering through the latter thereafter. If the article under test contains lecithin or oil, substitute Fluid B for Fluid A.

      Fluid C is used for rinsing or washing of the filtration membrane(s), in case the article under test contains petrolatum.

      Fluid D is used to dissolve or dilute ointments and oils soluble in isopropyl myristate.

      Procedure Either one or two filtering units may be used, and, after filtration, the membrane half, or the whole membrane, is transferred to each of the medium used.

      (A) LIQUIDS Aseptically transfer a small quantity (sufficient to moisten the membrane) of a suitable, sterile diluting fluid (Fluid A or Fluid B) onto the membrane and filter. Remove liquids from test containers of the article being examined with a sterile pipette or with a sterile syringe and needle. For each medium to be used, transfer to a membrane not less than the quantity that is prescribed in Table 3, if necessary after diluting to about 100 ml with a suitable sterile diluting fluid. Filter immediately. (If the article is a viscous liquid or suspension not adaptable to rapid filtration, aseptically add a sufficient quantity of diluting fluid to the pooled sample to increase the flow rate.) In case the liquid being tested has antimicrobial properties, or contains a preservative, use Fluid A, or Fluid B and proceed as directed for Membrane Filtration Method under Validation Tests for Bacteriostasis and Fungistasis, but exclude inoculation of the final rinse with challenge organisms. 

      Either transfer a membrane to each of the culture media used, or transfer each medium onto a membrane in the apparatus, and seal the apparatus so that the medium remains on the membrane.

      Alternatively, transfer the combined quantity of the article being examined for both media to the membrane, diluting if necessary, filtering and washing as above. Aseptically remove and cut the membrane into two approximately equal parts and transfer one of them to each medium used.

      Incubate the media for not less than 14 days unless otherwise prescribed in the monograph, at 30º to 35º in the test intended to detect bacteria and at 20º to 25º in the test intended to detect fungi. In some cases, where the liquid is highly viscous and not readily filterable through one or two membranes, more than two filter assemblies may be needed. In such cases, half the number of membranes used are incubated in each medium, provided that the volumes and requirements for numbers of containers per medium are complied with.

      (B) OILS AND OILY SOLUTIONS For each medium, use not less than the quantity of the article being examined, that is prescribed in Table 3, if necessary after diluting to about 100 ml with Fluid D. Oils or oily solutions of sufficiently low viscosity may be filtered, with or without dilution, through a dry membrane. Viscous oils may be diluted as necessary with Fluid D. Allow the oil to penetrate the membrane and filter, applying pressure or suction gradually. Wash the membrane by filtering through it at least two successive quantities, each of approximately 200 ml, of Fluid B, and then wash with 100 ml of Fluid A. Complete the test described under Liquids, except that the sterility test medium to be used contains 1 g of polysorbate 80 per litre.

      (C) SOLUBLE SOLIDS For each medium, dissolve not less than the quantity of the article being examined that is prescribed in Table 4 in a suitable sterile fluid such as Fluid A and carry out the test described under Liquids using a membrane or membranes appropriate to the chosen fluid.

      (D) OINTMENTS AND CREAMS For each medium, dissolve not less than 100 mg form each of not less than 20 containers in at least 100 ml of Fluid D, warming if necessary, to not more than 40º. In exceptional cases it may be necessary to heat to not more than 45º. Use warm solutions for washing. Filter as rapidly as possible and complete the test as described under Oils and Oily Solutions.

      If the article under test contains petrolatum, use Fluid C in place of Fluid D for washing and moisten the membrane(s) with approximately 200 μl of Fluid C before the filtration operation begins, and keep the membrane(s) covered with liquid throughout the filtration operation for maximum efficiency of the filter. Following filtration of the specimen, wash the membrane(s) with three 100-ml portions of Fluid C. Treat the test membrane(s) as directed above.

      (E) DEVICES Devices that are required to contain sterile pathways may be tested for sterility by the membrane filtration technique as follows. 

      Aseptically pass a sufficient volume of Fluid B through each device tested so that not less than 100 ml is recovered from each device. Collect the fluids in sterile containers, and filter the entire volume collected through membrane(s) as described under Liquids.

      Method II: Direct Inoculation For each medium, use the quantity of the article being examined that is prescribed in Table 2. Eliminate any antimicrobial properties as previously described for Direct Inoculation Method under Validation Tests for Bacteriostasis and Fungistasis. Transfer the article directly into the culture medium so that volume of the product is not more than 10 per cent of the volume of the medium, unless otherwise prescribed. (A larger volume may be required if antimicrobial properties are eliminated by dilution.) For those liquid articles where it is necessary to use a large volume of the article being examined, it may be preferable to use a concentrated culture medium prepared in such a way that it takes account of the subsequent dilution. In appropriate cases the concentrated medium may be added directly to the article in its container.

      (A) LIQUIDS Unless otherwise directed in the individual monograph, test 20 units of the article with each medium. Sterility tests are applied to individual discrete units or to composites of such units.

      For liquid articles from each unit, use not less than the volumes of article and medium specified in Table 3. If the contents are of sufficient quantity, they may be divided so that portions are added to the two specified media.

      Remove liquids from test containers with a sterile pipette or with a sterile syringe and needle. Aseptically transfer the specified volume of the material from each test container to a vessel of culture medium. Mix the liquid with the medium, but do not aerate excessively. Incubate in the specified media for not less than 14 days.

      Where the material being tested renders the medium turbid, so that the presence or absence of microbial growth cannot be determined readily by visual examination, transfer suitable portions of the medium to fresh vessels of the same medium between the third and seventh days after the test is started. Continue incubation of the original and of the transfer vessels for not less than 7 additional days after the transfer and for a total of not less than 14 days.

      (B) OILY LIQUIDS For oily liquids use media to which have been added 1 per cent w/v of polysorbate 80 or another suitable emulsifying agent in an appropriate concentration, shown not to have antimicrobial properties under the conditions of the test. Proceed as directed under Liquids.

      Aerobic cultures containing oily liquids should be shaken gently each day during the incubation period.

      (C) OINTMENTS AND OILS INSOLUBLE IN ISOPROPYL MYRISTATE Select 20 containers, assign them to two groups of 10 containers, and treat each group as follows. Aseptically transfer 100 mg from each of the 10 containers to a flask containing 200 ml of a sterile, aqueous vehicle capable of dispersing the test material homogeneously throughout the fluid mixture. (The choice of dispersing agent incorporated in the aqueous vehicle may differ according to the nature of the ointment or oil. Before use, test the dispersing agent to ascertain that in the concentration used it has no significant antimicrobial effect during the time interval for all transfers, using the test procedures set forth in Direct Inoculation Method under Validation Tests for Bacteriostasis and Fungistasis.) Mix 20 ml of the fluid mixture so obtained with 200 ml of medium, and proceed as directed under Liquids.

      (D) INSOLUBLE SOLIDS Transfer a quantity of the product in the form of a dry solid (or prepare a suspension of the product by adding sterile diluent to the immediate container), corresponding to not less than the quantity indicated in Table 4. Transfer the material so obtained to 200 ml of Fluid thioglycolate medium, and mix. Similarly, transfer the same quantity to 200 ml of Soybean-casein digest medium, and mix. Proceed as directed under Liquids.

      (E) COTTON, GAUZE, SURGICAL DRESSINGS, AND RELATED ARTICLES From each package of cotton, rolled gauze, or gauze bandage being tested, remove with sterile instruments two or more portions of 100 to 500 mg each from the innermost part of the sample. From individually packaged single-use materials such as gauze pads, remove a single portion of 250 to 500 mg or the entire article in the case of small, i.e. 25- by 75-mm or smaller, adhesive absorbent bandages.

      Aseptically transfer these portions of the article to the similar number of containers of each medium, and incubate them as described above.

      (F) SUTURES Place five containers of the sutures being examined in a suitable antimicrobial solution containing crystal violet or another suitable dye, for not less than 3 hours. Remove with sterile forceps, and if the containers show no evidence of leakage, hold under aseptic conditions prior to testing. Open the containers aseptically, and with sterile instruments transfer sutures to separate containers of appropriate media. Incubate them as described above for not less than 14 days.

      Carry out also the test for the presence of antimicrobial activity in the suture being examined and ensure the neutralization of any inhibitory effects which, for catgut and other sutures, may be due, in part, to the methods of sterilization used or to the constituents of the tubing fluid.

      (G) STERILIZED DEVICES Articles can be immersed intact or disassembled. To ensure that device pathways are also in contact with the media, immerse the appropriate number of units per medium in a volume of medium sufficient to immerse the device completely, and incubate them as described above for not less than 14 days.

      For catheters where the inside lumen and outside are required to be sterile, either cut them into pieces such that the medium is in contact with the entire lumen or fill the lumen with medium, and then immerse the intact unit.

      For extremely large devices, immerse those portions of the device that are to come into contact with the patient in a volume of medium sufficient to achieve complete immersion of those portions.

Observation and Interpretation of Results

      At intervals during the incubation period and at its conclusion, examine the contents of all of the vessels for macroscopic evidence of microbial growth, such as the development of turbidity. If no evidence of growth is found, the material tested meets the requirements of the test for sterility. If evidence of microbial growth is found, and confirmed microscopically, the material test fails to meet the requirements of the test for sterility, unless it can be demonstreated that microbial growth observed in the test was due to inadequate aseptic sampling and testing technique rather than to intrinsic contamination of the article, the test is invalid and must be repeated. If microbial growth is not observed, the material tested meets the requirements of the sterility test. If microbial growth is observed and confimed microscopically, the material tested does not meet the requirements of the sterility test.