How Much Penicillin To Filter During Sterility Test

APPENDIX 10 MICROBIOLOGICAL TESTS

x.1 STERILITY TEST

The exam 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 weather of the exam. Nevertheless, because the sample to exist tested is randomly selected from the detail batch information technology represents, information technology is thereafter assumed that the whole batch passes the sterility examination, which is at present the merely method available to the various regime 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 prove of microbial contamination is obtained by the procedure given in this Pharmacopoeia, the result so obtained is conclusive of failure of the commodity to encounter the requirements of the test.

Examination Conditions

Adventitious microbial growth that is transmitted to an article or to inoculated examination culture media from the environs during the grade of a sterility exam invalidates the results of the examination. Hence, it is necessary to demonstrate that the proper precautions accept been taken to exclude inapplicable micro-organisms throughout the examination 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 non be used during bodily testing operation. The test manipulations should exist carried out in a clean room (form 10,000) under a laminar flow hood, with operators dressed in sterilized, static-costless clothing, including caput- and foot-wears. The air pressure in the testing room should be more that of the outside surface area. The performance of the laminar menstruum 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 command test with known sterile preparations are besides advisable.

Civilization Media

The culture media used for sterility tests for leaner 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 than 1 culture medium will by and large be needed to fulfil these criteria.

The following culture media have been plant suitable for the test for Sterility. Fluid thioglycolate medium is intended primarily for the culture of anaerobic leaner only will also sustain the growth of aerobic bacteria. Soybean-casein assimilate medium is intended primarily for the civilisation of aerobic bacteria but volition besides sustain the growth of fungi.

A. Preparation

Culture media for the tests may exist prepared as described below, or dehydrated mixtures yielding similar formulations may be used, provided that, when reconstituted as directed past 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 thou
Sodium chloride	2.5 g
Dextrose monohydrate	5.5 g
Agar, granulated (moisture content not in excess of xv per cent)	0.75 g
Yeast extract (water-soluble)	5.0 g
Pancreatic assimilate of casein	15.0 g
Sodium thioglycolate (or thioglycolic acrid 0.iii ml)	0.5 g
Resazurin sodium Solution (0.01 per cent w/v, freshly prepared)	1.0 ml
H2o	one thousand ml

Mix and oestrus until solution is effected. Adjust the pH of the solution withsodium hydroxide TS so that, after sterilization, information technology will have a pH of vii.1±0.2. Filter while hot through a filter newspaper, if necessary. Transfer the medium to suitable containers that provide a ratio surface to depth of medium such that non more the upper half of the medium has undergone a colour change indicative of oxygen uptake at the finish of the incubation period, and sterilize every bit directed in a higher place. If more the upper i-third of the medium has a pink colour, the medium may exist restored once by heating the containers until the pink colour disappears. When set up for use, not more than the upper one-third of the medium in a container should have a pink colour.

Apply Fluid Thioglycolate Medium past 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 k
Dextrose monohydrate	5.5 m
Yeast extract (water-soluble)	5.0 yard
Pancreatic assimilate of casein	xv.0 one thousand
Sodium thioglycolate (or thioglycolic acid 0.3 ml)	0.5 thou
Water	1000 ml

Mix and heat until solution is effected. Adjust the pH of the solution withsodium hydroxide TS then that, after sterilization, it will have a pH of 7.1±0.ii. Filter, if necessary. Identify in suitable vessels, and sterilize past steam nether force per unit area (meet 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 volition clinch anaerobic weather condition for the duration of the incubation period.

III.SOYBEAN-CASEIN Assimilate MEDIUM

Pancreatic digest of casein	17.0 g
Papaic digest of soybean meal	3.0 thousand
Sodium chloride	5.0 g
Dipotassium hydrogenphosphate	2.v 1000
Dextrose monohydrate	2.v g
H2o	1000 ml

Deliquesce the solids withwater, heating slightly to effect a solution. Cool the solution to room temperature, and adjust the pH withsodium hydroxide VS then that, after sterilization, it will have a pH of vii.3±0.2. Filter, if necessary, and dispense into suitable containers. Sterilize as directed above or past a validated filtration process. Incubate under aerobic conditions.

B. Properties and suitability

All the media to exist used must comply with the following tests, carried out on each batch earlier or in parallel with the test on the commodity 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 xiv days or by incubating uninoculated containers as negative controls during a sterility examination procedure. No growth of micro-organisms occurs.

(2)GROWTH PROMOTION Inoculate duplicate test containers of each medium with ten to 100 viable microorganisms listed in Table ane, and incubate according to the conditions specified for it. The exam media are satisfactory if evidence of growth appears within v days. This examination tin be conducted simultaneously with the use of the media for sterility test purposes. However, the sterility examination is considered invalid if the sterility of the media or this growth promotion test is non successful.

(three)VALIDATION TESTS FOR BACTERIOSTASIS AND FUNGISTASIS This validation is performed when the test for sterility has to exist carried our on a new production 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 production 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 advisable 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 some other filter that has not been exposed to the specimen under test. This filter will serve every bit the positive control. Incubate these filters for not more than 7 days under the status 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 book 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 exam, using a larger number of rinses. Changes in the blazon of membrane filter used and in the utilise of neutralizing agents, if

Tabular array 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, keep with sterility test.

Direct Inoculation Method: Inoculate ii containers of each sterility test medium with less than 100 colony-forming units, using the volume of medium (encounter Tablet 3) for each appropriate micro-organism specified in Tabular array 1. Add together the specified portion of the article nether exam to one of the inoculated containers of each medium. The other inoculated container is the positive control. Repeat the procedure for each advisable 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 non visually comparable to that of the inoculated control container, the article is bacteriostatic or fungistatic. The use of a sterile neutralizing agent, such equally polysorbate lxxx, lecithin, azolectin, or β-lactamase, may be appropriate. If a neutralizing agent is non effective, found 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 all the same nowadays, keep with the sterility examination using the 2000 ml of medium. Volumes of medium more 2000 ml may be needed for testing medical devices to permit consummate immersion of the device.

Sampling of Test Samples

Unless otherwise directed in the private monographs, examination the number of articles specified in Table 2. If the contents of each commodity are of sufficient quantity (meet Tables 3 and 4), they may be divided then that equal appropriate portions are added to each of the specified media. If each commodity does not incorporate sufficient quantities for each medium, apply twice the number of manufactures 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 admission to the contents in suitable aseptic manner. If the vial contents are packaged under vacuum, acknowledge 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 private monograph, apply whenever possible the entire contents of each container, simply not less than the quantities specified in Tables iii and iv. 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 exam mixture for fourteen days with Fluid thioglycolate medium or Alternative thioglycolate medium, where then 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 non less than vii days, if the Membrane Filtration Method is used.

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

Table iii Quantities of Article for Liquid Products* 

Tabular array 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 advisable department. Method I is to be preferred whenever the nature of the commodity to be examined permits, that is, for filterable liquids and for those miscible with, or soluble in, aqueous or oily solvents that practise not have an antimicrobial effect under the conditions of the exam.

Method I: Membrane Filtration The sterility testing of the manufactures should exist performed when feasible past 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 commodity is an oil, an ointment, or a foam that can be put into solution with non-bacteriostatic or not-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 hygienic precautions are needed in the manipulations of the tests; the frequent use of negative controls is highly recommended.

Appliance A suitable unit consists of a airtight reservoir and a receptacle betwixt 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 0.45μm, and a bore of approximately 47 mm. Cellulose nitrate filters, for instance, 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 depression product binding characteristics that minimize inhibitory product residue may be needed for sure 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 information technology must permit the removal of the membrane for transfer to the culture medium or be suitable for carrying out the incubation after calculation the culture medium to the apparatus itself. The entire unit of measurement may be assembled and sterilized with the membrane(southward) in place prior to use in the test, or the membranes may be sterilized separately past steam under pressure, or by whatsoever 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 fluidsThe following fluids are to be used for dissolving, diluting or rinsing articles nether tests for sterility. They must be sterile and do not have antibacterial or antifungal properties.

FLUID A Dissolve i g of either peptic digest of animal tissue or dried meat peptone inh2o to make 1000 ml, filter or centrifuge to clarify, if necessary. Adapt to a pH of seven.ane±0.2, dispense into containers, and sterilize in an autoclave using a validated process.

FLUID B Fix Fluid B by adding 1 one thousand 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 assimilate of beast tissue or dried meat peptone, 3 one thousand of beefiness extract and 10 g ofpolysorbate 80 inwater to make thousand ml, filter or centrifuge to clarify, if necessary. Arrange to a pH of six.9±0.2, manipulate into flasks, and sterilize in an autoclave using a validated procedure.

FLUID D Adjust, if necessary, the pH of isopropyl myristate to be used as Fluid D to non less than 5.5, and sterilize by filtration through a 0.22-μ1000 membrane filter. Fluid D must also exist complimentary from antimicrobial properties.

By and large, Fluid A is used for dissolving watersoluble solids, for dilution before filtration the liquid article miscible with aqueous vehicles, and for washing the membrane(due south) past 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, afterwards 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 later diluting to nearly 100 ml with a suitable sterile diluting fluid. Filter immediately. (If the article is a sticky liquid or break not adaptable to rapid filtration, aseptically add a sufficient quantity of diluting fluid to the pooled sample to increase the menstruum rate.) In case the liquid existence tested has antimicrobial properties, or contains a preservative, utiliseFluid A, or Fluid B and proceed as directed for Membrane Filtration Method underValidation Tests for Bacteriostasis and Fungistasis, only exclude inoculation of the concluding rinse with challenge organisms.

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

Alternatively, transfer the combined quantity of the commodity being examined for both media to the membrane, diluting if necessary, filtering and washing equally to a higher place. 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 fourteen days unless otherwise prescribed in the monograph, at 30º to 35º in the exam intended to detect bacteria and at 20º to 25º in the exam intended to find fungi. In some cases, where the liquid is highly viscous and non readily filterable through one or two membranes, more than than ii filter assemblies may exist needed. In such cases, one-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 afterward diluting to nearly 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 equally necessary with Fluid D. Let 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 and so launder with 100 ml of Fluid A. Complete the test described under Liquids, except that the sterility test medium to be used contains 1 g ofpolysorbate 80 per litre.

(C)SOLUBLE SOLIDS For each medium, dissolve not less than the quantity of the commodity being examined that is prescribed in Tabular array four in a suitable sterile fluid such equally Fluid A and comport out the test described under Liquids using a membrane or membranes appropriate to the chosen fluid.

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

If the commodity nether examination contains petrolatum, utilise Fluid C in place of Fluid D for washing and moisten the membrane(s) with approximately 200μfifty of Fluid C before the filtration functioning 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(south) with three 100-ml portions of Fluid C. Treat the test membrane(due south) as directed above.

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

Aseptically laissez passer a sufficient book 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 Two: Directly Inoculation For each medium, use the quantity of the article being examined that is prescribed in Table 2. Eliminate any antimicrobial backdrop every bit previously described forDirect Inoculation Method underValidation Tests for Bacteriostasis and Fungistasis. Transfer the article directly into the culture medium and then that volume of the production is not more than 10 per cent of the book of the medium, unless otherwise prescribed. (A larger volume may be required if antimicrobial properties are eliminated by dilution.) For those liquid manufactures where it is necessary to utilize a large volume of the article being examined, it may be preferable to use a concentrated civilization medium prepared in such a way that it takes account of the subsequent dilution. In appropriate cases the concentrated medium may exist added directly to the article in its container.

(A)LIQUIDS Unless otherwise directed in the private monograph, exam twenty units of the article with each medium. Sterility tests are practical 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 exist 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 non 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 7th days after the test is started. Keep incubation of the original and of the transfer vessels for not less than 7 additional days after the transfer and for a full of not less than 14 days.

(B)OILY LIQUIDS For oily liquids utilise media to which have been added one per cent w/v of polysorbate lxxx or another suitable emulsifying agent in an appropriate concentration, shown non to have antimicrobial properties under the conditions of the examination. Proceed as directed nether Liquids.

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

(C)OINTMENTS AND OILS INSOLUBLE IN ISOPROPYL MYRISTATE Select 20 containers, assign them to two groups of x 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 pick of dispersing agent incorporated in the aqueous vehicle may differ according to the nature of the ointment or oil. Before use, examination the dispersing agent to ascertain that in the concentration used it has no significant antimicrobial effect during the fourth dimension interval for all transfers, using the test procedures set up forth inDirect Inoculation Method underValidation Tests forBacteriostasis and Fungistasis.) Mix 20 ml of the fluid mixture then 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 ready 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 cloth and then 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.

(East) COTTON, GAUZE, SURGICAL DRESSINGS, AND RELATED ARTICLES From each bundle 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-utilize materials such every bit 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 absorptive 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 Identify v containers of the sutures being examined in a suitable antimicrobial solution containingcrystal violet or another suitable dye, for not less than iii hours. Remove with sterile forceps, and if the containers show no testify of leakage, concur under aseptic conditions prior to testing. Open the containers aseptically, and with sterile instruments transfer sutures to carve up containers of appropriate media. Incubate them as described above for not less than xiv days.

Carry out also the examination for the presence of antimicrobial action in the suture existence examined and ensure the neutralization of any inhibitory furnishings 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.

(Thou)STERILIZED DEVICES Articles can exist immersed intact or disassembled. To ensure that device pathways are besides 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 fourteen days.

For catheters where the inside lumen and outside are required to be sterile, either cutting 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 big devices, immerse those portions of the device that are to come into contact with the patient in a book of medium sufficient to achieve complete immersion of those portions.

Observation and Interpretation of Results

At intervals during the incubation menstruum and at its conclusion, examine the contents of all of the vessels for macroscopic evidence of microbial growth, such equally the evolution of turbidity. If no evidence of growth is found, the textile tested meets the requirements of the test for sterility. If evidence of microbial growth is found, and confirmed microscopically, the textile exam fails to come across the requirements of the examination for sterility, unless information technology 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 cloth tested does not come across the requirements of the sterility test.