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10.2 MICROBIAL LIMIT TESTS

10.2 MICROBIAL LIMIT TESTS

Introduction​

      The hazard of microbiological contamination in non-sterile pharmaceuticals has been well realized, especially in those products of vegetable, animal and mineral origins and in those which lack good manufacturing practices (GMP). Microbiological attributes of non-sterile pharmaceutical products are described in Appendix 10.4.

      This appendix, therefore, comprises two parts of tests. Part I allows quantitative enumeration of mesophilic bacteria and fungi that may grow under aerobic condition and Part II allows determination of the absence or limited occurrence of specified micro-organisms that may be detected under the conditions described.

      The tests are designed primarily to determine whether a substance or preparation complies with an established specification for microbiological quality.

When used for such purposes, follow the instructions given below, including the number of samples to be taken, and interpret the results as stated below.

      The methods are not applicable to products containing viable micro-organisms as active ingredients.

      Alternative microbiological procedures, including automated methods, may be used, provided that their equivalence to the Pharmacopoeial method has been demonstrated.

      Acceptance criteria for microbiological quality of non-sterile pharmaceuticals are given in the table under “Limits for Microbial Contamination” (Appendix 10.5).

      In the following, the term “micro-organisms” is covering bacteria and fungi only; the term “pharmaceuticals” means pharmaceutical products of any kind, from raw materials to the finished forms; the term “growth” is used to designate the presence and presumed proliferation of micro-organisms.

Part I Microbial Enumeration Tests

PROCEDURE

      In preparing for and in applying the tests, precautions are taken so as to avoid the accidental microbial contamination of the product to be examined, as well as the inadvertent suppression of the growth of any microorganisms that are to be revealed in the test.

      If the product to be examined has antimicrobial activity, this is insofar as possible removed or neutralized. If inactivators are used for this purpose, their efficacy and their absence of toxicity for micro-organisms must be demonstrated.

      If surface-active substances are used for sample preparation, their absence of toxicity for micro-organisms and their compatibility with inactivators used must be demonstrated.

ENUMERATION METHODS

      Use the membrane filtration method or the platecount method, as prescribed. The most probable number (MPN) method is generally the least accurate method for microbial counts; however, for certain product groups with a very low bioburden, it may be the most appropriate method. The choice of method is based on factors such as the nature of the product and the required limit of micro-organisms. The chosen method must allow testing of a sufficient sample size to judge compliance with the specification. The suitability of the method chosen must be established.

GROWTH PROMOTION TEST, SUITABILITY OF THE COUNTING METHOD AND NEGATIVE CONTROLS

      The ability of the test to detect micro-organisms in the presence of product to be tested must be established. Suitability must be confirmed if a change in testing performance, or the product, which may affect the outcome of the test is introduced.

      Preparation of test strains

      Use standardized stable suspensions of test strains or prepare them as stated below. Seed lot culture maintenance techniques (seed-lot systems) are used so that the viable micro-organisms used for inoculation are not more than five passages removed from the original master seed-lot. Grow each of the bacterial and fungal test strains separately as described in Table 1.

      Use Buffered sodium chloride-peptone solution pH 7.0 or Phosphate buffer pH 7.2 to make test suspensions; to suspend Aspergillus niger spores, 0.05 per cent of polysorbate 80 may be added to the buffer. Use the suspensions within 2 hours or within 24 hours if stored at 2° to 8°. As an alternative to preparing and then diluting a fresh suspension of vegetative cells of A. niger or Bacillus subtilis, a stable spore suspension is prepared and then an appropriate volume of the spore suspension is used for test inoculation. The stable spore suspension may be maintained at 2° to 8° for a validated period of time.

      Negative control

      To verify testing conditions, a negative control is performed using the chosen diluent in place of the test preparation. There must be no growth of micro-organisms. A negative control is also performed when testing the products as described in Testing of Products. A failed negative control requires an investigation.

      Growth promotion of the media

      Test each batch of ready-prepared medium and each batch of medium, prepared either from dehydrated medium or from the ingredients described.

      Inoculate portions/plates of Soybean-casein digest broth and Soybean-casein digest agar with a small number (not more than 100 CFU) of the micro-organisms indicated in Table 1, using a separate portion/plate of medium for each. Inoculate plates of Sabouraud dextrose agar with a small number (not more than 100 CFU) of the micro-organisms indicated in Table 1, using a separate plate of medium for each. Incubate in the conditions described in Table 1.

      For solid media, growth obtained must not differ by a factor greater than 2 from the calculated value for a standardized inoculum. For a freshly prepared inoculum, growth of the micro-organisms comparable to that previously obtained with a previously tested and approved batch of medium occurs. Liquid media are suitable if clearly visible growth of the micro-organisms comparable to that previously obtained with a previously tested and approved batch of medium occurs. 

      Suitability of the counting method in the presence of product

      PREPARATION OF THE SAMPLE The method for sample preparation depends upon the physical characteristics of the product to be tested. If none of the procedures described below can be demonstrated to be satisfactory, an alternative procedure must be developed.

      Water-soluble products Dissolve or dilute (usually a 1 in 10 dilution is prepared) the product to be examined in Buffered sodium chloride-peptone solution pH 7.0, Phosphate buffer solution pH 7.2 or Soybean-casein digest broth. If necessary, adjust to pH 6 to 8. Further dilutions, where necessary, are prepared with the same diluent.

Table 1 Preparation and Use of Test Micro-organisms

*ATCC = American Type Culture Collection, USA; DMST = Department of Medical Sciences, Thailand; NCIMB = National Collection of Industrial and Marine Bactéria Ltd., Great Britain; C.I.P. = Collection de Bactéries de l’Institut Pasteur, France; NBRC = Biological Resource Center, Japan; NCPF = National Collection of Pathogenic Fungi, London School of Hygiene and Tropical Medicine, Great Britain; I.P. = Collection Nationale de Culture de Micro-organismes (C.N.C.M.) Institut Pasteur, France; IMI = International Mycological Institute, Great Britain

      Non-fatty products insoluble in water Suspend the product to be examined (usually a 1 in 10 dilution is prepared) in Buffered sodium chloride-peptone solution pH 7.0, Phosphate buffer solution pH 7.2 or Soybeancasein digest broth. A surface-active agent such as 0.1 per cent w/v of polysorbate 80 may be added to assist the suspension of poorly wettable substances. If necessary, adjust to pH 6 to 8. Further dilutions, where necessary, are prepared with the same diluent.

      Fatty products Dissolve in isopropyl myristate, sterilized by filtration or mix the product to be examined with the minimum necessary quantity of sterile polysorbate 80 or another non-inhibitory sterile surface-active agent, heated if necessary to not more than 40°, or in exceptional cases to not more than 45°. Mix carefully and if necessary maintain the temperature in a waterbath. Add sufficient of the pre-warmed chosen diluent to make a 1 in 10 dilution of the original sample. Mix carefully while maintaining the temperature for the shortest time necessary for the formation of an emulsion. Further serial tenfold dilutions may be prepared using the chosen diluent containing a suitable concentration of sterile polysorbate 80 or another non-inhibitory sterile surface-active agent.

      Fluids or solids in aerosol form Chill the container(s) for approximately 1 hour, cut open the container(s), and allow to reach room temperature, permitting the propellant to escape, or warming to drive off the propellant if feasible. Aseptically transfer the product into a membrane filter apparatus or a sterile container for further sampling. Use either the total contents or a defined number of metered doses from each of the containers tested.

      Transdermal patches Remove the protective cover sheets (“release liners”) of the transdermal patches and place them, adhesive side upwards, on sterile glass or plastic trays. Cover the adhesive surface with a sterile porous material, for example sterile gauze, to prevent the patches from sticking together, and transfer the patches to a suitable volume of the chosen diluent containing inactivators such as polysorbate 80 and/or lecithin. Shake the preparation vigorously for at least 30 minutes.

      INOCULATION AND DILUTION Add to the sample prepared as described in Preparation of the Sample and to a control (with no test material included) a sufficient volume of the microbial suspension to obtain an inoculum of not more than 100 CFU. The volume of the suspension of the inoculum should not exceed 1 per cent of the volume of diluted product.

      To demonstrate acceptable microbial recovery from the product, the lowest possible dilution factor of the prepared sample must be used for the test. Where this is not possible due to antimicrobial activity or poor solubility, further appropriate protocols must be developed. If inhibition of growth by the sample cannot otherwise be avoided, the aliquot of the microbial suspension may be added after neutralization, dilution or filtration.

      NEUTRALIZATION/REMOVAL OF ANTIMICROBIAL ACTIVITY The number of micro-organisms recovered from the prepared sample diluted as described in Inoculation and Dilution and incubated following the procedure described in Recovery of Micro-organism in the Presence of Product, is compared to the number of microorganisms recovered from the control preparation.

      If growth is inhibited (reduction by a factor greater than 2), then modify the procedure for the particular enumeration test to ensure the validity of the results. Modification of the procedure may include, for example, (1) an increase in the volume of the diluent or culture medium, (2) incorporation of specific or general neutralizing agents into the diluent such as Casein digest-soy lecithin polysorbate 20 broth, (3) membrane filtration, or (4) a combination of the above measures.

      Neutralizing agents Neutralizing agents may be used to neutralize the activity of antimicrobial agents (Table 2). They may be added to the chosen diluent or the medium preferably before sterilization. If used, their efficacy and their absence of toxicity for microorganisms must be demonstrated by carrying out a blank with the neutralizer and without the product.

Table 2 Common Neutralizing Agents for Interfering Substances

      If no suitable neutralizing method can be found, it can be assumed that the failure to isolate the inoculated organism is attributable to the microbicidal activity of the product. This information serves to indicate that the product is not likely to be contaminated with the given species of the micro-organism. However, it is possible that the product only inhibits some of the micro-organisms specified herein, but does not inhibit others not included amongst the test strains or for which the latter are not representative. Then, perform the test with the highest dilution factor compatible with microbial growth and the specific acceptance criterion.

      RECOVERY OF MICRO-ORGANISMS IN THE PRESENCE OF PRODUCT For each of the micro-organisms listed, separate tests are performed. Only micro-organisms of the added test strain are counted. 

      Membrane filtration Use membrane filters having a nominal pore size not greater than 0.45 μm. The type of filter material is chosen such that the bacteria-retaining efficiency is not affected by the components of the sample to be investigated. For each of the microorganisms listed, one membrane filter is used.

      Transfer a suitable amount of the sample prepared as described under Preparation of the Sample, under Inoculation and Dilution, and under Neutralization/ Removal of Antimicrobial Activity (preferably representing 1 g of the sample, or less if large numbers of CFU are expected) to the membrane filter, filter immediately and rinse the membrane filter with an appropriate volume of diluent.

      For the determination of total aerobic microbial count (TAMC), transfer the membrane filter to the surface of Soybean-casein digest agar. For the determination of total combined yeasts and moulds count (TYMC), transfer the membrane to the surface of Sabouraud dextrose agar. Incubate the plates as indicated in Table 1. Perform the counting.

      Plate-count methods Perform plate-count methods at least in duplicate for each medium and use the mean count of the result.

      Pour-plate method For Petri dishes 9 cm in diameter, add to the dish 1 ml of the sample prepared as described under Preparation of the Sample, under Inoculation and Dilution, and under Neutralization Removal of Antimicrobial Activity and 15 to 20 ml of Soybean-casein digest agar or Sabouraud dextrose agar, both media being at not more than 45°. If larger Petri dishes are used, the amount of agar medium is increased accordingly. For each of the micro-organisms listed in Table 1, at least two Petri dishes are used. Incubate the plates as indicated in Table 1. Take the arithmetic mean of the counts per medium and calculate the number of CFU in the original inoculum. 

      Surface-spread method For Petri dishes 9 cm in diameter, add 15 to 20 ml of Soybean-casein digest agar or Sabouraud dextrose agar at about 45° to each Petri dish and allow to solidify. If larger Petri dishes are used, the volume of the agar is increased accordingly. Dry the plates, for example in a laminar-air-flow cabinet or an incubator. For each of the micro-organisms listed in Table 1, at least two Petri dishes are used. Spread a measured volume of not less than 0.1 ml of the sample prepared as described under Preparation of the Sample, under Inoculation and Dilution, and under Neutralization/Removal of Antimicrobial Activity over the surface of the medium. Incubate and count as prescribed under Pour-plate Method.

      Most probable number (MPN) method The precision and accuracy of the MPN method is less than that of the membrane filtration method or the plate-count method. Unreliable results are obtained particularly for the enumeration of moulds. For these reason the MPN method is reserved for the enumeration of TAMC in situations where no other method is available. If the use of the method is justified, proceed as follows.

      Prepare a series of at least three serial tenfold dilutions of the product as described under Preparation of the Sample, under Inoculation and Dilution, and under Neutralization/Removal of Antimicrobial Activity. From each level of dilution, three aliquots of 1 g or 1 ml are used to inoculate three tubes with 9 to 10 ml of Soybean-casein digest broth. If necessary, a surfaceactive agent such as polysorbate 80 or an inactivator of antimicrobial agents may be added to the medium. Thus, if three levels of dilution are prepared, nine tubes are inoculated.

      Incubate all tubes at 30° to 35° for not more than 3 days. If reading of the results is difficult or uncertain owing to the nature of the product to be examined, subculture in the same broth, or in Soybean-casein digest agar, for 1 to 2 days at the same temperature and use these results. Determine the most probable number of micro-organisms per g or per ml of the product to be examined from Table 3.

      Results and interpretation

      When verifying the suitability of the membrane filtration method or the plate-count method, a mean count of any of the test organisms not differing by a factor greater than 2 from the value of the control defined in Inoculation and Dilution in the absence of the product must be obtained. When verifying the suitability of the MPN method the calculated value from the inoculum must be within 95 per cent confidence limits of the results obtained with the control.

      If the above criteria cannot be met for one or more of the organisms tested with any of the described methods, the method and test conditions that come closest to the criteria are used to test the product.

TESTING OF PRODUCTS

      Amount used for the test

      Unless otherwise prescribed, use 10 g or 10 ml of the product to be examined taken with the precautions referred to above. For fluids or solids in aerosol form, sample 10 containers. For transdermal patches, sample 10 patches.

      The amount to be tested may be reduced for active substances that will be formulated in the following conditions: the amount per dosage unit (e.g., tablet, capsule, injection) is less than or equal to 1 mg or the amount per g or per ml (for preparations not presented in dosage units) is less than 1 mg. In these cases, the amount to be tested is not less than the amount present in 10 dosage units or 10 g or 10 ml of the product.

      For materials used as active substances where sample quantity is limited or batch size is extremely small (i.e., less than 1000 ml or 1000 g), the amount tested shall be 1 per cent of the batch unless a lesser amount is prescribed or justified and authorized.

      For products where the total number of entities in a batch is less than 200 (e.g., samples used in clinical trials), the sample size may be reduced to 2 units, or 1 unit if the size is less than 100.

Table 3 Most Probable Number (MPN) Values of Micro-organisms

      Select the sample(s) at random from the bulk material or from the available containers of the preparation. To obtain the required quantity, mix the contents of a sufficient number of containers to provide the sample 

      Examination of the product

      MEMBRANE FILTRATION Use a filtration apparatus designed to allow the transfer of the filter to the medium. Prepare the sample using a method that has been shown suitable as described in Growth Promotion Test, Suitability of the Counting Method and Negative Controls. Transfer the appropriate amount to each of two membrane filters and filter immediately. Wash each filter following the procedure shown to be suitable.

      For the determination of TAMC, transfer one of the membrane filters to the surface of Soybean-casein digest agar. For the determination of TYMC, transfer the other membrane to the surface of Sabouraud dextrose agar. Incubate the plate of Soybean-casein digest agar at 30° to 35° for 3 to 5 days and the plate of Sabouraud dextrose agar at 20° to 25° for 5 to 7 days. Calculate the number or CFU per g or per ml of product.

      When examining transdermal patches, filter 10 per cent of the volume of the preparation described under Preparation of the Sample separately through each of two sterile filter membranes. Transfer one membrane to Soybean-casein digest agar for TAMC and the other membrane to Sabouraud dextrose agar for TYMC.

      PLATE-COUNT METHODS

      Pour-plate method Prepare the sample using a method that has been shown to be suitable as described in Growth Promotion Test, Suitability of the Counting Method and Negative Controls. Prepare for each medium at least two Petri dishes for each level of dilution. Incubate the plates of Soybean-casein digest agar at 30° to 35° for 3 to 5 days and the plates of Sabouraud dextrose agar at 20° to 25° for 5 to 7 days. Select the plates corresponding to a given dilution and showing the highest number of colonies less than 250 for TAMC and 50 for TYMC. Take the arithmetic mean per culture medium of the counts and calculate the number of CFU per g or per ml of product.

      Surface-spread method Prepare the sample using a method that has been shown to be suitable as described in Growth Promotion Test, Suitability of the Counting Method and Negative Controls. Prepare at least two Petri dishes for each medium and each level of dilution. For incubation and calculation of the number of CFU proceed as described for the pour-plate method.

      MOST PROBABLE NUMBER METHOD

      Prepare and dilute the sample using a method that has been shown to be suitable as described in Growth Promotion Test, Suitability of the Counting Method and Negative Controls. Incubate all tubes at 30° to 35° for 3 to 5 days. Subculture if necessary, using the procedure shown to be suitable. Record for each level of dilution the number of tubes showing microbial growth. Determine the most probable number of micro-organisms per g or per ml of the product to be examined from Table 3.

      Interpretation of the results

      The total aerobic microbial count (TAMC) is considered to be equal to the number of CFU found using Soybean-casein digest agar; if colonies of fungi are detected on this medium, they are counted as part of the TAMC. The total combined yeasts and moulds count (TYMC) is considered to be equal to the number of CFU found using Sabouraud dextrose agar; if colonies of bacteria are detected on this medium, they are counted as part of the TYMC. When the TYMC is expected to exceed the acceptance criterion due to the bacterial growth, Sabouraud dextrose agar with antibiotics may be used. If the count is carried out by the MPN method the calculated value is the TAMC.

      The recommended solutions and media are described in Part II.

      The limits prescribed in the “Limits for Microbial Contamination” (Appendix 10.5) are the maximum acceptable limits.

      Part II Test for Specified Micro-organisms 

PROCEDURE

      The preparation of the samples is carried out as described in Part I.

      If the product to be examined has antimicrobial activity, this is insofar as possible removed or neutralized as described in Part I.

      If surface-active substances are used for sample preparation, their absence of toxicity for micro-organisms and their compatibility with inactivators used must be demonstrated as described in Part I.

GROWTH-PROMOTING AND INHIBITORY PROPERTIES OF THE MEDIA, SUITABILITY OF THE TEST AND NEGATIVE CONTROLS

      The ability of the test to detect micro-organisms in the presence of the product to be tested must be established. Suitability must be confirmed if a change in testing performance, or the product, which may affect the outcome of the test is introduced. 

      Preparation of test strains

      Use standardized stable suspensions of test strains or prepare them as stated below. Seed lot culture maintenance techniques (seed-lot systems) are used so that the viable micro-organisms used for inoculation are not more than five passages removed from the original master seed-lot.

      AEROBIC MICRO-ORGANISMS Grow each of thebacterial test strains separately in Soybean-casein digest broth or on Soybean-casein digest agar at 30° to 35° for 18 to 24 hours. Grow the test strain for Candida albicans separately on Sabouraud dextrose agar or in Sabouraud dextrose broth at 20° to 25° for 2 to 3 days.

– Staphylococcus aureus such as ATCC 6538, DMST 8013, NCIMB 9518, C.I.P. 4.83 or NBRC 13276;

– Pseudomonas aeruginosa such as ATCC 9027, DMST 15501, NCIMB 8626, C.I.P. 82.118 or NBRC 13275;

– Escherichia coli such as ATCC 8739, DMST 15537, NCIMB 8545, C.I.P. 53.126 or NBRC 3972;

– Salmonella enterica subsp. enterica serovar Typhimurium, such as ATCC 14028, DMST 13311, or, as an alternative, Salmonella enterica subsp. enterica serovar Abony such as NCTC 6017, DMST 21863, C.I.P. 80.39, or NBRC 100797;

– Candida albicans such as ATCC 10231, DMST 5815, NCPF 3179, I.P. 48.72 or NBRC 1594.

Use Buffered sodium chloride-peptone solution pH 7.0 or Phosphate buffer pH 7.2 to make test suspensions. Use the suspensions within 2 hours or within 24 hours if stored at 2° to 8°. 

CLOSTRIDIUM SPP. Use Clostridium sporogenes such as ATCC 11437 (DMST 15536, NCIMB 12343, C.I.P. 100651, NBRC 14293) or ATCC 19404 (DMST 15282, NCTC 532, C.I.P. 79.03). Grow the clostridial test strain under anaerobic conditions in Reinforced medium for clostridia at 30° to 35° for 24 to 48 hours. As an alternative to preparing and then diluting down a fresh suspension of vegetative cells of Cl. sporogenes, a stable spore suspension is used for test inoculation. The stable spore suspension may be maintained at 2° to 8° for a validated period.

      Negative control

      To verify testing conditions, a negative control is performed using the chosen diluent in place of the test preparation. There must be no growth of micro-organisms. A negative control is also performed when testing the products as described in Testing of Products. A failed negative control requires an investigation.

      Growth promotion and inhibitory properties of the media

      Test each batch of ready-prepared medium and each batch of medium prepared either from dehydrated medium or from ingredients.

      Verify suitable properties of relevant media as described in Table 4.

      TEST FOR GROWTH PROMOTING PROPERTIES, LIQUID MEDIA: inoculate a portion of the appropriate medium with a small number (not more than 100 CFU) of the appropriate micro-organism. Incubate at the specified temperature for not more than the shortest period of time specified in the test. Clearly visible growth of the micro-organism comparable to that previously obtained with a previously tested and approved batch of medium occurs.

      TEST FOR GROWTH PROMOTING PROPERTIES, SOLID MEDIA: perform the surface-spread method, inoculating each plate with a small number (not more than 100 CFU) of the appropriate micro-organism. Incubate at the specified temperature for not more than the shortest period of time specified in the test. Growth of the micro-organism comparable to that previously obtained with a previously tested and approved batch of medium occurs.

      TEST FOR INHIBITORY PROPERTIES, LIQUID OR SOLID MEDIA: inoculate the appropriate medium with at least 100 CFU of the appropriate micro-organism. Incubate at the specified temperature for not less than the longest period of time specified in the test. No growth of the test micro-organism occurs.

      TEST FOR INDICATIVE PROPERTIES: perform the surface-spread method, inoculating each plate with a small number (not more than 100 CFU) of the appropriate micro-organism. Incubate at the specified temperature for a period of time within the range specified in the test. Colonies are comparable in appearance and indication reactions to those previously obtained with a previously tested and approved batch of medium.

      Suitability of the test method

      For each product to be tested, perform the sample preparation as described in the following paragraph in Testing of Products. Add each test strain at the time of mixing, in the prescribed growth medium. Inoculate the test strains individually. Use a number of microorganisms equivalent to not more than 100 CFU in the inoculated test preparation. Perform the test as described in the following paragraph in Testing of Products using the shortest incubation period prescribed.

      The specified micro-organisms must be detected with the indication reactions as described in Testing of Products.

      Any antimicrobial activity of the sample necessitates a modification of the test procedure described in Neutralization/Removal of Antimicrobial Activity under Part I.

      If for a given product the antimicrobial activity with respect to a micro-organism for which testing is prescribed cannot be neutralized, then it is to be assumed that the inhibited micro-organism will not be present in the product.

TESTING OF PRODUCTS

      Bile-tolerant gram-negative bacteria

      SAMPLE PREPARATION AND PRE-INCUBATION Prepare a sample using a 1 in 10 dilution of not less than 1 g of the product to be examined as described in Part I, but using Soybean-casein digest broth as the chosen diluent, mix and incubate at 20° to 25° for a time sufficient to resuscitate the bacteria but not sufficient to encourage multiplication of the organisms (usually 2 hours but not more than 5 hours).

      TEST FOR ABSENCE Use the volume corresponding to 1 g of the product, as prepared in Sample Preparation and Pre-incubation, to inoculate Enterobacteria enrichment broth-Mossel. Incubate at 30° to 35° for 24 to 48 hours. Subculture on plates of Violet red bile dextrose agar. Incubate at 30° to 35° for 18 to 24 hours. The product passes the test if there is no growth of colonies of Gram-negative bacteria on any plate.

      QUANTITATIVE TEST

      Selection and subculture Inoculate suitable quantities of Enterobacteria enrichment broth-Mossel with the preparation as described under Sample Preparation and Pre-incubation and/or dilutions of it containing respectively 0.1 g (or 0.1 ml), 0.01 g (or 0.01 ml), and 0.001 g

Table 4 Growth Promoting, Inhibitory and Indicative Properties of Media

(or 0.001 ml) of the sample to be examined. Incubate at 30° to 35° for 24 to 48 hours. Subculture each of the cultures on a plate of Violet red bile dextrose agar to obtain selective isolation. Incubate at 30° to 35° for 18 to 24 hours. 

      Interpretation Growth of well-developed colonies, generally red or reddish, of Gram-negative bacteria constitutes a positive result. Note the smallest quantity of the product that gives a positive result and the largest quantity that gives a negative result. Determine from Table 5 the probable number of bacteria.

      Salmonella species

      SAMPLE PREPARATION AND PRE-INCUBATION Prepare the product to be examined as described in Part I, and use the portion corresponding to not less than 10 g or 10 ml to inoculate a suitable amount (determined as described under Suitability of the Test Method) of Soybean-casein digest broth, mix and incubate at 30° to 35° for 18 to 24 hours. 

      SELECTION AND SUBCULTURE Separately transfer 0.1 ml and 1 ml of the enrichment culture to 10 ml of Rappaport-Vassiliadis broth and Tetrathionate bile brilliant green broth, respectively, mix and incubate at 30° to 35° for 18 to 24 hours. Subculture on plates of Xylose-lysine-deoxycholate agar, Brilliant green agar, and Bismuth sulfite agar. Cover and invert the dishes, and incubate at 30° to 35° for 18 to 48 hours.

      INTERPRETATION Upon examination, if none of the colonies conforms to the description given in Table 6, the product meets the requirements of the test for absence of the genus Salmonella. If colonies of Gramnegative rods matching the description in Table 6 are found, proceed with further identification.

      IDENTIFICATION Transfer representative suspect colonies individually, by means of an inoculating wire, to a butt-slant tube of Triple sugar-iron agar by first streaking the surface of the slant and then stabbing the wire well beneath the surface, and incubate. If the examination discloses no evidence of tubes having alkaline (red) slants and acid (yellow) butts (with or without concomitant blackening of the butt from hydrogen sulfide production), the product meets the requirements of the test for absence of the genus Salmonella. The presence of Salmonella may be confirmed by other suitable cultural or biochemical and serological tests, if necessary.

Table 5 Probable Number of Bacteria

Table 6 Morphology Characteristics of Salmonella Species on Selective Agar Media​

      Escherichia coli

      SAMPLE PREPARATION AND PRE-INCUBATION Prepare a sample using a 1 in 10 dilution of not less than 1 g of the product to be examined as described in Part I, and use 10 ml or the portion corresponding to 1 g or 1 ml to inoculate a suitable amount (determined as described under Suitability of the Test Method) of Soybean-casein digest broth, mix and incubate at 30° to 35° for 18 to 24 hours. 

      SELECTION AND SUBCULTURE Transfer 1 ml of the enrichment culture to 100 ml of MacConkey broth and incubate at 42° to 44° for 24 to 48 hours. Subculture on plates of MacConkey agar and incubate at 30° to 35° for 18 to 72 hours.

      INTERPRETATION Upon examination, if none of the colonies conforms to the description given in Table 7, the product meets the requirements of the test for absence of Escherichia coli. If colonies matching the description in Table 7 are found, proceed with further identification.

      IDENTIFICATION Transfer the suspect colonies individually, making subculture the suspect colonies individually on plates of Levine eosin-methylene blue agar, and incubate at 30° to 35° for 18 to 24 hours.

      Upon examination, if none of the colonies exhibits both a characteristic metallic sheen under reflected light and a blue-black appearance under transmitted light, the product meets the requirements of the test for absence of Escherichia coli. The presence of Escherichia coli may be confirmed by suitable cultural and, if necessary, biochemical tests. Further serological test may be performed.

      Staphylococcus aureus and Pseudomonas aeruginosa

      SAMPLE PREPARATION AND PRE-INCUBATION Prepare a sample using a 1 in 10 dilution of not less than 1 g of the product to be examined as described in Part I, and use 10 ml or the portion corresponding to 1 g or 1 ml to inoculate a suitable amount (determined as described under Suitability of the Test Method) of Soybean-casein digest broth and mix. When testing transdermal patches, filter the volume of sample corresponding to one patch of the preparation described under Preparation of the Sample in Part I through a sterile filter membrane and place in 100 ml of Soybean-casein digest broth. Incubate at 30° to 35° for 18 to 24 hours.

      SELECTION AND SUBCULTURE If growth is present, use an inoculating loop to streak a portion of the culture medium on the surface of Mannitol-salt agar, or BairdParker agar, or Vogel-Johnson agar and of Cetrimide agar, and incubate at 30° to 35° for 18 to 72 hours.

      INTERPRETATION Upon examination, if none of the plates contains colonies having the characteristics listed in Tables 8 and 9 for the media used, the product meets the requirements for the absence of Staphylococcus aureus and Pseudomonas aeruginosa. If colonies matching the description in Tables 8 and 9 are found, proceed with further identification.

      IDENTIFICATION

      Coagulase test (for Staphylococcus aureus) With the aid of an inoculating loop, transfer representative suspect colonies from the agar surfaces of the Mannitolsalt agar (or Baird-Parker agar or Vogel-Johnson agar) to individual tubes, each containing 0.5 ml of mammalian, preferably rabbit or horse, plasma with or without suitable additives. Incubate in a water-bath at 37°, examining the tubes at 3 hours and subsequently at suitable intervals up to 24 hours. Test positive and negative controls simultaneously with the unknown products. If no coagulation in any degree is observed, the product meets the requirements of the test for absence of Staphylococcus aureus.

      Oxidase and pigment tests (for Pseudomonas aeruginosa) With the aid of an inoculating loop, streak representative suspect colonies from the agar surfaces of Cetrimide agar on the agar surface of Pseudomonas agar for detection of fluorescin and Pseudomonas agar for detection of pyocyanin contained in Petri dishes. Cover and invert the inoculated media, and incubate at

Table 7 Morphology Characteristics of Escherichia coli on MacConkey Agar

Table 8 Morphology Characteristics of Staphylococcus aureus on Selective Agar Media​

Table 9 Morphology and Diagnostic Characteristics of Pseudomonas aeruginosa on Selective Agar Media​

30° to 35° for not less than 3 days. Examine the streaked surfaces under UV light. Examine the plates to determine whether colonies having the characteristics listed in Table 9 are present.

      Confirm any suspect colonial growth on one or more of the media as Pseudomonas aeruginosa by means of the oxidase test. Upon the colonial growth, place or transfer colonies to strips or discs of filter paper that previously has been impregnated with N,N-dimethyl-pphenylenediamine dihydrochloride. If there is no development of a pink colour, changing to purple, the product meets the requirements of the test for the absence of Pseudomonas aeruginosa. The presence of Pseudomonas aeruginosa may be confirmed by suitable cultural and, if necessary, biochemical tests.

      Candida albicans

      SAMPLE PREPARATION AND PRE-INCUBATION Prepare the product to be examined as described under Preparation of the Sample and use 10 ml or the portion corresponding to 1 g or 1 ml to inoculate a suitable amount (determined as described under Suitability of the Test Method) of Sabouraud dextrose broth and mix. Incubate at 30° to 35° for 3 to 5 days.

      SELECTION AND SUBCULTURE Subculture on a plate of Sabouraud dextrose agar and incubate at 30° to 35° for 24 to 48 hours.

      INTERPRETATION When growth of white colonies may indicate the presence of Candida albicans occurs, proceed with further identification.

      IDENTIFICATION Transfer the suspect colonies individually, making subculture the suspect colonies individually on plates of a suitable selective medium1 .

      Upon examination, the product passes the test if there is no growth of colonies of Candida albicans on any plate.

      Clostridium spp.

      SAMPLE PREPARATION AND HEAT TREATMENT Prepare the product to be examined as described under Preparation of the Sample in Part I. Use two 10-ml portions each corresponding to 1 g or 1 ml of the product to be examined to inoculate a suitable amount (determined as described under Suitability of the Test Method) of Reinforced medium for clostridia. Heat one portion at 80° for 10 minutes and cool rapidly. Do not heat the other portion. Incubate both containers under anaerobic conditions at 30° to 35° for 48 hours.

      SELECTION AND SUBCULTURE After incubation, make subcultures from each container on plates of Columbia agar to which gentamicin has been added and incubate under anaerobic conditions at 30° to 35° for 48 to 72 hours.

      INTERPRETATION If no growth occurs, the product passes the test for absence of Clostridium spp. When growth of rods (with or without endospores) giving a negative catalase reaction occurs, subculture each distinct colony from on plates of Columbia agar, without gentamicin, and incubate at 30° to 35° for 48 to 72 hours, one plate anaerobically and the other aerobically, to check that the organism will not grow under aerobic condition.

      Examine the appearance of only anaerobic growth of Gram-positive bacilli giving a negative catalase reaction together with the extent of hemolysis, by making subculture on a plate of Defibrinated sheep blood agar, and also examine microscopically for spore formation, using Gram stain or spore stain technique and confirmed by further suitable biochemical and biological tests. The description in Table 10 gives the characteristics of some Clostridium species on Defibrinated sheep blood agar. 

Table 10 Characteristics of Clostridium Species on Defribrinated Sheep Blood Agar

Buffer Solution and Media​

       Culture media may be prepared as follows, or dehydrated culture media may be used if they have similar or comparable nutritive and selective properties for the micro-organisms to be tested for.

      In preparing the media according to the formulae set forth herein, dissolve the soluble solids in the water, using heat, if necessary, to effect complete solution, and add other ingredients. Add, if necessary, a solution of hydrochloric acid or sodium hydroxide in quantities sufficient to yield the desired pH in the medium when it is ready for use. Determine the pH at 25°±2°.

      Where agar is called for in a formula, use agar that has a moisture content of not more than 15 per cent.

      Unless otherwise indicated, the buffer solution and media should be dispensed and sterilized by heating in an autoclave at 121°±2° for not less than 15 minutes, depending on the volume to be sterilized. Store under refrigeration.


1Biggy agar, CHROMagar Candida, or Candida isolation agar is recommended.

BUFFER SOLUTION

      Stock buffer solution​

      Place 34 g of potassium dihydrogenphosphate in a 1000-ml volumetric flask, dissolve in 500 ml of water, adjust to pH 7.2±0.2 with sodium hydroxide, dilute to 1000.0 ml with water and mix. Dispense into containers and sterilize. Store at 2° to 8°.

      Phosphate buffer pH 7.2

      Prepare a mixture of 1 volume of stock buffer solution and 800 volumes of water and sterilize.

      Buffered sodium chloride-peptone solution pH 7.0

Potassium dihydrogenphosphate 3.6 g
Disodium hydrogenphosphate dihydrate 7.2 g
Sodium chloride 4.30 g
Peptone, dried 1.0 g
Water 1000 ml 

      Polysorbate 20 or 80 may be added to obtain a 0.1 to 1.0 per cent w/v solution.

      pH after sterilization: 7.0±0.1.

MEDIA

      Baird-Parker agar

Pancreatic digest of casein 10.0 g
Beef extract 5.0 g
Yeast extract 1.0 g
Lithium chloride 5.0 g
Agar 20.0 g
Glycine 12.0 g
Sodium pyruvate 10.0 g
Water 950 ml

      Heat with frequent agitation, and boil for 1 minute. Sterilize, cool to between 45° and 50°, and add 10 ml of a sterile, 1 per cent w/v solution of potassium tellurate(IV) and 50 ml of egg-yolk emulsion. Mix intimately but gently, and pour into plates.

      pH after sterilization: 6.8±0.2.

      Preparation of the egg-yolk emulsion: Disinfect the surface of whole shell eggs, aseptically crack the eggs, and separate out intact yolks into a sterile graduated cylinder. Add saline TS to obtain a 3 to 7 ratio of eggyolk to saline. Add to a sterile blender cup, and mix at high speed for 5 seconds.

      Bismuth sulfite agar

Beef extract 5.0 g
Pancreatic digest of casein 5.0 g
Peptic digest of animal tissue 5.0 g
Dextrose monohydrate  5.0 g
Disodium hydrogenphosphate heptahydrate 4.0 g
Iron(II) sulfate 0.3 g
Bismuth sulfite indicator 8.0 g
Agar 20.0 g
Brilliant green 25.0 mg
Water 1000 ml

      Heat the mixture of solids and water, with swirling, just to the boiling point. Do not overheat or sterilize. Transfer at once to a water-bath maintained at about 50°, and pour into plates as soon as the medium has cooled.

      Final pH: 7.6±0.2.

      Brilliant green agar

Yeast extract 3.0 g
Peptic digest of animal tissue 5.0 g
Pancreatic digest of casein 5.0 g
Lactose 10.0 g
Sodium chloride 5.0 g
Sucrose 10.0 g
Phenol red 80.0 mg
Agar 20.0 g
Brilliant green 12.5 mg
Water 1000 ml

      Boil the solution of solids for 1 minute. Sterilize just prior to use. Melt the medium, pour into Petri dishes, and allow to cool.

      pH after sterilization: 6.9±0.2.

      Casein digest-soy lecithin polysorbate 20 broth

Pancreatic digest of casein 20.0 g
Soy lecithin 5.0 g
Polysorbate 20 40 ml
Water 960 ml 

      Dissolve pancreatic digest of casein and soy lecithin in 960 ml of water, heating in a water-bath at 48° to 50° for about 30 minutes to effect solution. Add 40 ml of polysorbate 20. Mix and dispense as desired.

      pH after sterilization: 7.3±0.2. 

      Cetrimide agar

Pancreatic digest of casein 20.0 g
Magnesium chloride 1.4 g
Potassium sulfate 10.0 g
Agar 13.6 g
Cetrimide 0.3 g
Glycerol 10.0 ml
Water 1000 ml

      Dissolve all solid components in water, and add glycerol. Heat, with frequent agitation, and boil for 1 minute to effect solution.

      pH after sterilization: 7.2±0.2. 

      Columbia agar

Pancreatic digest of casein 10.0 g
Peptic digest of animal tissue 5.0 g
Heart pancreatic digest 3.0 g
Yeast extract 5.0 g
Maize starch 1.0 g
Sodium chloride  5.0 g
Agar, according to gelling power 10.0 to 15.0 g
Water 1000 ml

      Hydrate the agar, and dissolve by heating to boiling with continuous stirring. Sterilize, cool to between 45° and 50° and add, where necessary, gentamicin sulfate corresponding to 20 mg of gentamicin base. Pour into Petri dishes.

      pH after sterilization: 7.3±0.2.

      Defibrinated sheep blood agar (Blood agar)

      Heat Soybean-casein digest agar and cool to 45° to 50° in a water-bath. Add sufficient amount of defibrinated sheep blood to make 5 per cent and mix.

      Enterobacteria enrichment broth-Mossel

Pancreatic digest of gelatin 10.0 g
Dextrose monohydrate 5.0 g
Dehydrated ox bile 20.0 g
Potassium dihydrogenphosphate 2.0 g
Disodium hydrogenphosphate dihydrate 8.0 g
Brilliant green 15.0 mg
Water 1000 ml

      Mix and heat at 100° for 30 minutes to sterilize and cool immediately. Do not autoclave.

      Final pH: 7.2±0.2.

      Lactose broth

Beef extract 3.0 g
Pancreatic digest of gelatin 5.0 g
Lactose 5.0 g
Water 1000 ml

      Cool as quickly as possible after sterilization.

      pH after sterilization: 6.9±0.2.

      Levine eosin-methylene blue agar

Pancreatic digest of gelatin 10.0 g
Dipotassium hydrogenphosphate 2.0 g
Agar 15.0 g
Lactose 10.0 g
Eosin Y 0.4 g
Methylene blue 65.0 mg
Water 1000 ml

      Dissolve pancreatic digest of gelatin, dipotassium hydrogenphosphate and agar in water, with warming, and allow to cool. Just prior to use, liquefy the gelled agar solution, add the remaining ingredients, as solutions, in the following amounts, and mix: for each 100 ml of the liquefied agar solution 5 ml of a 20 per cent w/v solution of lactose, 2 ml of a 2 per cent w/v solution of eosin Y, and 2 ml of a 0.33 per cent w/v solution of methylene blue. The finished medium may not be clear.

      pH after sterilization: 7.1±0.2.

      MacConkey agar

Pancreatic digest of gelatin 17.0 g
Pancreatic digest of casein 1.5 g
Peptic digest of animal tissue 1.5 g
Lactose 10.0 g
Bile salts mixture 1.5 g
Sodium chloride 5.0 g
Agar 13.5 g
Neutral red 30.0 mg
Crystal violet 1.0 mg 
Water 1000 ml 

      Boil the mixture of solids and water for 1 minute to effect solution.

      pH after sterilization: 7.1±0.2.

      MacConkey broth

Pancreatic digest of gelatin 20.0 g
Lactose 10.0 g
Dehydrated ox bile 5.0 g
Bromocresol purple 10.0 mg
Water 1000 ml

      Prepare as directed under Buffer Solution and Media.

      pH after sterilization: 7.3±0.2. 

      Mannitol-salt agar

Pancreatic digest of casein 5.0 g
Papaic digest of animal tissue 5.0 g
Beef extract 1.0 g
Mannitol 10.0 g
Sodium chloride 75.0 g
Agar 15.0 g
Phenol red 25.0 mg
Water 1000 ml

      Mix, then heat with frequent agitation, and boil for 1 minute to effect solution.

      pH after sterilization: 7.4±0.2.

      Potato dextrose agar

      Cook 300 g of peeled and diced potatoes in 500 ml of water prepared by distillation, filter through cheesecloth, add water prepared by distillation to make 1000 ml, and add the following:

Agar 15.0 g
Dextrose monohydrate 20.0 g

      Dissolve by heating and sterilize.

      pH after sterilization: 5.6±0.2.

      For use, just prior to pouring the plates, adjust the melted and cooled to 45° medium with a sterile 10 per cent w/v solution of tartaric acid to a pH of 3.5±0.1. Do not reheat the pH 3.5 medium.

      Pseudomonas agar for detection of fluorescin

Pancreatic digest of casein 10.0 g
Peptic digest of animal tissue 10.0 g
Dipotassium hydrogenphosphate 1.5 g
Magnesium sulfate 1.5 g
Agar 15.0 g
Glycerol 10.0 ml
Water 1000 ml

      Dissolve the solid components in water before adding glycerol. Heat, with frequent agitation, and boil for 1 minute to effect solution.

      pH after sterilization: 7.2±0.2.

      Pseudomonas agar for detection of pyocyanin

Pancreatic digest of gelatin 20.0 g
Magnesium chloride 3.0 g
Potassium sulfate 10.0 g
Agar 15.0 g
Glycerol 10.0 ml
Water 1000 ml

      Dissolve the solid components in water before adding glycerol. Heat, with frequent agitation, and boil for 1 minute to effect solution.

      pH after sterilization: 7.2±0.2.

      Rappaport-Vassiliadis broth

Soya peptone 4.5 g
Sodium chloride 8.0 g
Dipotassium phosphate 0.4 g
Potassium dihydrogenphosphate 0.6 g
Magnesium chloride 29.0 g
Malachite green 36.0 mg
Water 1000 ml

      Mix and heat to effect solution.

      pH after sterilization: 5.2±0.2. 

      Reinforced medium for clostridia

Beef extract 10.0 g
Peptone 10.0 g
Yeast extract 3.0 g
Soluble starch 1.0 g
Dextrose monohydrate 5.0 g
Cysteine hydrochloride 0.5 g
Sodium chloride 5.0 g
Sodium acetate 3.0 g
Agar 0.5 g
Water 1000 ml 

      Hydrate the agar, and dissolve by heating to boiling with continuous stirring.

      pH after sterilization: 6.8±0.2.

      Sabouraud dextrose agar

Dextrose monohydrate 40.0 g
Mixture of equal parts of peptic digest of animal tissue and pancreatic digest of casein 10.0 g
Agar 15.0 g
Water 1000 ml

      Mix and boil to effect solution. 

      pH after sterilization: 5.6±0.2.

      Sabouraud dextrose agar with antibiotics

Dextrose monohydrate 40.0 g
Mixture of equal parts of peptic digest of animal tissue and pancreatic digest of casein 10.0 g
Agar 15.0 g
Water 1000 ml

      Mix and boil to effect solution. Immediately before use, add 0.10 g of benzylpenicillin sodium and 0.10 g of tetracycline per litre of medium as sterile solutions or alternatively, add 50 mg of chloramphenicol per litre of medium before sterilization.

      pH after sterilization: 5.6±0.2.

      (Note Other antibiotics can all be used, individually or in combination.)

      Sabouraud dextrose broth

Dextrose monohydrate 20.0 g
Mixture of equal parts of peptic digest of animal tissue and pancreatic digest of casein 10.0 g
Water 1000 ml

      Prepare as directed under Buffer Solution and Media.

      pH after sterilization: 5.6±0.2.

      Soybean-casein digest agar

Pancreatic digest of casein 15.0 g
Papaic digest of soybean meal 5.0 g
Sodium chloride 5.0 g
Agar 15.0 g
Water 1000 ml

      Prepare as directed under Buffer Solution and Media.

      pH after sterilization: 7.3±0.2.

      Soybean-casein digest broth

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

      Prepare as directed under Buffer Solution and Media.

      pH after sterilization: 7.3±0.2. 

Tetrathionate bile brilliant green broth

Peptone 8.6 g
Ox bile, dried 8.0 g
Sodium chloride 6.4 g
Calcium carbonate 20.0 g
Potassium tetrathionate 20.0 g
Brilliant green 70.0 mg 
Water 1000 ml

      Heat the solution of solids to boiling. Do not reheat.

      Final pH: 7.0±0.2.

      Triple sugar-iron-agar

Pancreatic digest of casein 10.0 g
Pancreatic digest of animal tissue 10.0 g
Lactose 10.0 g
Sucrose 10.0 g
Dextrose monohydrate 1.0 g
Ammonium iron(II) sulfate 0.2 g
Sodium chloride 5.0 g
Sodium thiosulfate 0.2 g
Agar 13.0 g
Phenol red 25.0 mg
Water 1000 ml

      Prepare as directed under Buffer Solution and Media.

      pH after sterilization: 7.3±0.2.

      Violet red bile dextrose agar

Yeast extract 3.0 g
Pancreatic digest of gelatin 7.0 g
Bile salts mixture 1.5 g
Lactose 10.0 g
Sodium chloride 5.0 g
Dextrose monohydrate 10.0 g
Agar 15.0 g
Neutral red 30.0 mg
Crystal violet 2.0 mg
Water 1000 ml

      Mix and heat to boiling. Do not overheat or sterilize. Transfer at once to a water-bath maintained at about 50°, and pour into plates as soon as the medium has cooled.

      Final pH: 7.4±0.2.

      Vogel-Johnson agar

Pancreatic digest of casein 10.0 g
Yeast extract 5.0 g
Mannitol 10.0 g
Dipotassium hydrogenphosphate 5.0 g
Lithium chloride 5.0 g
Glycine 10.0 g
Agar 16.0 g
Phenol red 25.0 mg
Water 1000 ml

      Boil the solution of solids for 1 minute. Sterilize, cool to between 45° and 50°, and add 20 ml of a sterile 1 per cent w/v solution of potassium tellurate(IV).

      pH after sterilization: 7.2±0.2.

      Xylose-lysine-deoxycholate agar

Xylose 3.5 g
L-Lysine 5.0 g
Lactose 7.5 g
Sucrose 7.5 g
Sodium chloride 5.0 g
Yeast extract 3.0 g
Agar 13.5 g
Sodium desoxycholate 2.5 g
Sodium thiosulfate 6.8 g
Ammonium iron(III) citrate 0.8 g
Phenol red 80.0 mg
Water 1000 ml 

      Heat the mixture of solids and water, with swirling, just to the boiling point. Do not overheat or sterilize. Transfer at once to a water-bath maintained at about 50°, and pour into plates as soon as the medium has cooled. 

       Final pH: 7.4±0.2.

APPENDICES • 10.2 MICROBIAL LIMIT TESTS
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