Appendix XVI B. Microbiological Examination of Non-sterile Products

(Ph. Eur. method 2.6.13)

The tests described hereafter will allow 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.

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

The preparation of samples is carried out as described in general chapter 2.6.12.

If the product to be examined has antimicrobial activity, this is insofar as possible removed or neutralised as described in general chapter 2.6.12.

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 general chapter 2.6.12.

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.

Use standardised 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 5 passages removed from the original master seed-lot.

3-1-1 Aerobic micro-organisms Grow each of the bacterial test strains separately in casein soya bean digest broth or on casein soya bean digest agar at 30-35 °C for 18-24 h. Grow the test strain for Candida albicans separately on Sabouraud-dextrose agar or in Sabouraud-dextrose broth at 20-25 °C for 2-3 days.

• — Staphylococcus aureus such as ATCC 6538, NCIMB 9518, CIP 4.83 or NBRC 13276;

• — Pseudomonas aeruginosa such as ATCC 9027, NCIMB 8626, CIP 82.118 or NBRC 13275;

• — Escherichia coli such as ATCC 8739, NCIMB 8545, CIP 53.126 or NBRC 3972;

• — Salmonella enterica subsp. enterica serovar Typhimurium, such as ATCC 14028 or, as an alternative, Salmonella enterica subsp. enterica serovar Abony such as NBRC 100797, NCTC 6017 or CIP 80.39;

• — Candida albicans such as ATCC 10231, NCPF 3179, IP 48.72 or NBRC 1594.

Use buffered sodium chloride-peptone solution pH 7.0 or phosphate buffer solution pH 7.2 to make test suspensions. Use the suspensions within 2 h or within 24 h if stored at 2-8 °C.

3-1-2 Clostridia Use Clostridium sporogenes such as ATCC 11437 (NBRC 14293, NCIMB 12343, CIP 100651) or ATCC 19404 (NCTC 532 or CIP 79.03) or NBRC 14293. Grow the clostridial test strain under anaerobic conditions in reinforced medium for clostridia at 30-35 °C for 24-48 h. 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 2-8 °C for a validated period.

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 section 4. A failed negative control requires an investigation.

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 2.6.13.-1.

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.

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.

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.

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.

For each product to be tested, perform the sample preparation as described in the relevant paragraph in section 4. Add each test strain at the time of mixing, in the prescribed growth medium. Inoculate the test strains individually. Use a number of micro-organisms equivalent to not more than 100 CFU in the inoculated test preparation.

Perform the test as described in the relevant paragraph in section 4 using the shortest incubation period prescribed.

The specified micro-organisms must be detected with the indication reactions as described in section 4.

Any antimicrobial activity of the product necessitates a modification of the test procedure (see 4-5-3 of general chapter 2.6.12).

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

4-1-1 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 general chapter 2.6.12, but using casein soya bean digest broth as the chosen diluent, mix and incubate at 20-25 °C for a time sufficient to resuscitate the bacteria but not sufficient to encourage multiplication of the organisms (usually 2 h but not more than 5 h).

4-1-2 Test for absence Unless otherwise prescribed, use the volume corresponding to 1 g of the product, as prepared in 4-1-1, to inoculate enterobacteria enrichment broth-Mossel. Incubate at 30-35 °C for 24-48 h. Subculture on plates of violet red bile glucose agar. Incubate at 30-35 °C for 18-24 h.

The product complies with the test if there is no growth of colonies.

4-1-3 Quantitative test

4-1-3-1 Selection and subculture Inoculate suitable quantities of enterobacteria enrichment broth-Mossel with the preparation as described under 4-1-1 and/or dilutions of it containing respectively 0.1 g, 0.01 g and 0.001 g (or 0.1 mL, 0.01 mL and 0.001 mL) of the product to be examined. Incubate at 30-35 °C for 24-48 h. Subculture each of the cultures on a plate of violet red bile glucose agar. Incubate at 30-35 °C for 18-24 h.

4-1-3-2 Interpretation Growth of colonies 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 2.6.13.-2 the probable number of bacteria.

4-2-1 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 general chapter 2.6.12, and use 10 mL or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount (determined as described under 3-4) of casein soya bean digest broth, mix and incubate at 30-35 °C for 18-24 h.

4-2-2 Selection and subculture Shake the container, transfer 1 mL of casein soya bean digest broth to 100 mL of MacConkey broth and incubate at 42-44 °C for 24-48 h. Subculture on a plate of MacConkey agar at 30-35 °C for 18-72 h.

4-2-3 Interpretation Growth of colonies indicates the possible presence of E. coli. This is confirmed by identification tests.

The product complies with the test if no colonies are present or if the identification tests are negative.

4-3-1 Sample preparation and pre-incubation Prepare the product to be examined as described in general chapter 2.6.12, and use the quantity corresponding to not less than 10 g or 10 mL to inoculate a suitable amount (determined as described under 3-4) of casein soya bean digest broth, mix and incubate at 30-35 °C for 18-24 h.

4-3-2 Selection and subculture Transfer 0.1 mL of casein soya bean digest broth to 10 mL of Rappaport Vassiliadis Salmonella enrichment broth and incubate at 30-35 °C for 18-24 h. Subculture on plates of xylose, lysine, deoxycholate agar. Incubate at 30-35 °C for 18-48 h.

4-3-3 Interpretation The possible presence of Salmonella is indicated by the growth of well-developed, red colonies, with or without black centres. This is confirmed by identification tests.

The product complies with the test if colonies of the types described are not present or if the confirmatory identification tests are negative.

4-4-1 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 general chapter 2.6.12, and use 10 mL or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount (determined as described under 3-4) of casein soya bean digest broth and mix. When testing transdermal patches, filter the volume of sample corresponding to 1 patch of the preparation described under 4-5-1 in general chapter 2.6.12 through a sterile filter membrane and place in 100 mL of casein soya bean digest broth. Incubate at 30-35 °C for 18-24 h.

4-4-2 Selection and subculture Subculture on a plate of cetrimide agar and incubate at 30-35 °C for 18-72 h.

4-4-3 Interpretation Growth of colonies indicates the possible presence of P. aeruginosa. This is confirmed by identification tests.

The product complies with the test if colonies are not present or if the confirmatory identification tests are negative.

4-5-1 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 general chapter 2.6.12, and use 10 mL or the quantity corresponding to 1 g or 1 mL to inoculate a suitable amount (determined as described under 3-4) of casein soya bean digest broth and mix. When testing transdermal patches, filter the volume of sample corresponding to 1 patch of the preparation described under 4-5-1 in general chapter 2.6.12 through a sterile filter membrane and place in 100 mL of casein soya bean digest broth. Incubate at 30-35 °C for 18-24 h.

4-5-2 Selection and subculture Subculture on a plate of mannitol salt agar and incubate at 30-35 °C for 18-72 h.

4-5-3 Interpretation The possible presence of S. aureus is indicated by the growth of yellow/white colonies surrounded by a yellow zone. This is confirmed by identification tests.

The product complies with the test if colonies of the types described are not present or if the confirmatory identification tests are negative.

4-6-1 Sample preparation and heat treatment Prepare a sample using a 1 in 10 dilution (with a minimum total volume of 20 mL) of not less than 2 g or 2 mL of the product to be examined as described in general chapter 2.6.12. Divide the sample into 2 portions of at least 10 mL. Heat 1 portion at 80 °C for 10 min and cool rapidly. Do not heat the other portion.

4-6-2 Selection and subculture Use 10 mL or the quantity corresponding to 1 g or 1 mL of the product to be examined of both portions to inoculate suitable amounts (determined as described under 3-4) of reinforced medium for clostridia. Incubate under anaerobic conditions at 30-35 °C for 48 h. After incubation, make subcultures from each container on Columbia agar and incubate under anaerobic conditions at 30-35 °C for 48-72 h.

4-6-3 Interpretation The occurrence of anaerobic growth of rods (with or without endospores) giving a negative catalase reaction indicates the presence of clostridia. This is confirmed by identification tests.

The product complies with the test if colonies of the types described are not present or if the confirmatory identification tests are negative.

4-7-1 Sample preparation and pre-incubation Prepare the product to be examined as described in general chapter 2.6.12, and use 10 mL or the quantity corresponding to not less than 1 g or 1 mL to inoculate 100 mL of Sabouraud-dextrose broth and mix. Incubate at 30-35 °C for 3-5 days.

4-7-2 Selection and subculture Subculture on a plate of Sabouraud-dextrose agar and incubate at 30-35 °C for 24-48 h.

4-7-3 Interpretation Growth of white colonies may indicate the presence of C. albicans. This is confirmed by identification tests.

The product complies with the test if such colonies are not present or if the confirmatory identification tests are negative.

The following section is given for information.

The following solutions and culture media have been found to be satisfactory for the purposes for which they are prescribed in the test for microbial contamination in the Pharmacopoeia. Other media may be used provided that their suitability can be demonstrated.

Stock buffer solution Place 34 g of potassium dihydrogen phosphate in a 1000 mL volumetric flask, dissolve in 500 mL of purified water, adjust to pH 7.2 ± 0.2 with sodium hydroxide, dilute to 1000.0 mL with purified water and mix. Dispense into containers and sterilise. Store at 2-8 °C.

Phosphate buffer solution pH 7.2 Prepare a mixture of stock buffer solution and purified water (1:800 V/V) and sterilise.

Potassium dihydrogen phosphate	3.6 g
Disodium hydrogen phosphate dihydrate	7.2 g, equivalent to 0.067 M phosphate
Sodium chloride	4.3 g
Peptone (meat or casein)	1.0 g
Purified water	1000 mL

Sterilise in an autoclave using a validated cycle.

Pancreatic digest of casein	17.0 g
Papaic digest of soya bean	3.0 g
Sodium chloride	5.0 g
Dipotassium hydrogen phosphate	2.5 g
Glucose monohydrate	2.5 g
Purified water	1000 mL

Adjust the pH so that after sterilisation it is 7.3 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.

Pancreatic digest of casein	15.0 g
Papaic digest of soya bean	5.0 g
Sodium chloride	5.0 g
Agar	15.0 g
Purified water	1000 mL

Adjust the pH so that after sterilisation it is 7.3 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.

Dextrose	40.0 g
Mixture of peptic digest of animal tissue and pancreatic digest of casein (1:1)	10.0 g
Agar	15.0 g
Purified water	1000 mL

Adjust the pH so that after sterilisation it is 5.6 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.

Infusion from potatoes	200 g
Dextrose	20.0 g
Agar	15.0 g
Purified water	1000 mL

Adjust the pH so that after sterilisation it is 5.6 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.

Dextrose	20.0 g
Mixture of peptic digest of animal tissue and pancreatic digest of casein (1:1)	10.0 g
Purified water	1000 mL

Adjust the pH so that after sterilisation it is 5.6 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.

Pancreatic digest of gelatin	10.0 g
Glucose monohydrate	5.0 g
Dehydrated ox bile	20.0 g
Potassium dihydrogen phosphate	2.0 g
Disodium hydrogen phosphate dihydrate	8.0 g
Brilliant green	15 mg
Purified water	1000 mL

Adjust the pH so that after heating it is 7.2 ± 0.2 at 25 °C. Heat at 100 °C for 30 min and cool immediately.

Yeast extract	3.0 g
Pancreatic digest of gelatin	7.0 g
Bile salts	1.5 g
Sodium chloride	5.0 g
Glucose monohydrate	10.0 g
Agar	15.0 g
Neutral red	30 mg
Crystal violet	2 mg
Purified water	1000 mL

Adjust the pH so that after heating it is 7.4 ± 0.2 at 25 °C. Heat to boiling; do not heat in an autoclave.

Pancreatic digest of gelatin	20.0 g
Lactose monohydrate	10.0 g
Dehydrated ox bile	5.0 g
Bromocresol purple	10 mg
Purified water	1000 mL

Adjust the pH so that after sterilisation it is 7.3 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.

Pancreatic digest of gelatin	17.0 g
Peptones (meat and casein)	3.0 g
Lactose monohydrate	10.0 g
Sodium chloride	5.0 g
Bile salts	1.5 g
Agar	13.5 g
Neutral red	30.0 mg
Crystal violet	1 mg
Purified water	1000 mL

Adjust the pH so that after sterilisation it is 7.1 ± 0.2 at 25 °C. Boil for 1 min with constant shaking then sterilise in an autoclave using a validated cycle.

Soya peptone	4.5 g
Magnesium chloride hexahydrate	29.0 g
Sodium chloride	8.0 g
Dipotassium phosphate	0.4 g
Potassium dihydrogen phosphate	0.6 g
Malachite green	0.036 g
Purified water	1000 mL

Dissolve, warming gently. Sterilise in an autoclave using a validated cycle, at a temperature not exceeding 115 °C. The pH is to be 5.2 ± 0.2 at 25 °C after heating and autoclaving.

Xylose	3.5 g
l-Lysine	5.0 g
Lactose monohydrate	7.5 g
Sucrose	7.5 g
Sodium chloride	5.0 g
Yeast extract	3.0 g
Phenol red	80 mg
Agar	13.5 g
Sodium deoxycholate	2.5 g
Sodium thiosulfate	6.8 g
Ferric ammonium citrate	0.8 g
Purified water	1000 mL

Adjust the pH so that after heating it is 7.4 ± 0.2 at 25 °C. Heat to boiling, cool to 50 °C and pour into Petri dishes. Do not heat in an autoclave.

Pancreatic digest of gelatin	20.0 g
Magnesium chloride	1.4 g
Dipotassium sulfate	10.0 g
Cetrimide	0.3 g
Agar	13.6 g
Purified water	1000 mL
Glycerol	10.0 mL

Heat to boiling for 1 min with shaking. Adjust the pH so that after sterilisation it is 7.2 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.

Pancreatic digest of casein	5.0 g
Peptic digest of animal tissue	5.0 g
Beef extract	1.0 g
d-Mannitol	10.0 g
Sodium chloride	75.0 g
Agar	15.0 g
Phenol red	0.025 g
Purified water	1000 mL

Heat to boiling for 1 min with shaking. Adjust the pH so that after sterilisation it is 7.4 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.

Beef extract	10.0 g
Peptone	10.0 g
Yeast extract	3.0 g
Soluble starch	1.0 g
Glucose monohydrate	5.0 g
Cysteine hydrochloride	0.5 g
Sodium chloride	5.0 g
Sodium acetate	3.0 g
Agar	0.5 g
Purified water	1000 mL

Hydrate the agar, dissolve by heating to boiling with continuous stirring. If necessary, adjust the pH so that after sterilisation it is 6.8 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle.

Pancreatic digest of casein	10.0 g
Meat peptic digest	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	10.0-15.0 g
Purified water	1000 mL

Hydrate the agar, dissolve by heating to boiling with continuous stirring. If necessary, adjust the pH so that after sterilisation it is 7.3 ± 0.2 at 25 °C. Sterilise in an autoclave using a validated cycle. Allow to cool to 45-50 °C; add, where necessary, gentamicin sulfate corresponding to 20 mg of gentamicin base and pour into Petri dishes.

(Ph. Eur. method 2.6.12)

The tests described hereafter will allow quantitative enumeration of mesophilic bacteria and fungi that may grow under aerobic conditions.

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 Pharmacopoeia method has been demonstrated.

Carry out the determination under conditions designed to avoid extrinsic microbial contamination of the product to be examined. The precautions taken to avoid contamination must be such that they do not affect any micro-organisms that are to be revealed in the test.

If the product to be examined has antimicrobial activity, this is insofar as possible removed or neutralised. 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.

Use the membrane filtration method or the plate-count methods, 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.

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.

Use standardised 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 5 passages removed from the original master seed-lot. Grow each of the bacterial and fungal test strains separately as described in Table 2.6.12.-1.

Use buffered sodium chloride-peptone solution pH 7.0 or phosphate buffer solution pH 7.2 to make test suspensions; to suspend A. brasiliensis spores, 0.05 per cent of polysorbate 80 may be added to the buffer. Use the suspensions within 2 h or within 24 h if stored at 2-8 °C. As an alternative to preparing and then diluting a fresh suspension of vegetative cells of A. brasiliensis or B. 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 2-8 °C for a validated period of time.

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 section 5. A failed negative control requires an investigation.

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 casein soya bean digest broth and casein soya bean digest agar with a small number (not more than 100 CFU) of the micro-organisms indicated in Table 2.6.12.-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 2.6.12.-1, using a separate plate of medium for each. Incubate in the conditions described in Table 2.6.12.-1.

For solid media, growth obtained must not differ by a factor greater than 2 from the calculated value for a standardised 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.

4-5-1 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 casein soya bean digest broth. If necessary, adjust to pH 6-8. Further dilutions, where necessary, are prepared with the same diluent.

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 casein soya bean digest broth. A surface-active agent such as 1 g/L of polysorbate 80 may be added to assist the suspension of poorly wettable substances. If necessary, adjust to pH 6-8. Further dilutions, where necessary, are prepared with the same diluent.

Fatty products Dissolve in isopropyl myristate, sterilised 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 °C, or in exceptional cases to not more than 45 °C. Mix carefully and if necessary maintain the temperature in a water-bath. Add sufficient of the pre-warmed chosen diluent to make a 1 in 10 dilution of the original product. Mix carefully whilst 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 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 min.

4-5-2 Inoculation and dilution Add to the sample prepared as described above (4-5-1) 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 neutralisation, dilution or filtration.

4-5-3 Neutralisation/removal of antimicrobial activity The number of micro-organisms recovered from the prepared sample diluted as described in 4-5-2 and incubated following the procedure described in 4-5-4, is compared to the number of micro-organisms 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 neutralising agents into the diluent, (3) membrane filtration, or (4) a combination of the above measures.

Neutralising agents Neutralising agents may be used to neutralise the activity of antimicrobial agents (Table 2.6.12.-2). They may be added to the chosen diluent or the medium preferably before sterilisation. If used, their efficacy and their absence of toxicity for micro-organisms must be demonstrated by carrying out a blank with neutraliser and without product.

If no suitable neutralising 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.

4-5-4 Recovery of micro-organism 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.

4-5-4-1 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 micro-organisms listed, one membrane filter is used.

Transfer a suitable amount of the sample prepared as described under 4-5-1 to 4-5-3 (preferably representing 1 g of the product, 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 casein soya bean digest agar. For the determination of total combined yeasts/moulds count (TYMC), transfer the membrane to the surface of Sabouraud-dextrose agar. Incubate the plates as indicated in Table 2.6.12.-1. Perform the counting.

4-5-4-2 Plate-count methods Perform plate-count methods at least in duplicate for each medium and use the mean count of the result.

4-5-4-2-1 Pour-plate method

For Petri dishes 9 cm in diameter, add to the dish 1 mL of the sample prepared as described under 4-5-1 to 4-5-3 and 15-20 mL of casein soya bean digest agar or Sabouraud-dextrose agar, both media being at not more than 45 °C. If larger Petri dishes are used, the amount of agar medium is increased accordingly. For each of the micro-organisms listed in Table 2.6.12.-1, at least 2 Petri dishes are used. Incubate the plates as indicated in Table 2.6.12.-1. Take the arithmetic mean of the counts per medium and calculate the number of CFU in the original inoculum.

4-5-4-2-2 Surface-spread method

For Petri dishes 9 cm in diameter, add 15-20 mL of casein soya bean digest agar or Sabouraud-dextrose agar at about 45 °C 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 2.6.12.-1, at least 2 Petri dishes are used. Spread a measured volume of not less than 0.1 mL of the sample prepared as described under 4-5-1 to 4-5-3 over the surface of the medium. Incubate and count as prescribed under 4-5-4-2-1

4-5-4-3 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 reasons 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 3 serial tenfold dilutions of the product as described under 4-5-1 to 4-5-3. From each level of dilution, 3 aliquots of 1 g or 1 mL are used to inoculate 3 tubes with 9-10 mL of casein soya bean digest broth. If necessary, a surface-active agent such as polysorbate 80 or an inactivator of antimicrobial agents may be added to the medium. Thus, if 3 levels of dilution are prepared, 9 tubes are inoculated.

Incubate all tubes at 30-35 °C 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 casein soya bean digest agar, for 1-2 days at the same temperature and use these results. Determine the most probable number of micro-organisms per gram or millilitre of the product to be examined from Table 2.6.12.-3.

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 4-5-2 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.

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 gram or millilitre (for preparations not presented in dose 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 authorised.

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.

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.

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 section 4 and transfer the appropriate amount to each of 2 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 casein soya bean digest agar. For the determination of TYMC, transfer the other membrane to the surface of Sabouraud-dextrose agar. Incubate the plate of casein soya bean digest agar at 30-35 °C for 3-5 days and the plate of Sabouraud-dextrose agar at 20-25 °C for 5-7 days. Calculate the number of CFU per gram or per millilitre of product.

When examining transdermal patches, filter 10 per cent of the volume of the preparation described under 4-5-1 separately through each of 2 sterile filter membranes. Transfer one membrane to casein soya bean digest agar for TAMC and the other membrane to Sabouraud-dextrose agar for TYMC.

5-2-2-1 Pour-plate method

Prepare the sample using a method that has been shown to be suitable as described in section 4. Prepare for each medium at least 2 Petri dishes for each level of dilution. Incubate the plates of casein soya bean digest agar at 30-35 °C for 3-5 days and the plates of Sabouraud-dextrose agar at 20-25 °C for 5-7 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 gram or per millilitre of product.

5-2-2-2 Surface-spread method

Prepare the sample using a method that has been shown to be suitable as described in section 4. Prepare at least 2 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.

Prepare and dilute the sample using a method that has been shown to be suitable as described in section 4. Incubate all tubes at 30-35 °C for 3-5 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 gram or millilitre of the product to be examined from Table 2.6.12.-3.

The total aerobic microbial count (TAMC) is considered to be equal to the number of CFU found using casein soya bean digest agar; if colonies of fungi are detected on this medium, they are counted as part of the TAMC. The total combined yeasts/mould 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 containing antibiotics may be used. If the count is carried out by the MPN method the calculated value is the TAMC.

When an acceptance criterion for microbiological quality is prescribed it is interpreted as follows:

• — 10¹ CFU: maximum acceptable count = 20;

• — 10² CFU: maximum acceptable count = 200;

• — 10³ CFU: maximum acceptable count = 2000, and so forth.

The recommended solutions and media are described in general chapter 2.6.13.

(Ph. Eur. method 2.6.7 as applied to vaccines for human use)

Where the test for mycoplasmas is prescribed for a master cell bank, for a working cell bank, for a virus seed lot or for control cells, both the culture method and the indicator cell culture method are used. Where the test for mycoplasmas is prescribed for a virus harvest, for a bulk vaccine or for the final lot (batch), the culture method is used. The indicator cell culture method may also be used, where necessary, for screening of media.

Nucleic acid amplification techniques (NAT) may be used as an alternative to one or both of the other methods after suitable validation.

The test is carried out using a sufficient number of both solid and liquid media to ensure growth in the chosen incubation conditions of small numbers of mycoplasmas that may be present in the product to be examined. Liquid media must contain phenol red. The range of media chosen is shown to have satisfactory nutritive properties for at least the micro-organisms shown below. The nutritive properties of each new batch of medium are verified for the appropriate micro-organisms in the list. When testing for mycoplasmas in the product to be examined, at least 1 of the following species will be included as a positive control:

• — Acholeplasma laidlawii (vaccines for human and veterinary use where an antibiotic has been used during production);

• — Mycoplasma gallisepticum (where avian material has been used during production or where the vaccine is intended for use in poultry);

• — Mycoplasma hyorhinis (non-avian veterinary vaccines);

• — Mycoplasma orale (vaccines for human and veterinary use);

• — Mycoplasma pneumoniae (vaccines for human use) or other suitable species of d-glucose fermenter such as Mycoplasma fermentans;

• — Mycoplasma synoviae (where avian material has been used during production or where the vaccine is intended for use in poultry).

The test strains are field isolates having undergone a limited number of subcultures (not more than 15), and are stored frozen or freeze-dried. After cloning, the strains are identified as being of the required species by comparison with type cultures, for example:

A. laidlawii	NCTC 10116	CIP 75.27	ATCC 23206
M. gallisepticum	NCTC 10115	CIP 104967	ATCC 19610
M. fermentans	NCTC 10117	CIP 105680	ATCC 19989
M. hyorhinis	NCTC 10130	CIP 104968	ATCC 17981
M. orale	NCTC 10112	CIP 104969	ATCC 23714
M. pneumoniae	NCTC 10119	CIP 103766	ATCC 15531
M. synoviae	NCTC 10124	CIP 104970	ATCC 25204

Acholeplasma laidlawii BRP, Mycoplasma fermentans BRP, Mycoplasma hyorhinis BRP, Mycoplasma orale BRP and Mycoplasma synoviae BRP are suitable for use as low-passage reference strains.

Incubate liquid media in tightly stoppered containers at 35-38 °C. Incubate solid media in microaerophilic conditions (nitrogen containing 5-10 per cent of carbon dioxide and sufficient humidity to prevent desiccation of the agar surface) at 35-38 °C.

Carry out the test for nutritive properties for each new batch of medium. Inoculate the chosen media with the appropriate test micro-organisms; use not more than 100 CFU (colony-forming units) per 60 mm diameter plate containing 9 mL of solid medium and per 100 mL container of liquid medium; use a separate plate and container for each species of micro-organism. Incubate the media and make subcultures from 0.2 mL of liquid medium to solid medium at the specified intervals (see below under Test for mycoplasmas in the product to be examined). The solid medium complies with the test if adequate growth is found for each test micro-organism (growth obtained does not differ by a factor greater than 5 from the value calculated with respect to the inoculum). The liquid medium complies with the test if growth on agar plates subcultured from the broth is found for at least 1 subculture for each test micro-organism.

The test for inhibitory substances is carried out once for a given product and is repeated whenever there is a change in production method that may affect the detection of mycoplasmas.

To demonstrate absence of inhibitory substances, carry out the test for nutritive properties in the presence and absence of the product to be examined. If growth of a test micro-organism occurs more than 1 subculture sooner in the absence of the product to be examined than in its presence, or if plates directly inoculated with the product to be examined have fewer than 1/5 of the number of colonies of those inoculated without the product to be examined, inhibitory substances are present and they must be neutralised or their effect otherwise countered, for example by passage in substrates not containing inhibitors or dilution in a larger volume of medium before the test. If dilution is used, larger medium volumes may be used or the inoculum volume may be divided among several 100 mL flasks. The effectiveness of the neutralisation or other process is checked by repeating the test for inhibitory substances after neutralisation.

Inoculate 10 mL of the product to be examined per 100 mL of each liquid medium. If it has been found that a significant pH change occurs upon the addition of the product to be examined, the liquid medium is restored to its original pH value by the addition of a solution of either sodium hydroxide or hydrochloric acid. Inoculate 0.2 mL of the product to be examined on each plate of each solid medium. Incubate liquid media for 20-21 days. Incubate solid media for not less than 14 days, except those corresponding to the 20-21 day subculture, which are incubated for 7 days. At the same time incubate an uninoculated 100 mL portion of each liquid medium and agar plates, as a negative control. On days 2-4 after inoculation, subculture each liquid medium by inoculating 0.2 mL on at least 1 plate of each solid medium. Repeat the procedure between the 6th and 8th days, again between the 13th and 15th days and again between the 19th and 21st days of the test. Observe the liquid media every 2 or 3 days and if a colour change occurs, subculture. If a liquid medium shows bacterial or fungal contamination, the test is invalid. The test is valid if at least 1 plate per medium and per inoculation day can be read. Include in the test positive controls prepared by inoculation of not more than 100 CFU of at least 1 test micro-organism on agar medium or into broth medium. Where the test for mycoplasmas is carried out regularly and where possible, it is recommended to use the test micro-organisms in regular rotation. The test micro-organisms used are those listed under Choice of culture media.

At the end of the prescribed incubation period, examine all inoculated solid media microscopically for the presence of mycoplasma colonies. The product complies with the test if growth of typical mycoplasma colonies has not occurred. The product does not comply with the test if growth of typical mycoplasma colonies has occurred on any of the solid media. The test is invalid if 1 or more of the positive controls do not show growth of mycoplasmas on at least 1 subculture plate. The test is invalid if 1 or more of the negative controls show growth of mycoplasmas. If suspect colonies are observed, a suitable validated method may be used to determine whether they are due to mycoplasmas.

The following section is published for information.

The following media are recommended. Other media may be used, provided that their ability to sustain the growth of mycoplasmas has been demonstrated on each batch in the presence and absence of the product to be examined.

Liquid medium
Beef heart infusion broth (1)		90.0 mL
Horse serum (unheated)		20.0 mL
Yeast extract (250 g/L)		10.0 mL
Phenol red (0.6 g/L solution)		5.0 mL
Penicillin (20 000 IU/mL)		0.25 mL
Deoxyribonucleic acid (2 g/L solution)		1.2 mL
Adjust to pH 7.8.

Prepare as described above replacing beef heart infusion broth by beef heart infusion agar containing 15 g/L of agar.

Beef heart infusion broth (1)	90.0 mL
Essential vitamins (2)	0.025 mL
Glucose monohydrate (500 g/L solution)	2.0 mL
Swine serum (inactivated at 56 °C for 30 min)	12.0 mL
β-Nicotinamide adenine dinucleotide (10 g/L solution)	1.0 mL
Cysteine hydrochloride (10 g/L solution)	1.0 mL
Phenol red (0.6 g/L solution)	5.0 mL
Penicillin (20 000 IU/mL)	0.25 mL

Mix the solutions of β-nicotinamide adenine dinucleotide and cysteine hydrochloride and after 10 min add to the other ingredients. Adjust to pH 7.8.

Beef heart infusion broth (1)	90.0 mL
Agar, purified (3)	1.4 g

Adjust to pH 7.8, sterilise by autoclaving then add:

Essential vitamins (2)	0.025 mL
Glucose monohydrate (500 g/L solution)	2.0 mL
Swine serum (unheated)	12.0 mL
β-Nicotinamide adenine dinucleotide (10 g/L solution)	1.0 mL
Cysteine hydrochloride (10 g/L solution)	1.0 mL
Phenol red (0.6 g/L solution)	5.0 mL
Penicillin (20 000 IU/mL)	0.25 mL

Hanks' balanced salt solution (modified) (4)	800 mL
Distilled water	67 mL
Brain heart infusion (5)	135 mL
PPLO Broth (6)	248 mL
Yeast extract (170 g/L)	60 mL
Bacitracin	250 mg
Meticillin	250 mg
Phenol red (5 g/L)	4.5 mL
Horse serum	165 mL
Swine serum	165 mL

Adjust to pH 7.40-7.45.

Hanks' balanced salt solution (modified) (4)	200 mL
DEAE-dextran	200 mg
Agar, purified (3)	15.65 g

Mix well and sterilise by autoclaving. Cool to 100 °C. Add to 1740 mL of liquid medium as described above.

(1) Beef heart infusion broth

Beef heart (for preparation of the infusion)	500 g
Peptone	10 g
Sodium chloride	5 g
Distilled water	to 1000 mL

Sterilise by autoclaving.

(2) Essential vitamins

Biotin	100 mg
Calcium pantothenate	100 mg
Choline chloride	100 mg
Folic acid	100 mg
i-Inositol	200 mg
Nicotinamide	100 mg
Pyridoxal hydrochloride	100 mg
Riboflavine	10 mg
Thiamine hydrochloride	100 mg
Distilled water	to 1000 mL

(3) Agar, purified

A highly refined agar for use in microbiology and immunology, prepared by an ion-exchange procedure that results in a product having superior purity, clarity and gel strength. It contains about:

Water	12.2 per cent
Ash	1.5 per cent
Acid-insoluble ash	0.2 per cent
Chlorine	0
Phosphate (calculated as P2O5)	0.3 per cent
Total nitrogen	0.3 per cent
Copper	8 ppm
Iron	170 ppm
Calcium	0.28 per cent
Magnesium	0.32 per cent

(4) Hanks' balanced salt solution (modified)

Sodium chloride	6.4 g
Potassium chloride	0.32 g
Magnesium sulfate heptahydrate	0.08 g
Magnesium chloride hexahydrate	0.08 g
Calcium chloride, anhydrous	0.112 g
Disodium hydrogen phosphate dihydrate	0.0596 g
Potassium dihydrogen phosphate, anhydrous	0.048 g
Distilled water	to 800 mL

(5) Brain heart infusion

Calf-brain infusion	200 g
Beef-heart infusion	250 g
Proteose peptone	10 g
Glucose monohydrate	2 g
Sodium chloride	5 g
Disodium hydrogen phosphate, anhydrous	2.5 g
Distilled water	to 1000 mL

(6) PPLO broth

Beef-heart infusion	50 g
Peptone	10 g
Sodium chloride	5 g
Distilled water	to 1000 mL

Cell cultures are stained with a fluorescent dye that binds to DNA. Mycoplasmas are detected by their characteristic particulate or filamentous pattern of fluorescence on the cell surface and, if contamination is heavy, in surrounding areas. Mitochondria in the cytoplasm may be stained but are readily distinguished from mycoplasmas.

If for viral suspensions the interpretation of results is affected by marked cytopathic effects, the virus may be neutralised using a specific antiserum that has no inhibitory effects on mycoplasmas or a cell culture substrate that does not allow growth of the virus may be used. To demonstrate the absence of inhibitory effects of serum, carry out the positive control tests in the presence and absence of the antiserum.

Use Vero cells or another cell culture (for example, the production cell line) that is equivalent in effectiveness for detecting mycoplasmas. Test the effectiveness of the cells to be used by applying the procedure shown below and inoculating not more than 100 CFU or CFU-like micro-organisms of suitable reference strains of M. hyorhinis and M. orale. The following strains have been found to be suitable:

M. hyorhinis			ATCC 29052
M. orale	NCTC 10112	CIP 104969	ATCC 23714

The cells are suitable if both reference strains are detected.

The indicator cells must be subcultured without an antibiotic before use in the test.

1. Seed the indicator cell culture at a suitable density (for example, 2 × 10⁴ to 2 × 10⁵ cells/mL, 4 × 10³ to 2.5 × 10⁴ cells/cm²) that will yield confluence after 3 days of growth. Inoculate 1 mL of the product to be examined into the cell culture vessel and incubate at 35-38 °C.

2. After at least 3 days of incubation, when the cells have grown to confluence, make a subculture on cover slips in suitable containers or on some other surface (for example, chambered slides) suitable for the test procedure. Seed the cells at low density so that they reach 50 per cent confluence after 3-5 days of incubation. Complete confluence impairs visualisation of mycoplasmas after staining and must be avoided.

3. Remove the medium and rinse the indicator cells with phosphate buffered saline pH 7.4 R, then add a suitable fixing solution (a freshly prepared mixture of 1 volume of glacial acetic acid R and 3 volumes of methanol R is suitable when bisbenzimide R is used for staining).

4. Remove the fixing solution and wash the cells with sterile water R. Dry the slides completely if they are to be stained more than 1 h later (particular care is needed for staining of slides after drying owing to artefacts that may be produced).

5. Add a suitable DNA stain and allow to stand for a suitable time (bisbenzimide working solution R and a standing time of 10 min are suitable).

6. Remove the stain and rinse the monolayer with water R.

7. Mount each coverslip, where applicable (a mixture of equal volumes of glycerol R and phosphate-citrate buffer solution pH 5.5 R is suitable for mounting). Examine by fluorescence (for bisbenzimide stain a 330 nm/380 nm excitation filter and an LP 440 nm barrier filter are suitable) at 400 × magnification or greater.

8. Compare the microscopic appearance of the test cultures with that of the negative and positive controls, examining for extranuclear fluorescence. Mycoplasmas produce pinpoints or filaments over the indicator cell cytoplasm. They may also produce pinpoints and filaments in the intercellular spaces. Multiple microscopic fields are examined according to the protocol established during validation.

The product to be examined complies with the test if fluorescence typical of mycoplasmas is not present. The test is invalid if the positive controls do not show fluorescence typical of mycoplasmas. The test is invalid if the negative controls show fluorescence typical of mycoplasmas.

NAT (2.6.21) may be used for detection of mycoplasmas by amplification of nucleic acids extracted from a test sample with specific primers that reveal the presence of the target nucleic acid. NAT indicate the presence of a particular nucleic acid sequence and not necessarily the presence of viable mycoplasmas. A number of different techniques are available. This general chapter does not prescribe a particular method for the test. The procedure applied must be validated as described, taking account of the guidelines presented at the end of this section. Where a commercial kit is used, certain elements of the validation may be carried out by the manufacturer and information provided to the user but it must be remembered that full information on the primers may not be available and that production of the kit may be modified or discontinued.

NAT are applied where prescribed in a monograph. They may also be used instead of the culture method and the indicator cell culture method after suitable validation.

Direct NAT can be applied in the presence of cytotoxic material and where a rapid method is needed.

Cell-culture enrichment followed by NAT The test sample and a suitable cell substrate (as described under the indicator cell-culture method) are cultured together for a suitable period; the nucleic acids are then extracted from cells and supernatant and used for detection by NAT.

Reference standards are required at various stages during validation and for use as controls during routine application of the test. The reference standards may be mycoplasmas or nucleic acids.

For validation of the limit of detection, the following species represent an optimal selection in terms of the frequency of occurrence as contaminants and phylogenetic relationships:

• — A. laidlawii;

• — M. fermentans;

• — M. hyorhinis (where cell-culture enrichment is used, a fastidious strain such as ATCC 29052 is included);

• — M. orale;

• — M. pneumoniae or M. gallisepticum;

• — M. synoviae (where there is use of or exposure to avian material during production);

• — Mycoplasma arginini;

• — Spiroplasma citri (where there is use of or exposure to insect or plant material during production).

Demonstration of specificity requires the use of a suitable range of bacterial species other than mycoplasmas. Bacterial genera with close phylogenetic relation to mycoplasmas are most appropriate for this validation; these include Clostridium, Lactobacillus and Streptococcus.

Comparability studies for use of NAT as an alternative method For each mycoplasma test species:

• — as an alternative to the culture method: the NAT test system must be shown to detect 10 CFU/mL;

• — as an alternative to the indicator cell culture method: the NAT test system must be shown to detect 100 CFU/mL;

or an equivalent limit of detection in terms of the number of copies of mycoplasma nucleic acid in the test sample (using suitable reference standards of mycoplasma nucleic acid).

Internal controls Internal controls are necessary for routine verification of absence of inhibition. The internal control may contain the primer binding-site, or some other suitable sequence may be used. It is preferably added to the test material before isolating the nucleic acid and therefore acts as an overall control (extraction, reverse transcription, amplification, detection).

External controls The external positive control contains a defined number of target-sequence copies or CFUs from 1 or more suitable species of mycoplasma chosen from those used during validation of the test conditions. 1 of the positive controls is set close to the positive cut-off point to demonstrate that the expected sensitivity is achieved. The external negative control contains no target sequence but does not necessarily represent the same matrix as the test article.

The primers used may also amplify non-mycoplasmal bacterial nucleic acid, leading to false positive results. Procedures are established at the time of validation for dealing with confirmation of positive results, where necessary.

The following section is published for information.

Nucleic acid amplification techniques (NAT) are either qualitative or quantitative tests for the presence of nucleic acid. For the detection of mycoplasma contamination of various samples such as vaccines and cell substrates, qualitative tests are adequate and may be considered to be limit tests.

These guidelines describe methods to validate qualitative nucleic acid amplification analytical procedures for assessing mycoplasma contamination. They may also be applicable for real-time NAT used as limit tests for the control of contaminants.

The 2 characteristics regarded as the most important for validation of the analytical procedure are the specificity and the detection limit. In addition, the robustness of the analytical procedure should be evaluated.

For the purpose of this document, an analytical procedure is defined as the complete procedure from extraction of nucleic acid to detection of the amplified products.

Where commercial kits are used for part or all of the analytical procedure, documented validation points already covered by the kit manufacturer can replace validation by the user. Nevertheless, the performance of the kit with respect to its intended use has to be demonstrated by the user (e.g. detection limit, robustness, cross-detection of other classes of bacteria).

NAT may be used as:

• — a complementary test (for example, for cytotoxic viral suspensions) or for in-process control purposes;

• — an alternative method to replace an official method (indicator cell culture method or culture method).

These guidelines will thus separate these 2 objectives by presenting first a guideline for the validation of the NAT themselves, and second, a guideline for a comparability study between NAT and official methods.

3 parameters should be evaluated: specificity, detection limit and robustness.

2-1 Specificity Specificity is the ability to unequivocally assess target nucleic acid in the presence of components that may be expected to be present.

The specificity of NAT is dependent on the choice of primers, the choice of probe (for analysis of the final product) and the stringency of the test conditions (for both the amplification and detection steps).

The ability of the NAT to detect a large panel of mycoplasma species will depend on the choice of primers, probes and method parameters. This ability should be demonstrated using characterised reference panels (e.g. reference strains provided by the EDQM). Since NAT systems are usually based on a mix of primers, the theoretical analysis of primers and probes by comparison with databases is not recommended, because interpretation of the results may be quite complex and may not reflect the experimental results.

Moreover, as it is likely that the primers will detect other bacterial species, the potential cross-detection should be documented in the validation study. Bacterial genera such as gram-positive bacteria with close phylogenetic relation to mycoplasmas are most appropriate for this validation; these include Clostridium, Lactobacillus and Streptococcus. However, this is not an exhaustive list and species to be tested will depend on the theoretical ability (based on primers/probes sequences) of the NAT system to detect such other species.

Based on the results from this validation of the specificity, if a gap in the specificity of the method is identified (such as detection of non-mycoplasmal bacterial nucleic acid), an appropriate strategy must be proposed in the validation study to allow interpretation of positive results on a routine basis. For example, a second test may be performed using an alternative method without this specificity gap or using an official method.

2-2 Detection limit. The detection limit of an individual analytical procedure is the lowest amount of target nucleic acid in a sample that can be detected but not necessarily quantitated as an exact value.

For establishment of the detection limit, a positive cut-off point should be determined for the nucleic acid amplification analytical procedure. The positive cut-off point (as defined in general chapter 2.6.21) is the minimum number of target sequence copies per volume of sample that can be detected in 95 per cent of test runs. This positive cut-off point is influenced by the distribution of mycoplasmal genomes in the individual samples being tested and by factors such as enzyme efficiency, and can result in different 95 per cent cut-off values for individual analytical test runs.

To determine the positive cut-off point, a dilution series of characterised and calibrated (either in CFUs or nucleic acid copies) in-house working strains or EDQM standards should be tested on different days to examine variation between test runs.

For validation of the limit of detection, the following species represent an optimal selection in terms of the frequency of occurrence as contaminants and phylogenetic relationships:

• — A. laidlawii;

• — M. fermentans;

• — M. hyorhinis;

• — M. orale;

• — M. pneumoniae or M. gallisepticum;

• — M. synoviae (where there is use of or exposure to avian material during production);

• — M. arginini;

• — S. citri (where there is use of or exposure to insect or plant material during production).

For each strain, at least 3 independent 10-fold dilution series should be tested, with a sufficient number of replicates at each dilution to give a total number of 24 test results for each dilution, to enable a statistical analysis of the results.

For example, a laboratory may test 3 dilution series on different days with 8 replicates for each dilution, 4 dilution series on different days with 6 replicates for each dilution, or 6 dilution series on different days with 4 replicates for each dilution. In order to keep the number of dilutions at a manageable level, a preliminary test should be performed to obtain a preliminary value for the positive cut-off point (i.e. the highest dilution giving a positive signal). The range of dilutions can then be chosen around the predetermined preliminary cut-off point. The concentration of mycoplasmas (CFUs or copies) that can be detected in 95 per cent of test runs can then be calculated using an appropriate statistical evaluation.

These results may also serve to evaluate the variability of the analytical procedure.

2-3 Robustness The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters, and provides an indication of its reliability during normal usage.

The evaluation of robustness should be considered during the development phase. It should show the reliability of the analytical procedure with respect to deliberate variations in method parameters. For NAT, small variations in the method parameters can be crucial. However, the robustness of the method can be demonstrated during its development when small variations in the concentrations of reagents (e.g. MgCl₂, primers or deoxyribonucleotides) are tested. Modifications of extraction kits or extraction procedures as well as different thermal cycler types may also be evaluated.

Finally, robustness of the method can be evaluated through collaborative studies.

NAT may be used instead of official methods (indicator cell culture method and/or culture method). In this case a comparability study should be carried out. This comparability study should include mainly a comparison of the respective detection limits of the alternative method and official methods. However, specificity (mycoplasma panel detected, putative false positive results) should also be considered.

For the detection limit, acceptability criteria are defined as follows:

• — if the alternative method is proposed to replace the culture method, the NAT system must be shown to detect 10 CFU/mL for each mycoplasma test species described in paragraph 2-2;

• — if the alternative method is proposed to replace the indicator cell culture method, the NAT system must be shown to detect 100 CFU/mL for each mycoplasma test species described in paragraph 2-2.

For both cases, suitable standards calibrated for the number of nucleic acid copies and the number of CFUs may be used for establishing that these acceptability criteria are reached. The relation between CFUs and nucleic acid copies for the reference preparations should be previously established to compare the performance of the alternative NAT method with the performance of the official methods.

1 of the following 2 strategies can be used to perform this comparability study:

• — perform the NAT alternative method in parallel with the official method(s) to evaluate simultaneously the detection limit of both methods using the same samples of calibrated strains;

• — compare the performance of the NAT alternative method using previously obtained data from official method validation. In this case, calibration of standards used for both validations as well as their stabilities should be documented carefully.

Comparability study reports should describe all the validation elements described in section 2 (specificity, limit of detection and variability, as well as robustness) in order to assess all the advantages and/or disadvantages of the alternative NAT method compared to official methods.

(Ph. Eur. method 2.6.2)

If the sample to be examined may be contaminated by micro-organisms other than mycobacteria, treat it with a suitable decontamination solution, such as acetylcysteine-sodium hydroxide solution or sodium laurilsulfate solution.

Inoculate 0.2 mL of the sample in triplicate onto each of 2 suitable solid media (Löwenstein-Jensen medium and Middlebrook 7H10 medium are considered suitable). Inoculate 0.5 mL in triplicate into a suitable liquid medium. Incubate all media at 37 °C for 56 days.

Establish the fertility of the media in the presence of the preparation to be examined by inoculation of a suitable strain of a Mycobacterium sp. such as BCG and if necessary use a suitable neutralising substance.

If contaminating micro-organisms develop during the first 8 days of incubation, repeat the test and carry out at the same time a bacteriological sterility test.

If at the end of the incubation time no growth of mycobacteria occurs in any of the test media, the preparation complies with the test.

(Ph. Eur. method 2.6.16)

In those tests that require prior neutralisation of the virus, use specific antibodies of non-human, non-simian origin; if the virus has been propagated in avian tissues, the antibodies must also be of non-avian origin. To prepare antiserum, use an immunising antigen produced in cell culture from a species different from that used for the production of the vaccine and free from extraneous agents. Where the use of SPF eggs is prescribed, the eggs are obtained from a flock free from specified pathogens (5.2.2).

Take samples of the virus seed lot at the time of harvesting and, if they are not tested immediately, keep them at a temperature below - 40 °C.

Adult mice Inoculate each of not fewer than 10 adult mice, each weighing 15-20 g, intracerebrally with 0.03 mL and intraperitoneally with 0.5 mL of the virus seed lot. Observe the mice for at least 21 days. Carry out an autopsy of all mice that die after the first 24 h of the test or that show signs of illness, and examine for evidence of viral infection, both by direct macroscopical observation and by subinoculation of appropriate tissue suspensions by the intracerebral and intraperitoneal routes into not fewer than 5 additional mice, which are observed for 21 days. The virus seed lot complies with the test if no mouse shows evidence of infection attributable to the seed lot. The test is not valid unless at least 80 per cent of the original inoculated mice survive the observation period.

Suckling mice Inoculate each of not fewer than 20 mice, each less than 24 h old, intracerebrally with 0.01 mL and intraperitoneally with at least 0.1 mL of the virus seed lot. Observe the mice daily for at least 14 days. Carry out an autopsy of all mice that die after the first 24 h of the test or that show signs of illness, and examine for evidence of viral infection, both by direct macroscopical observation and by subinoculation of appropriate tissue suspensions by the intracerebral and intraperitoneal routes into not fewer than 5 additional suckling mice, which are observed daily for 14 days. The virus seed lot passes the test if no mouse shows evidence of infection attributable to the seed lot. The test is not valid unless at least 80 per cent of the original inoculated mice survive the observation period.

Guinea-pigs Inoculate intraperitoneally into each of not fewer than 5 guinea pigs, each weighing 350-450 g, 5.0 mL of the virus seed lot. Observe the animals for at least 42 days for signs of disease. Carry out an autopsy of all guinea-pigs that die after the first 24 h of the test or that show signs of illness, and examine macroscopically; examine the tissues both microscopically and culturally for evidence of infection. Euthanise animals that survive the observation period and examine in a similar manner. The virus seed lot passes the test if no guinea-pig shows evidence of infection attributable to the seed lot. The test is not valid unless at least 80 per cent of the guinea-pigs survive the observation period.

Spiroplasmas Virus seed lots produced in insect cells are demonstrated by a validated method approved by the competent authority to be free of spiroplasmas.

Take samples at the time of harvesting and, if not tested immediately, keep them at a temperature below - 40 °C.

Bacterial and fungal sterility A 10 mL sample complies with the test for sterility (2.6.1).

Mycoplasmas A 10 mL sample complies with the test for mycoplasmas (2.6.7).

Mycobacteria (2.6.2) A 5 mL sample is tested for the presence of Mycobacterium spp. by culture methods known to be sensitive for the detection of these organisms.

Test in cell culture for other extraneous agents Neutralised samples equivalent, unless otherwise prescribed, to 500 human doses of vaccine or 50 mL, whichever is the greater, are tested for the presence of extraneous agents by inoculation into continuous simian kidney and human cell cultures. If the virus is grown in simian or human cells, the neutralised virus harvest is tested on a separate culture of these cells. If the virus is grown in a mammalian or avian cell system other than simian or human, cells of that species, but from a separate batch, are also inoculated. The cells are incubated at 36 ± 1 °C and observed for a period of 14 days. The virus seed lot or harvest passes the tests if none of the cell cultures show evidence of the presence of any extraneous agents. The test is not valid unless at least 80 per cent of the cell cultures remain viable.

Avian viruses (only required for virus seed lot propagated in avian tissues and for virus harvest propagated in primary avian tissues) Neutralise a sample equivalent to 100 human doses of vaccine or 10 mL, whichever is the greater. Using 0.5 mL per egg, inoculate a group of fertilised SPF eggs, 9-11 days old, by the allantoic route and a second group, 5-7 days old, into the yolk sac. Incubate for 7 days. The virus seed lot or harvest complies with the test if the allantoic and yolk sac fluids show no sign of the presence of any haemagglutinating agent and if all embryos and chorio-allantoic membranes, examined for gross pathology, are normal. The test is not valid unless at least 80 per cent of the inoculated eggs survive for 7 days.

Insect viruses (only required for virus propagated in insect cells) Neutralised samples equivalent, unless otherwise prescribed, to 500 human doses of vaccine or 50 mL, whichever is the greater, are tested for the presence of extraneous agents by inoculation into at least one cell culture different from that used in production and permissible to insect viruses, and that allows detection of human arboviruses. The choice of cells is approved by the competent authority and takes into account the origin of the production cells and the likely contaminants that may be detected by the chosen cells. The cells are incubated at 27 ± 1 °C and observed for a period of 14 days. The virus seed lot or harvest passes the tests if none of the cell cultures show evidence of the presence of any extraneous agents. The test is not valid unless at least 80 per cent of the cell cultures remain viable.

Examine the control cells microscopically for freedom from any virus causing cytopathic degeneration throughout the time of incubation of the inoculated production cell cultures or for not less than 14 days beyond the time of inoculation of the production vessels, whichever is the longer. The test is not valid unless at least 80 per cent of the control cell cultures survive to the end of the observation period.

At 14 days or at the time of the last virus harvest, whichever is the longer, carry out the tests described below.

Haemadsorbing viruses Examine not fewer than 25 per cent of the control cultures for the presence of haemadsorbing viruses by the addition of guinea-pig red blood cells. If the test for haemadsorbing viruses is not feasible, carry out a test for haemagglutination viruses. If the guinea-pig red blood cells have been stored, they shall have been stored at 5 ± 3 °C for not more than 7 days. Read half of the cultures after incubation at 5 ± 3 °C for 30 min and the other half after incubation at 20-25 °C for 30 min. No evidence of haemadsorbing agents is found.

Tests in cell cultures for other extraneous agents Pool the supernatant fluids from the control cells and examine for the presence of extraneous agents by inoculation of simian kidney and human cell cultures. If the virus is grown in a mammalian cell system other than simian or human, cells of that species, but from a separate batch, are also inoculated. In each cell system, at least 5 mL is tested. Incubate the inoculated cultures at 36 ± 1 °C and observe for a period of 14 days. No evidence of extraneous agents is found.

If the production cell culture is maintained at a temperature different from 36 ± 1 °C, a supplementary test for extraneous agents is carried out at the production temperature using the same type of cells as used for growth of the virus.

If the virus is grown in insect cells the pooled supernatant is also inoculated into at least one cell culture different from that used in production and permissible to insect viruses, and that allows detection of human arboviruses. The cells are incubated at 27 ± 1 °C for 14 days. No evidence of extraneous agents is found.

Avian leucosis viruses (required only if the virus is propagated in primary avian tissues) Carry out a test for avian leucosis viruses using 5 mL of the supernatant fluid from the control cells.

Haemagglutinating agents Examine 0.25 mL of the allantoic fluid from each egg for haemagglutinating agents by mixing directly with chicken red blood cells and after a passage in SPF eggs carried out as follows: inoculate a 5 mL sample of the pooled amniotic fluids from the control eggs in 0.5 mL volumes into the allantoic cavity and into the amniotic cavity of SPF eggs. The control eggs comply with the test if no evidence of the presence of haemagglutinating agents is found in either test.

Avian leucosis viruses Use a 10 mL sample of the pooled amniotic fluids from the control eggs. Carry out amplification by 5 passages in leucosis-free chick-embryo cell cultures; carry out a test for avian leucosis using cells from the 5^th passage. The control eggs comply with the test if no evidence of the presence of avian leucosis viruses is found.

Other extraneous agents Inoculate 5 mL samples of the pooled amniotic fluids from the control eggs into human and simian cell cultures. Observe the cell cultures for 14 days. The control eggs comply with the test if no evidence of the presence of extraneous agents is found. The test is not valid unless 80 per cent of the inoculated cultures survive to the end of the observation period.

¹ This chapter has undergone pharmacopoeial harmonisation. See Chapter 5.8. Parmacopoeial harmonisation.

¹ This chapter has undergone pharmacopoeial harmonisation. See chapter 5.8. Pharmacopoeial harmonisation.Microbiological Examination of Non-Sterile Products: Microbiological Enumeration Tests