EUROPEAN PHARMACOPOEIA 6.7 2.4.14.

Sulphated ash

04/2010:20414 until the residue is completely incinerated. Ensure that

flames are not produced at any time during the procedure.
Allow the crucible to cool in a desiccator over silica gel or
2.4.14. SULPHATED ASH(2) other suitable desiccant, weigh it again and calculate the
Ignite a suitable crucible (for example, silica, platinum, percentage of residue.
porcelain or quartz) at 600 ± 50 °C for 30 min, allow to cool If the amount of the residue so obtained exceeds the
in a desiccator over silica gel or other suitable desiccant prescribed limit, repeat the moistening with sulphuric
and weigh. Place the prescribed amount of the substance acid R and ignition, as previously, for 30 min periods until
to be examined in the crucible and weigh. Moisten the 2 consecutive weighings do not differ by more than 0.5 mg or
substance to be examined with a small amount of sulphuric until the percentage of residue complies with the prescribed
acid R (usually 1 ml) and heat gently at as low a temperature limit.
as practicable until the sample is thoroughly charred. The amount of substance used for the test (usually 1-2 g)
After cooling, moisten the residue with a small amount of is chosen so that at the prescribed limit the mass of the
sulphuric acid R (usually 1 ml), heat gently until white residue (usually about 1 mg) can be measured with sufficient
fumes are no longer evolved and ignite at 600 ± 50 °C accuracy.

(2) This chapter has undergone pharmacopoeial harmonisation. See chapter 5.8. Pharmacopoeial harmonisation.

General Notices (1) apply to all monographs and other texts 5427
 EUROPEAN PHARMACOPOEIA 6.7

5428 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.7

2.6. BIOLOGICAL TESTS

2.6.12. Microbiological examination of non-sterile products : 2.6.30. Monocyte-activation test...........................................5440
microbial enumeration tests................................................ 5431 2.6.31. Microbiological examination of herbal medicinal
2.6.13. Microbiological examination of non-sterile products : products for oral use.............................................................5445
test for specified micro-organisms.. ...................................5435

General Notices (1) apply to all monographs and other texts 5429
 EUROPEAN PHARMACOPOEIA 6.7

5430 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.7 2.6.12. Microbial enumeration tests

04/2010:20612 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.
2.6.12. MICROBIOLOGICAL
EXAMINATION OF NON-STERILE Use buffered sodium chloride-peptone solution pH 7.0 or
phosphate buffer solution pH 7.2 to make test suspensions ;
PRODUCTS : MICROBIAL to suspend A. niger spores, 0.05 per cent of polysorbate 80
ENUMERATION TESTS(3) 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
1. INTRODUCTION and then diluting a fresh suspension of vegetative cells of
The tests described hereafter will allow quantitative A. niger or B. subtilis, a stable spore suspension is prepared
enumeration of mesophilic bacteria and fungi that may grow and then an appropriate volume of the spore suspension is
under aerobic conditions. used for test inoculation. The stable spore suspension may
be maintained at 2-8 °C for a validated period of time.
The tests are designed primarily to determine whether a
substance or preparation complies with an established 4-3. NEGATIVE CONTROL
specification for microbiological quality. When used for such To verify testing conditions, a negative control is performed
purposes follow the instructions given below, including the using the chosen diluent in place of the test preparation.
number of samples to be taken, and interpret the results as There must be no growth of micro-organisms. A negative
stated below. control is also performed when testing the products as
described in section 5. A failed negative control requires an
The methods are not applicable to products containing investigation.
viable micro-organisms as active ingredients.
Alternative microbiological procedures, including automated 4-4. GROWTH PROMOTION OF THE MEDIA
methods, may be used, provided that their equivalence to the Test each batch of ready-prepared medium and each batch of
Pharmacopoeia method has been demonstrated. medium, prepared either from dehydrated medium or from
the ingredients described.
2. GENERAL PROCEDURES Inoculate portions/plates of casein soya bean digest broth
Carry out the determination under conditions designed to and casein soya bean digest agar with a small number (not
avoid extrinsic microbial contamination of the product to be more than 100 CFU) of the micro-organisms indicated in
examined. The precautions taken to avoid contamination Table 2.6.12.-1, using a separate portion/plate of medium
must be such that they do not affect any micro-organisms for each. Inoculate plates of Sabouraud-dextrose agar
that are to be revealed in the test. with a small number (not more than 100 CFU) of the
If the product to be examined has antimicrobial activity, this micro-organisms indicated in Table 2.6.12.-1, using a separate
is insofar as possible removed or neutralised. If inactivators plate of medium for each. Incubate in the conditions
are used for this purpose, their efficacy and their absence of described in Table 2.6.12.-1.
toxicity for micro-organisms must be demonstrated. For solid media, growth obtained must not differ by a factor
If surface-active substances are used for sample preparation, greater than 2 from the calculated value for a standardised
their absence of toxicity for micro-organisms and their inoculum. For a freshly prepared inoculum, growth of the
compatibility with inactivators used must be demonstrated. micro-organisms comparable to that previously obtained
with a previously tested and approved batch of medium
3. ENUMERATION METHODS occurs. Liquid media are suitable if clearly visible growth of
Use the membrane filtration method or the plate-count the micro-organisms comparable to that previously obtained
methods, as prescribed. The most-probable-number (MPN) with a previously tested and approved batch of medium
method is generally the least accurate method for microbial occurs.
counts, however, for certain product groups with a very low 4-5. SUITABILITY OF THE COUNTING METHOD IN THE
bioburden, it may be the most appropriate method. PRESENCE OF PRODUCT
The choice of method is based on factors such as the nature 4-5-1. Preparation of the sample. The method for sample
of the product and the required limit of micro-organisms. The preparation depends upon the physical characteristics of the
chosen method must allow testing of a sufficient sample size product to be tested. If none of the procedures described
to judge compliance with the specification. The suitability of below can be demonstrated to be satisfactory, an alternative
the method chosen must be established. procedure must be developed.
4. GROWTH PROMOTION TEST, SUITABILITY OF THE Water-soluble products. Dissolve or dilute (usually a 1 in
COUNTING METHOD AND NEGATIVE CONTROLS 10 dilution is prepared) the product to be examined in
buffered sodium chloride-peptone solution pH 7.0, phosphate
4-1. GENERAL CONSIDERATIONS buffer solution pH 7.2 or casein soya bean digest broth.
The ability of the test to detect micro-organisms in the If necessary, adjust to pH 6-8. Further dilutions, where
presence of product to be tested must be established. necessary, are prepared with the same diluent.
Suitability must be confirmed if a change in testing
Non-fatty products insoluble in water. Suspend the product
performance, or the product, which may affect the outcome
to be examined (usually a 1 in 10 dilution is prepared) in
of the test is introduced.
buffered sodium chloride-peptone solution pH 7.0, phosphate
4-2. PREPARATION OF TEST STRAINS buffer solution pH 7.2 or casein soya bean digest broth. A
Use standardised stable suspensions of test strains or surface-active agent such as 1 g/l of polysorbate 80 may be
prepare them as stated below. Seed lot culture maintenance added to assist the suspension of poorly wettable substances.
techniques (seed-lot systems) are used so that the viable If necessary, adjust to pH 6-8. Further dilutions, where
micro-organisms used for inoculation are not more than necessary, are prepared with the same diluent.
(3) This chapter has undergone pharmacopoeial harmonisation. See chapter 5.8. Pharmacopoeial harmonisation.

General Notices (1) apply to all monographs and other texts 5431