PREPARATION OF THE SAMPLE
The method for sample preparation depends on the physical characteristics of the product to be tested. If none of the procedures described below can be demonstrated to be satisfactory, a suitable 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 ChloridePeptone Solution pH 7.0, Phosphate Buffer Solution pH 7.2, or SoybeanCasein Digest Broth. If necessary, adjust to a pH of 6 to 8. Further dilutions, where necessary, are prepared with the same diluent.
Nonfatty Products Insoluble in Water
Suspend the product to be examined (usually a 1 in 10 dilution is prepared) in Buffered Sodium ChloridePeptone Solution pH 7.0, Phosphate Buffer Solution pH 7.2, or SoybeanCasein Digest Broth. A surface-active agent such as 1 g per L of polysorbate 80 may be added to assist the suspension of poorly wettable substances. If necessary, adjust to a pH of 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 noninhibitory sterile surface-active reagent 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 water bath. Add a sufficient quantity of the prewarmed chosen diluent to make a 1 in 10 dilution of the original product. Mix carefully, while maintaining the temperature for the shortest time necessary for the formation of an emulsion. Further serial 10-fold dilutions may be prepared using the chosen diluent containing a suitable concentration of sterile polysorbate 80 or another noninhibitory sterile surface-active reagent.
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 suitable sterile porous material (e.g., 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 directed above and to a control (with no test material included) a sufficient volume of the microbial suspension to obtain an inoculum of not more than than 100 cfu. The volume of the suspension of the inoculum should not exceed 1% 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 microorganisms recovered from the prepared sample diluted as described in Inoculation and Dilution and incubated following the procedure described in Recovery of Microorganisms 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,
-
An increase in the volume of the diluent or culture medium;
-
Incorporation of a specific or general neutralizing agents into the diluent;
-
Membrane filtration; or
-
A combination of the above measures.
Neutralizing Agents
Neutralizing agents may be used to neutralize the activity of antimicrobial agents (see
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 neutralizer and without product.
Table 2. Common Neutralizing Agents/Methods for
Interfering Substances
Interfering Substance |
Potential Neutralizing Agents/ Method |
Glutaraldehyde, mercurials |
Sodium hydrogen sulfite (Sodium bisulfite) |
Phenolics, alcohol, aldehydes, sorbate |
Dilution |
Aldehydes |
Glycine |
Quaternary ammonium compounds (QACs), parahydroxybenzoates (parabens), bis-biguanides |
Lecithin |
QACs, iodine, parabens |
Polysorbate |
Mercurials |
Thioglycollate |
Mercurials, halogens, aldehydes |
Thiosulfate |
EDTA (edetate) |
Mg or Ca ions |
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 article is not likely to be contaminated with the given species of the microorganism. However, it is possible that the product inhibits only some of the microorganisms specified herein, but does not inhibit others not included among the test strains or those 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 microorganisms in the presence of product
For each of the microorganisms listed, separate tests are performed. Only microorganisms 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 in such a way 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 quantity of the sample prepared as described under Preparation of the Sample, Inoculation and Dilution, and Neutralization/Removal of Antimicrobial Activity (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 the
SoybeanCasein Digest Agar. For the determination of total combined yeasts and molds count (TYMC), transfer the membrane to the surface of the
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, Inoculation and Dilution, and
Neutralization/Removal of Antimicrobial Activity and 15 to 20 mL of
SoybeanCasein Digest Agar or
Sabouraud Dextrose Agar, both media maintained at not more than 45
. If larger Petri dishes are used, the amount of agar medium is increased accordingly. For each of the microorganisms 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
SoybeanCasein 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-airflow cabinet or in an incubator. For each of the microorganisms 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 directed under
Preparation of the Sample, Inoculation and Dilution, and
Neutralization/Removal of Antimicrobial Activity over the surface of the medium. Incubate and count as directed for
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 molds. 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 three serial 10-fold dilutions of the product as described for Preparation of the Sample, Inoculation and Dilution, and 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 SoybeanCasein 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 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
SoybeanCasein Digest Agar for 1 to 2 days at the same temperature, and use these results. From
Table 3, determine the most probable number of microorganisms per g or mL of the product to be examined.
Table 3. Most-Probable-Number Values of Microorganisms
Observed Combinations of Numbers of Tubes Showing Growth in Each Set |
MPN per g or per mL of Product |
95% Confidence Limits |
Number of g or mL of Product per Tube |
|
|
0.1 |
0.01 |
0.001 |
0 |
0 |
0 |
<3 |
09.4 |
0 |
0 |
1 |
3 |
0.19.5 |
0 |
1 |
0 |
3 |
0.110 |
0 |
1 |
1 |
6.1 |
1.217 |
0 |
2 |
0 |
6.2 |
1.217 |
0 |
3 |
0 |
9.4 |
3.535 |
1 |
0 |
0 |
3.6 |
0.217 |
1 |
0 |
1 |
7.2 |
1.217 |
1 |
0 |
2 |
11 |
435 |
1 |
1 |
0 |
7.4 |
1.320 |
1 |
1 |
1 |
11 |
435 |
1 |
2 |
0 |
11 |
435 |
1 |
2 |
1 |
15 |
538 |
1 |
3 |
0 |
16 |
538 |
2 |
0 |
0 |
9.2 |
1.535 |
2 |
0 |
1 |
14 |
435 |
2 |
0 |
2 |
20 |
538 |
2 |
1 |
0 |
15 |
438 |
2 |
1 |
1 |
20 |
538 |
2 |
1 |
2 |
27 |
994 |
2 |
2 |
0 |
21 |
540 |
2 |
2 |
1 |
28 |
994 |
2 |
2 |
2 |
35 |
994 |
2 |
3 |
0 |
29 |
994 |
2 |
3 |
1 |
36 |
994 |
3 |
0 |
0 |
23 |
594 |
3 |
0 |
1 |
38 |
9104 |
3 |
0 |
2 |
64 |
16181 |
3 |
1 |
0 |
43 |
9181 |
3 |
1 |
1 |
75 |
17199 |
3 |
1 |
2 |
120 |
30360 |
3 |
1 |
3 |
160 |
30380 |
3 |
2 |
0 |
93 |
18360 |
3 |
2 |
1 |
150 |
30380 |
3 |
2 |
2 |
210 |
30400 |
3 |
2 |
3 |
290 |
90990 |
3 |
3 |
0 |
240 |
40990 |
3 |
3 |
1 |
460 |
901980 |
3 |
3 |
2 |
1100 |
2004000 |
3 |
3 |
3 |
> 1100 |
|
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 product must be obtained. When verifying the suitability of the MPN Method, the calculated value from the inoculum must be within 95% confidence limits of the results obtained with the control.
If the above criteria cannot be met for one of 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.