The Microbiology section is the national lead centre for microbial quality analysis of homoeopathic medicines. Homeopathic medicines are often prepared from a wide range plant materials (roots, stems, leaves, flowers, bark, pollen, lichen, moss, ferns and algae), microorganisms (fungi, bacteria, viruses) , animal origin (tissues, secretions and cell lines), human origin (tissues, secretions, hormones, and cell lines) and natural or synthetic sources (minerals and chemicals. The homeopathic stock or its potencies may consist of processed or unprocessed substances. Raw material used in homeopathic medicines are may have a higher level contamination with microorganisms (bioburden), toxins, insects, pesticides, agrochemical residues and heavy metals. Due to the faulty post harvesting methods, inaccurate storage condition the herbal drugs may be contaminated with saprophytic or pathogenic microorganisms. Cross contamination is also possible from extraneous materials such as plastics, glass and other materials which come in contact with herbal drugs.
The therapeutic efficacy of herbal drugs depends on the active ingredients which are effective against the specific malady. In case of high microbial contamination in herbal drugs it may be feasible that active ingredients of drugs may deteriorate.
The quality of source material and the procedure is important in the manufacturing of homeopathic medicines. The Good Manufacturing Practices for Homoeopathic Drugs as described in Sub-Rule (2) of Rule 85E of Drugs & Cosmetics Rules, 1945 with conditions as specified in Schedule ‘MI‘ GMP are to ensure that:
- Raw materials used in the manufacture of drugs are authentic, of prescribed quality and are free from contamination;
- The manufacturing process is as has been prescribed to maintain the standards;
- Adequate quality control measures are adopted;
- The manufactured drug which is released for sale is of acceptable quality;
- To achieve the objectives listed above, each licensee shall evolve methodology and procedures for following the prescribed process of manufacture of drugs which should be documented as a manual and kept for reference and inspection.
Microbiology is an essential section to carry out the quality control tests and standardization of the Homoeopathic Drugs. The microbiology section accomplishes the sterility test and microbial limit test (MLT) for Homoeopathic drugs as per Drugs & Cosmetic Act. The section is also focusing on the quality control and standardization of nosodes as per HPI. The microbiology laboratory is equipped with all essential laboratory instruments and equipments. The laboratory is designed in such a manner that the risk of contamination will be very low. The laboratory is categorized in to following different area to maintain the aseptic condition to carry out the quality control tests and standardization of the Homoeopathic Drugs:
- Sample receipt and holding area:
This part of the laboratory is dedicated to receive the samples and keeping their records.
- Media preparation area:
This area is specifically designed for maintaining and preparing the different culture media to carry out the quality control tests and standardization of the Homoeopathic Drugs. This area is equipped with:
- Electronic balance
- Top pan balance.
The media preparation area of the laboratory is maintaining the stocks of:
- Introduction Culture Media
- Complex culture media
- Enriched media
- Defined culture media
- Selective media
This area is specifically designed for cleaning and sterilizing the laboratory glassware, equipments and to decontaminate the used utensils during laboratory experiment. This area is equipped with highly specific sterilization device:
- Vertical Autoclave
- Hot air Oven
This area is specifically designed to carry out the microbial culture experiments and inoculation of Drug samples under aseptic environment. This area is equipped with:
- Biosaftey cabinet
- Laminar air flow
This area is specifically designed for providing the suitable condition for observation of Microbial growth. This are of the laboratory is equipped with highly specific device:
- Biological oxygen demand Incubator (BOD)
- Rotary shaker
The test for sterility is carried out under aseptic conditions. In order to achieve such conditions, the test environment has to be adapted to the way in which the sterility test is performed. The precautions taken to avoid contamination are such that they do not affect any microorganisms which are to be revealed in the test. The working conditions in which the tests are performed are monitored regularly by appropriate sampling of the working area and by carrying out appropriate controls.
CULTURE MEDIA AND INCUBATION TEMPERATURES:
Media for the test may be prepared as described below, or equivalent commercial media may be used provided that they comply with the growth promotion test. The following culture media have been found to be suitable for the test for sterility. Fluid thioglycollate medium is primarily intended for the culture of anaerobic bacteria; however, it will also detect aerobic bacteria. Soya-bean casein digest medium is suitable for the culture of both fungi and aerobic bacteria.
Fluid thioglycollate mediumL-Cystine 0.5 g Agar 0.75 g Sodium chloride 2.5 g Glucose monohydrate/anhydrous 5.5/5.0 g Yeast extract (water-soluble) 5.0 g Pancreatic digest of casein 15.0 g Sodium thioglycollate or 0.5 g Thioglycollic acid 0.3 ml Resazurin sodium solution (1 g/l of resazurin sodium), freshly prepared 1.0 ml Water 1000 ml pH after sterilization 6.9 to 7.3.
Mix the L-cystine, agar, sodium chloride, glucose, water-soluble yeast extract and pancreatic digest of casein with the water and heat until solution is effected. Dissolve the sodium thioglycollate or thioglycollic acid in the solution and, if necessary, add sodium hydroxide (1 mol/l) VS so that, after sterilization, the solution will have a pH of 9 to 7.3. If filtration is necessary heat the solution again without boiling and filter while hot through moistened filter paper. Add the resazurin sodium solution, mix and place the medium in suitable vessels which provide a ratio of surface to depth of medium such that not more than the upper half of the medium has undergone a colour change indicative of oxygen uptake at the end of the incubation period. Sterilize using a validated process. If the medium is stored, store at a temperature between 2 °C and 25 °C in a sterile, airtight container. If more than the upper one-third of the medium has acquired a pink colour, the medium may be restored once by heating the containers in a water-bath or in free-flowing steam until the pink colour disappears and cooling quickly, taking care to prevent the introduction of non-sterile air into the container. Do not use the medium for a longer storage period than has been validated.
Fluid thioglycollate medium is to be incubated at 30–35 °C
For products containing a mercurial preservative that cannot be tested by the membrane-filtration method, fluid thioglycollate medium incubated at 20–25 °C may be used instead of soya-bean casein digest medium provided that it has been validated as described in growth promotion test.. Alternative thioglycollate medium The following alternative thioglycollate medium might be used. Prepare a mixture having the same composition as that of the fluid thioglycollate medium, but omitting the agar and the resazurin sodium solution, sterilize as directed above. The pH after sterilization is 6.9 to 7.3. Heat in a water-bath prior to use and incubate at 30–35 °C under anaerobic conditions.
Soya-bean casein digest mediumPancreatic digest of casein 17.0 g Papaic digest of soya-bean meal 3.0 g Sodium chloride 5.0 g Dipotassium hydrogen phosphate 2.5 g Glucose monohydrate/anhydrous 2.5/2.3 g Water 1 000 ml pH after sterilization 7.1 to 7.5.
Dissolve the solids in water, warming slightly to effect solution. Cool the solution to room temperature. Add sodium hydroxide (1 mol/l) VS, if necessary, so that after sterilization the solution will have a pH of 7.1 to 7.5. Filter, if necessary, to clarify, distribute into suitable vessels and sterilize using a validated process. Store at a temperature between 2 °C and 25 °C in a sterile well-closed container, unless it is intended for immediate use. Do not use the medium for a longer storage period than has been validated.
Soya-bean casein digest medium is to be incubated at 20–25 °C.
The media used comply with the following tests, carried out before or in parallel with the test on the product to be examined.
Incubate portions of the media for 14 days. No growth of microorganisms occurs.
Growth promotion test of aerobes, anaerobes and fungi:
Test each batch of ready-prepared medium and each batch of medium prepared either from dehydrated medium or from ingredients. Inoculate portions of fluid thioglycollate medium with a small number (not more than 100 CFU) of the following microorganisms, using a separate portion of medium for each of the following species of microorganism: Clostridium sporogenes, Pseudomonas aeruginosa, Staphylococcus aureus. Inoculate portions of soya-bean casein digest medium with a small number (not more than 100 CFU) of the following microorganisms, using a separate portion of medium for each of the following species of microorganism: Aspergillus brasiliensis, Bacillus subtilis, Candida albicans. Incubate for not more than 3 days in the case of bacteria and not more than 5 days in the case of fungi. The media are suitable if a clearly visible growth of the microorganisms occurs.
METHOD SUITABILITY TEST:
Carry out a test as described below under Test for sterility of the product to be examined using exactly the same methods except for the following modifications.
After transferring the content of the container or containers to be tested to the membrane add an inoculum of a small number of viable microorganisms (not more than 100 CFU) to the final portion of sterile diluent used to rinse the filter.
After transferring the contents of the container or containers to be tested to the culture medium add an inoculum of a small number of viable microorganisms (not more than 100 CFU) to the medium.
In both cases use the same microorganisms as those described above under Growth promotion test of aerobes, anaerobes and fungi. Perform a growth promotion test as a positive control. Incubate all the containers containing medium for not more than 5 days.
If clearly visible growth of microorganisms is obtained after the incubation, visually comparable to that in the control vessel without product, either the product possesses no antimicrobial activity under the conditions of the test or such activity has been satisfactorily eliminated. The test for sterility may then be carried out without further modification.
If clearly visible growth is not obtained in the presence of the product to be tested, visually comparable to that in the control vessels without product, the product possesses antimicrobial activity that has not been satisfactorily eliminated under the conditions of the test. Modify the conditions in order to eliminate the antimicrobial activity and repeat the method suitability test.
This method suitability is performed:
- When the test for sterility has to be carried out on a new product;
- Whenever there is a change in the experimental conditions of the test.
The method suitability may be performed simultaneously with the Test for sterility of the product to be examined.
TEST FOR STERILITY OF THE PRODUCT TO BE EXAMINED:
The test may be carried out using the technique of membrane filtration or by direct inoculation of the culture media with the product to be examined. Appropriate negative controls are included. The technique of membrane filtration is used whenever the nature of the product permits, that is, for filterable aqueous preparations, for alcoholic or oily preparations and for preparations miscible with or soluble in aqueous or oily solvents provided these solvents do not have an antimicrobial effect in the conditions of the test.
- Membrane filtration. Use membrane filters having a nominal pore size not greater than 0.45 μm whose effectiveness to retain microorganisms has been established. Cellulose nitrate filters, for example, are used for aqueous, oily and weakly alcoholic solutions and cellulose acetate filters, for example, for strongly alcoholic solutions. Specially adapted filters may be needed for certain products, e.g. for antibiotics. The technique described below assumes that membranes about 50 mm in diameter will be used. If filters of a different diameter are used the volumes of the dilutions and the washings should be adjusted accordingly. The filtration apparatus and membrane are sterilized by appropriate means. The apparatus is designed so that the solution to be examined can be introduced and filtered under aseptic conditions; it permits the aseptic removal of the membrane for transfer to the medium or it is suitable for carrying out the incubation after adding the medium to the apparatus itself.
- Direct inoculation of the culture medium. Transfer the quantity of the preparation to be examined directly into the culture medium so that the volume of the product is not more than 10% of the volume of the medium, unless otherwise prescribed. If the product to be examined has antimicrobial activity, carry out the test after neutralizing this with a suitable neutralizing substance or by dilution in a sufficient quantity of culture medium. When it is necessary to use a large volume of the product it may be preferable to use a concentrated culture medium prepared in such a way that it takes account of the subsequent dilution. Where appropriate the concentrated medium may be added directly to the product in its container.
Apart from the contribution of microbiology section in quality control tests and standardization of the Homoeopathic Drugs, the section also provides hands on training on microbiological analysis of Homoeopathic Drugs to drugs inspectors, asst. drugs controllers, Govt. analyst, faculty and student of various colleges across the Nation.
Dr. M.Ramesh, Scientific Officer (Microbiology), Head of the Section
Dr. Akhilesh Tiwari, Scientific Assistant (Microbiology)