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1.0 OBJECTIVE : The aim of this SOP is to provide a general guideline for the preliminary identification of the common bacterial and fungal isolates frequently found in pharmaceutical manufacturing area.
2.0 SCOPE : This SOP is applicable for the unknown microbial isolates found in the (Company Name).
3.0 RESPONSIBILITY
3.1 Microbiologist/ QC officer shall be responsible for carrying out the procedure.
3.2 Sr. officer shall be responsible to ensure proper execution of the procedure.
3.3 Overall responsibility for training, implementation & follow-up with the QC Manager or his nominee.
4.0 ACCOUNTABILITY
4.1 Head of Quality.
5.0 PRECAUTIONS
5.1 Staining procedure should be done very carefully.
5.2 Biochemical tests should be performed according to the manual.
6.0 PROCEDURE
6.1 Identification of Bacteria
6.1.1 Microorganisms found in pharmaceutical ingredients, water for pharmaceutical use, the manufacturing environment, intermediate and finished products are frequently identified to assist in product investigations. Identification of the organisms at species and strain level is very critical and requires many phenotypic and genotypic characteristics determination and that is not feasible to perform in microbiology laboratory of a pharmaceutical industry. So, our wish is to determine the organism at genus level. In the pharmaceutical manufacturing area of track-II, the morphological characteristics of the identified microorganisms shall be studied once in each year.
6.1.2 To determine the bacterial genus, only phenotypic characteristics will be considered here. For the identification of bacteria at species level or strain level if required, sample will be sent to recognized contract microbiology laboratory.
6.1.3 The first step for identification is to isolate a pure colony for analysis. This purification is normally accomplished by subculturing one or more times to get isolated colony by streaking on solid media. The primarily isolated culture on the microbial limit test or environmental monitoring plates also can be taken for identification.
6.1.4 The cultural or colony morphology and colour, cellular morphology, differential staining, biochemical reaction and different substrate utilization will be used for the identification of bacteria.
6.2 Cultural characteristics
6.2.1 Different bacteria vary from each other by colony color, size, texture and shape. So note down the colony characteristics of the particular colonies of interest grown on solid agar media.
6.3 Staining
6.3.1 After observing the colony characteristics observe the cellular morphology of the bacteria and determine the Gram reaction.
6.3.2 Perform the Gram staining by following the SOP No.: QC106 (Gram staining).
6.3.3 On the basis of Gram reaction and cellular morphology bacteria can be differentiated into four major groups. The Gram staining is a critical step for the phenotypic identification scheme. The total identification procedure is presented in a schematic way in the Annexure-I.
6.4 Biochemical characteristics
6.4.1 Numerous biochemical tests are usually used for the identification of bacteria.
6.4.2 If Gram positive cocci are found perform Catalase test to separates Gram positive cocci into two different groups: catalase negative and catalase positive bacteria.
6.4.3 Catalase negative Gram positive cocci in chain distribution can be assumed as bacteria of Streptococcus genus.
6.4.4 Catalase positive Gram positive cocci in cluster distribution can be assumed as bacteria of Staphylococcus genus. Staphylococcus aureus can be confirmed by carry out the procedure described for S. aureus in GTP No.: GTP/007.
6.4.5 Gram positive rod shaped bacteria and Gram negative cocci shaped bacteria sample is to be sent to contract lab for identification.
6.4.6 Inoculate a Microbact GNB 24E microplate with Gram negative rod shaped bacterial isolate and incubate as per procedure mentioned by the manufacturer.
6.4.7 After incubation check the growth pattern of the organisms on the microplate and match the growth pattern with standard table and record the corresponding bacterial name.
6.4.8 In addition to inoculate the Microbact GNB 24E carry out the test for Pseudomonas sp with the same colony by following the GTP No.: GTP/007.
6.4.9 For identification of Salmonella sp, Escherichia sp, follow the procedure described for them in GTP No.: GTP/007.
6.5 Identification of Fungi
6.5.1 Classical identification method based on morphology is generally used.
6.5.2 Identification of fungi requires more expertise, but some of the common features make some fungi very easily recognizable.
6.5.3 Yeast can be easily identified by their cell morphology. Yeast cells are generally spherical or ellipsoidal shaped and budding is a common feature of yeast cells. Budding is a process in which a daughter cell arises from the parent as a localized outgrowth or bud.
6.5.4 Aspergillus and Penicellium can be easily identified by their colony morphology, aerial hyphae and conidia distribution.
6.5.5 A mature colony of Aspergillus genus is generally black in color sorrounded by white mass of mycellial growth. Lines radiating from the centre to the periphery is found on the underside of the colony and this is a characteristic feature of Aspergillus.The conidiophore arises from the foot cells and at the apex, the conidiophore inflates to form a vesicle- that is sterigmata. Conidia are produced within the sterigmata and extruded to form spore chains- which is characteristic feature of Aspergillus.
6.5.6 A mature colony of Penicellium genus is generally green or bluish greeen in color. Conidiophores are developed from aerial hyphae, may be in branched and have brushlike heads bearing spores. Cluster of sterigmata are usually in one place, and from each is formed a chain of conidia.
