Microbial identification plays a central role in the cleanroom control program (24). The method of identification, however, must be wedded to the need. For example, any organism isolated from the critical aseptic processing area must be identified to great detail, while those from class D/ISO 8/100,000 areas might only be characterized to the genus level. The key concern is providing sufficient detail to assist in the tracking of the state of control of the facility.
Most identification schemes still rely on the Gram stain, a differential staining technique developed in the late 1800’s by Christian Gram (15). This differential counterstaining technique is very good at distinguishing a real difference in cellular morphology. Unfortunately, this method is prone to a significant level of operator error, which has encouraged the development of alternate methods for showing the difference in cell structure (17, 23). Traditional methods of identification also consider a variety of phenotypic characteristics.
Phenotypic methods typically incorporate reactions to different chemicals or different biochemical markers. The API strip is basically a prepackaging of the standard method that required racks of test tubes into a convenient bubble-wrap. This method was further refined in the Vitek automated system which miniaturized the process (2, 20). This system has recently been enhanced to provide greater resolution of microorganisms (9, 10). A second phenotypic system is offered by Biolog, Inc. The fundamental unit in this system is a 96- well plate that has different carbohydrate sources in each well, with a tetrazolium redox dye. If the microorganism is capable of utilizing the carbohydrate the well turns dark indicating reduction of the dye (14, 19). The end-result is a pattern of wells (a “metabolic fingerprint”) that allows the user to identify the unknown microorganism. This method has recently been extended to include the identification of molds and filamentous fungi with a proprietary software package. The use of cellular fatty acid (FA) composition to identify the genus and species has been popular for several years (1, 5). The fatty acids are extracted from the cell cultures and then the patterns of fatty acid esters are determined by gas chromatography (22).
There are some new methods under development for the pharmaceutical QC lab. These include Fourier- Transform Infrared (FTIR) microscopy (16) and Matrix- Assisted Laser Desorption Ionization–Time of Flight (MALDI-TOF) mass spectroscopy (8, 18). However, these have not seen widespread use in the QC lab as of yet.
The FDA has recently elevated the use of genotypic identification methods with the release of the revised aseptic processing guidance document late in 2004 (6).
The Riboprinter is fundamentally an automated Southern Blot apparatus using labeled ssDNA probe from the 16sRNA codon. The resulting pattern is then used to identify the unknown microorganism (4, 12). If the initial banding pattern is inconclusive, then the restriction endonuclease can be changed to provide an extraordinary level of strain discrimination (3). Another genotypic identification system on the market is the MicroSeq 500 16S rDNA Bacterial Sequencing Kit which is offered by Applied Biosystems. As the name implies, it provides the materials needed to sequence the first 500 base pairs of the unknown microorganism’s 16s ribosomal RNA codon (7). The technology involves amplification of the 16S codon by PCR, followed by automated sequencing.
A final genotypic method that is being marketed into the QC pharmaceutical laboratory is the Bacterial Barcodes system (11). This system is also based on PCR technology, using as a primer a sequence homologous to a repetitive sequence in the bacterial genome. The amplified sequence is then separated by gel electrophoresis and visualized to give the “barcode” specific to that strain.
Qualicon markets the BAX system to the food industry that contains primers for Salmonella, Listeria or E. coli O157:H7 (13). This system has promise for determination of the absence of specified organisms in the product. Other genetic methods have been published in the literature, although few are available to the pharmaceutical market (21).
There are a variety of identification technologies available. When choosing one for the lab you must bear in mind the strengths, and weaknesses, of the various methodologies. For example, the recently released aseptic processing guidance document (FDA 2004) strongly recommends the use of genotypically based methods. However, if you choose PCR based methods or DNA sequencing, there is potentially an associated cost in facilities, labor (highly skilled technicians) and maintenance that is not present with the more traditional methods.
The most direct approach to deciding the appropriate technology is to research the choices fully based on an understanding of what your requirements may be. I recommend the development of a User Requirements Specification (URS) document to drive this process. This is a formal Quality document, similar in concept to a Design Qualification document. Different companies will have different formats for these documents, but the essential features of the document will be that it has the essential requirements and that it has upper management sign-off (for a variety of reasons it is a good idea to document upper management commitment).
A partial list of topics to be covered in any URS designed for an identification system should include:
In short, there are a wide variety of choices available to help with the identification of unknown organisms. It is important to define your specific requirements and to purchase the appropriate system to meet those needs.
Experts at the Microbiology Network are ready to assist with consulting or training to meet your needs. Have customized, in-house assistance with your questions from our recognized experts at your schedule either through consulting agreements, in-house training or customized webinars – contact us using the “Let’s Talk” communication found in the upper right of this page.