How Are Microbial Characterizations For Microbiology Testing
What is microbial characterization, and why is it important for regulatory testing?
Drug substances, excipients, water for pharmaceutical use, manufacturing environments, intermediates, and finished drug products are all tested for the presence of microorganisms during the production of medical products. Often positive identification of microbes in a sample will result in microorganism characterization tests for microbe identification. Microorganism characterization tests may include determining colony morphology, cellular morphology (rods, cocci, cell groupings, modes of sporulation, etc.), Gram reaction, spore staining, and certain key biochemical reactions (e.g., oxidase, catalase, and coagulase activity).
Microbial characterization to the level described above is sufficient for many risk-assessment purposes in non-sterile pharmaceutical manufacturing operations and some sterile product manufacturing environments. In other sterile product manufacturing environment microbial tests, the identification of microbe genus and species will be needed. Microorganisms can also be assessed for their “strain-level,” which supports identifying the sources of the microorganisms in a manufacturing environment or process. Overall, microbial identification is important when an unusually high rate of microbes is recovered from a product or a controlled manufacturing environment (such as aseptic processing). Microbial identification is accomplished by matching characteristics (genotypic and phenotypic) to an established standard (reference) organism such as a type strain. If a microorganism does not have a reference standard or is not included in the database, it will not be identified. Thus, manufacturers should review the breadth of the microbial database of the identification system they plan to use and its applicability to their needs.
How are microbes characterized in microbiology testing?
Microorganisms from raw materials, medical products, packaging, manufacturing environment, or other processes are physiologically stressed and in “survival” metabolic states. For microbiology testing, these sample microorganisms are placed in nutrient-rich environments that optimize cellular growth and reproduction. In preparation for microbe identification, representative colonies from the primary isolation media are streaked for colonies of a single microorganism type onto solid media. Then the isolated colonies are stained for Gram reaction, assessed for cellular morphology, and (in some cases) assessed for biochemical reactions of the bacteria isolates. If the wrong microbial characteristics are assigned to an isolate, incorrect identification of the microbes in a sample can occur. Gram staining, spore staining, phenotypic methods, and genotypic methods are standard microbial screening tests for microbial characterization.
Gram Staining
Gram staining methods include a four-step method and a three-step method. The four-step process uses crystal violet (primary stain), iodine (mordant), alcohol or alcohol– acetone (decolorizer), and safranin (counterstain). The three-step method is the same as the four-step method except that the decolorization (alcohol) and counterstaining (safranin) steps are combined. Gram-positive organisms retain the crystal violet stain and appear blue-violet. Gram-negative organisms lose the crystal violet stain and appear red. Some bacteria may be Gram-variable and exhibit both blue/violet and red stains. Gram staining isn’t perfect. Indeed, heat fixation may cause Gram-positive cells to stain Gram-negative, and older microbial cultures may yield Gram-variable results.
Additionally, too much decolorizer can result in a false Gram-negative result, and not using enough decolorizer may yield a false Gram-positive result. In some cases, alcohol fixation instead of heat fixation may give more consistent Gram stain results. Gram-positive and Gram-negative controls are included to allow for the identification of errors in staining. Gram-staining and cellular morphology are often determined simultaneously since gram-staining is read under a microscope.
Spore Staining
Spore staining uses a malachite green stain for bacterial spores. A positive control should be included when performing spore staining.
Biochemical Screening
Traditional biochemical screening tests include:
- The oxidase test to separate Gram-negative, rod-shaped bacteria into oxidase-positive and enteric (oxidase negative) bacteria
- The catalase test to separate Staphylococci (catalase-positive) from Streptococci (catalase-negative)
- The coagulase test to separate Staphylococci into coagulase-negative (presumptively nonpathogenic) and coagulase-positive (more likely pathogenic) Staphylococci
The biochemical screen described above provides sufficient information for surveying manufacturing environmental bioburdens. However, an in-depth microbial assessment is needed (such as identifying the genus, species, or strain) to provide insights into the nature and source of environmental bioburden.
What are phenotypic methods, and how are they used to identify microorganisms for microbiology testing?
Phenotypic microbial identification methods enable microbiologists to make informed decisions regarding microbial risk to the product and recognize changes in environmental microflora. In most quality control investigations, phenotypic microbial identification alone is sufficient to recommend appropriate corrective actions. Phenotypic methods identify microbes by expressed gene products such as size, shape, color, metabolite preference, etc., to identify different microorganisms. Phenotypic identification can be tricky for stressed microorganisms that may not express their total phenotypic properties.
What are genotypic methods, and how are they used to identify microorganisms for microbiology testing?
Genotypic microbial identification methods are more reliable than phenotypic methods because nucleic acid sequences are conserved in most microbial species. Genotypic methods for microbial identification include DNA–DNA hybridization, PCR, 16S and 23S rRNA sequencing, multi-locus sequence typing (MLST), pyrosequencing, DNA probes, and analytical ribotyping. Genotypic methods are technically challenging and require more expensive analytical equipment. For these reasons, genotypic identification methods are limited to critical microbiological investigations such as product failure investigations.
DNA sequencing using 16S rRNA sequence helps identify the species level but may not provide sufficient power to differentiate between closely related species or strains of the same species. In contrast, southern hybridization of restriction endonuclease digests is effective in demonstrating differences between the two strains. Nucleic acid-based methods can also be used to screen for specific microorganisms. Nucleic acid-based methods are completed through the following steps: sample collection, nucleic acid extraction, target amplification, hybridization, and detection. Issues with amplifying DNA from nonviable bacterial cells can be overcome by using reverse transcription to convert rRNA that is transitional to DNA for PCR amplification.
Summary
Overall, positive identification of microbes in a sample often results in microorganism characterization tests for microbe identification. Microorganism characterization tests may include determining colony morphology, cellular morphology (rods, cocci, cell groupings, modes of sporulation, etc.), Gram reaction, spore staining, and specific key biochemical reactions (e.g., oxidase, catalase, and coagulase activity). Identification of specific microbial contaminants and the strain level of the contaminating microbes can support manufacturers in eliminating microbial contaminants from medical products. When performing microbiology testing, ensure you choose a contract testing organization to help your unique medical device or product needs.
Ethide Labs is a contract testing organization specializing in Particulate Matter Testing and Microbiology Testing. Ethide Labs also offers EO Residual Testing, Sterility Testing, Cytotoxicity Testing, Bacterial Endotoxin Testing, Bioburden Testing, Package Integrity Testing & Environmental Monitoring services for medical device companies and allied industries. Ethide is an ISO 13485 certified facility.
References
United States Pharmacopeial Convention. <1113> Microbial Characterization, Identification, And Strain Typing. Rockville, MD, USA. 2021. (USPC <1113>).
