Morphology and purity
When a new strain arrives at ATCC, it is our goal to ensure that it matches the depositor’s description, that it is pure, and that its classification is consistent with the description. The first step in authentication is to check the growth, purity, and morphology of each culture that arrives at ATCC for deposit. If these match the description given by the depositor, cultures then undergo further characterization to verify the classification of the new deposit.
When evaluating cultures for morphology, we observe attributes at both the cellular and colony level. Bacterial species come in a number of cellular morphologies and aggregations. At ATCC, we use different staining techniques to identify cell wall characteristics, general shape, cellular arrangement, and the presence and localization of flagella. At the colony level, we assess common characteristics such as shape, margin, elevation, size, appearance, optical property, texture, and pigmentation.
Historically, suites of biochemical and physiological tests were used to gather sets of phenotypic traits and dozens of schema were developed. Numerical taxonomy combined the traits to yield an identity for the organism. These biochemical tests are still instrumental for authenticating many important phenotypic properties of ATCC microbes; however, with advancements in technology and methodology, we have moved toward using automated and rapid testing methods.
Commercial test methods such as API® strips and VITEK® 2 cards (bioMérieux), the Remel RapID™ panels (Thermo Fisher Scientific), and the Biolog Gen III Microbial ID System (Biolog) are frequently used at ATCC for prokaryotic authentication. API strips and Remel RapID panels have replaced a number of biochemical tests that had been required in the past, and they provide rapid and reliable identification of hundreds of species. The VITEK 2 Microbial Identification system further automates the quality control process while minimizing manual steps, and the Biolog Gen III Microbial ID System is used for the analysis of environmental isolates.
Another method used for prokaryotic identification at ATCC is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which is a proteotypic method that identifies proteins using peptide mass matching.2 Here, the mass-spectral pattern of intact prokaryotic cells is obtained through the analysis of ionized components, creating a unique spectrum that represents the protein profile of each sample. These spectral patterns are then compared to a database of stored profiles from known organisms, allowing for the quick and accurate identification of a species.
Antimicrobial resistance testing
We also test specific phenotypes—such as the antibiotic susceptibility profile of a clinical isolate—when they are important for the item being deposited. For evaluating antibiotic susceptibility/resistance, we employ a variety of methods such as Kirby-Bauer disk diffusion, the Modified Hodge Test, and VITEK® 2 AST Cards (bioMérieux). Which test(s) is used is dependent on the strain. For example, if the item is considered to be a quality control strain by the Clinical Laboratory Standards Institute (CLIS), we evaluate the strain against antibiotics that are considered to be clinically relevant by CLSI. For these analyses, we use the testing method specified by CLSI. For other items that are characterized as multidrug-resistant, we use VITEK® 2 AST Cards to rapidly evaluate the strain against an array of antibiotics simultaneously. In some cases, if a strain is found to be resistant to a particular antibiotic, we perform an ETEST® (bioMérieux) to determine the minimum inhibitory concentration.