Bacterial Authentication

Bacterial Culture Guide Bacterial Authentication


Table of Contents

Authentication

When preparing a bacterial strain for distribution, ATCC performs numerous quality control (QC) assays to guarantee that the product is of the highest standard before it reaches the consumer. All bacterial strains undergo thorough phenotypic and genotypic examinations to ensure that strain identification is accurate, the culture is pure, and that all biochemical results are consistent. To assist in maintaining these standards, all equipment and biochemical tests required in the QC process are similarly evaluated for quality assurance. Described below are several tests that ATCC commonly performs on bacterial strains during QC analysis.

Phenotypic Characterization

API strips

When bacterial strains are initially received by ATCC, they are analyzed for characteristic colony and bacterial morphology; this is observed via colony growth and Gram staining, respectively. Additionally, samples are tested for culture purity on blood agar plates. The presence of more than one colony type is often, but not always, indicative of a mixed culture.

In addition to purity and morphology, strains are also examined for consistency in sugar metabolism, antibiotic susceptibility, and/or broad-spectrum biochemical reactivity. To confirm the bacterial species identity based on these associated phenotypic traits, ATCC commonly uses appropriate API® strip tests and VITEK® cards. Both aforementioned techniques employ over 20 different biochemical tests in reaction cells to assess the growth and viability of the bacterial strain.30 The API® strip test characterizes bacterial identity through a manual, micro-method that generates a seven-digit code based upon the reaction of the individual biochemical assays. This generated code is the identifier number linked to a specific bacterial species. In contrast, the VITEK® system provides an automated, computer-based method of species identification via the measurement of light attenuation associated with each biochemical reaction.30, 31 For this latter system, ATCC requires a ≥ 90% probability of organism identification.

Genotypic and Proteotypic Characterization

Ribo Printer

ATCC genotypically confirms bacterial species primarily through 16S ribosomal RNA (rRNA) sequencing. These sequences are commonly used to analyze bacterial phylogeny and taxonomy as they are highly conserved and universal among prokaryotic species. In general, the 16S rRNA gene sequence is composed of both variable and conserved regions, the comparison of these sequences can allow for differentiation at least to the genus level.32, 33 A secondary approach is ribotyping, which involves the comparative analysis of discrete-sized genome fragments generated by restriction digestion of rRNA operons.34, 35

Another method of prokaryotic identification is through matrix-assisted laser desorption/ionization timeof-flight mass spectrometry (MALDI-TOF MS), which is a proteotypic method that identifies proteins using peptide mass matching.36 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.