Authors: Cara N. Wilder, PhD; Jennifer Murdock, MS; and Dev Mittar, PhD
This study will demonstrate the use of ATCC Minis as quality control strains for microbial identification on the VITEK 2 platform (bioMérieux), employing strains recommended for use with the Anaerobic and Corynebacteria (ANC), Bacillus (BCL), Corynebacteria (CBC), Gram-Positive (GP), Gram-Negative (GN), Neisseria-Haemophilus (NH), and Yeast (YST) Streamlined QC Sets as examples.
Commercial firms and research laboratories look to ATCC to provide top-quality microbial strains needed to maintain outstanding quality control programs for instruments, reagents, media, etc. To help aid in the rapid identification of microbial strains, ATCC now provides the same trusted quality control strains in a convenient, single-use, “mini” format that saves precious time and resources. ATCC Minis are a six pack of glass-free cryovials containing ready-to-use strains in glycerol stock, complete with peel-off labels for fast and reliable recordkeeping. Further, similar to their lyophilized counterparts, ATCC Minis are backed by polyphasic testing to establish identity, viability, and purity, and are maintained using a seed stock system to minimize subculturing.
To provide convenient reference materials for fast, accurate microbial identification using the VITEK 2 system, ATCC has expanded its collection of quality control strains to include bacterial and fungal isolates in the ATCC Minis frozen format. In the following study, we demonstrate the application of ATCC Minis as quality control strains for microbial identification on the VITEK 2 platform using ANC, BCL, CBC, GP, GN, NH, and YST Streamlined QC Sets as examples.
Materials and methods
ATCC Minis (Table 1) were thawed and cultured according to the recommended propagation procedure described on the product sheet. Following growth in broth, strains were streaked for isolation on the recommended agar medium and incubated under the appropriate temperature and atmospheric conditions until growth was established. To create the sample inoculum, isolated colonies were selected using a sterile cotton swab and suspended into 3.0 mL of 0.45% saline solution within a 12 mm x 75 mm polystyrene test tube. Inoculums were adjusted accordingly with the assistance of a calibrated DensiCHECK Plus (bioMérieux) to reach the McFarland Standard density range as recommended in the VITEK 2 Systems Product Information manual.1 The sample inoculum and associated VITEK 2 card were then inserted into a VITEK 2 Cassette and processed according to instructions found within the VITEK 2 Compact System and OBSERVA Computer System (bioMérieux) manuals. For this study, microbial identity was confirmed using the VITEK 2 ANC, BCL, CBC, GP, GN, NH, and YST cards, which provide species-level identification of anaerobes, bacilli, corynebacteria, Gram-positive bacteria, Gram-negative bacteria, Neisseria and Haemophilus strains, and yeasts, respectively.
Table 1. ATCC Minis
|QC set||ATCC number||Species|
|VITEK 2 ANC Streamlined QC Set||BAA-1296-MINI-PACK||Bacteroides ovatus (ATCC BAA-1296)|
|12464-MINI-PACK||Clostridium septicum (ATCC 12464)|
|VITEK 2 BCL Streamlined QC Set||51663-MINI-PACK||Brevibacillus agri (ATCC 51663)|
|VITEK 2 CBC Streamlined QC Set||43044-MINI-PACK||Corynebacterium urealyticum (ATCC 43044)|
|15829-MINI-PACK||Microbacterium testaceum (ATCC 15829)|
|VITEK 2 GP Streamlined QC Set||700327-MINI-PACK||Enterococcus casseliflavus (ATCC 700327)|
|BAA-750-MINI-PACK||Staphylococcus saprophyticus (ATCC BAA-750)|
|VITEK 2 GN Streamlined QC Set||700323-MINI-PACK||Enterobacter hormaechei (ATCC 700323)|
|17666-MINI-PACK||Stenotrophomonas maltophilia (ATCC 17666)|
|VITEK 2 NH Streamlined QC Set||BAA-1152-MINI-PACK||Eikenella corrodens (ATCC BAA-1152)|
|VITEK 2 YST Streamlined QC Set||14053-MINI-PACK||Candida albicans (ATCC 14053)|
Results and discussion
As part of the microbial identification process, the VITEK 2 software compares the resulting biochemical reaction outcomes of organisms present in each sample to the expected set of results for organisms that have been previously identified by the database.1 These biochemical test outcomes are represented as a bionumber, a unique species identification pattern generated from the conversion of test results into positive or negative test probabilities. The identity percent probability is based on the similarity of the observed reactions compared to that of the expected reactions of an organism. Here, the possible range of percent probabilities for a single organism match is 85-99%, with values closer to 99% indicating a perfect match between the test reaction pattern and the unique reaction pattern of a known species.1
From this analysis, each of the ATCC Minis were identified using the appropriate VITEK 2 cards at a percent probability ranging from 91-99%, reflecting confidence levels of low, good, very good, and excellent (Table 2). Further, the results obtained using the ATCC Minis quality control strains corresponded with that of their lyophilized counterparts, indicating that the frozen format does not affect the biochemical phenotype of the strains (data not shown).
BioMérieux selects quality control strains based on their biochemical characteristics. In some cases, these quality control strains exhibit expected biochemical results, but are unidentified or misidentified in the VITEK 2 system.1 As an example, the data for Brevibacillus agri ATCC 51633-MINI-PACK reflected an identity probability of 96% and a low confidence interval on the BCL card. This particular species belongs to a pseudoslashline taxa with Brevibacillus brevis. Thus, it is possible for the two species to exhibit the same biopattern, which results in low discrimination between B. agri and B. brevis.
In some instances, VITEK 2 cards may be limited in the number of individual species within a given genera that can be identified.1 For example, the CBC card is capable of identifying Microbacterium spp. and Microbacterium lacticum, but not M. testaceum specifically. Accordingly, though the analysis of ATCC 15829-MINI-PACK, which is a strain of Microbacterium testaceum, exhibited a percent probability of 91% and a good confidence interval, it was identified only at the genus level. Therefore, in this instance, ATCC 15829-MINI-PACK performed as expected and was correctly identified within the identification constraints of the CBC card.1
Table 2. VITEK 2 test results
|ATCC number||VITEK 2 card||VITEK 2 lot number||Analysis time (hours)||Percent probability||Bionumber||Confidence level||Species confirmed|
|700327-MINI-PACK||GP||242342010||4.75||93%||524213665777731||Very good||Enterococcus casseliflavus|
|BAA-750-MINI-PACK||GP||242351110||6.00||94%||030002056670131||Very good||Staphylococcus saprophyticus|
Overall, this study demonstrates that ATCC Minis exhibit results consistent with those expected for each respective VITEK 2 card and are identical to their lyophilized counterparts, making them ideal for use as quality control strains for the identification of unknown microbial strains using the VITEK 2 system.
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