Sample Submission Concerns
On the Identification Request Form (IRF), what does "Marketed" product mean and what samples should be indicated as being recovered from a marketed product?
‘Marketed’ product refers to any product that is either already on the market or post-filing (NDA, ANDA, etc.) with either the FDA or any other regulatory agency. If you have filed for licensure for a product, it should be considered ‘Marketed.’ An organism should be identified as originating from marketed product if it is affiliated with a marketed product or is being used to make any decisions about a marketed product. If the box is not checked, Charles River assumes that it was not isolated from a marketed product.
How are the final reports provided?
We provide final electronic identification reports via email or fax. Please indicate your preference on the Identification Request Form. Charles River automatically emails 21 CFR Part 11 compliant reports on their due date. Printed reports are available upon request. Fees may apply.
What permit(s) are required to ship live bacterial or fungal cultures within the US?
No permits are required to ship unknown environmental samples within the United States; however, all live cultures should be packaged appropriately. If your company requires a CDC permit for shipment or if you are located outside the United States, we can provide that information for you.
What if I can’t tell if it’s a bacteria or yeast? How should I submit the sample?
Prior to submitting your samples, prepare a wet mount and look at the size of the organism. If the organism is in the 10 µM range it is a yeast, and if it is in the 1 µM range it is a bacterium. Please let us know if you have this concern and the results of your wet mount when samples are submitted. You can also submit your sample and indicate “possible yeast” in the comments column on the IRF, and we can perform a wet mount upon arrival.
How do I ship anaerobic bacteria?
We recommended that the samples are sent in with a BD Gaspak. Also, write “anaerobe” in the comments section on the IRF, this way if a sample requires a subculture, we will know the oxygen requirement.
Determining an Appropriate Test
Does Charles River use MicroSEQ® for identification?
MicroSEQ® is a commercially available sequence-based identification method that was developed by Applied BioSystems. We use a similar PCR-based amplification and sequence methodology; however, the methods used for analysis and data interpretation are different, and the library against which sequences are compared for ID are significantly different.
How does 16S rDNA sequence-based ID compare to ribotyping or sequence typing?
For the purpose of bacterial identification, 16S rDNA sequencing is more accurate and reproducible. Questions regarding sub-species differentiation, strain typing or determining if two isolates are the same or not are best answered by utilizing a DNA sequence-based method such as multi or single locus typing (MLST or SLST).
If two organisms have the same 16S sequence, are they the same strain?
They could be the same strain, but 16S rRNA gene sequences are highly conserved at the species level and are generally not useful for strain level differentiation. However, if two isolates have different 16S rRNA gene sequences, they can be considered to be different strains. This can be clarified by submitting the isolates for testing by SLST/MLST or on the RiboPrinter® system.
DNA Sequencing Libraries and Data Interpretation
Why have I have received a different species level identification for an isolate previously identified?
There are a few reasons why this may happen. Organism names can change when it is shown by phylogenetic analysis that an organism was previously classified incorrectly (probably based on phenotypic characteristics). The organism is then reclassified with the appropriate name based on phylogenetic taxonomy, and the name change is published. We update the names of organisms in our library to reflect these published changes in current taxonomy. You could also receive a different result for an isolate if the isolate is a newly identified species, but was previously thought to be part of another existing species.
Will the price for those services you offer change with the release of the new libraries?
No. Unlike other services providers, we do not charge for library updates.
When can I expect to see identification reports produced utilizing the most recent version of the library?
Any identification reports produced on or after the library launch date will use the new library. The date and time (Eastern Time) of report generation is displayed within the electronic signatures on each report, and we publish the date of the new library launches.
What do I do if an organism I need to identify is not represented in your libraries?
Please contact our Technical Support Department to explore other ways we can help you gain valuable information about your sample. The sequence data we generated for your organism can provide very useful information for you. Also, if the organism of interest is validly published, we can add it to the library.
Why is my identification result different than the ATCC culture identification?
The Accugenix® libraries contain sequences of the type strain for each species (when available). There have been instances where culture collections, such as the ATCC, have not updated their strains to reflect current taxonomy.
A Gram-positive organism was sent, but the identification report received was for a Gram-negative. How could this happen?
Accurate Gram staining is often difficult and depends on many factors. In our studies, we have seen that Gram stains can be incorrect a high percentage of the time. If you have questions, please consider repeating the Gram stain with a fresh culture. If the results are still discrepant please call Technical Support for further recommendations.
The GenBank database is so large. Why not use it for identification?
The GenBank database does contain hundreds of thousands of 16S sequences from microorganisms. However, there are several issues that must be considered before using it. First of all, many of the sequences are from new and unusual organisms isolated from extreme environments, or even cloned 16S sequences of organisms that have never been cultured in the laboratory. In those cases, these sequences are not associated with a particular genus and species name. There is little utility, for the purpose of identifying an organism, to show that the unknown is 96% identical to a sequence cloned from the environment that has never been cultured or described. Also, GenBank often has limited coverage for routinely isolated organisms, so it can be misleading for the purpose of identification. One of the most important factors to consider is that the GenBank database is not curated, or quality controlled, so any individual can submit a 16S sequence to GenBank, with no assurance that the data are correct. There is no process for verifying the accuracy of the sequence, or that it was derived from the genus and species name associated with it. Also, it is not cGMP-compliant. The size of the database also limits its utility, because the algorithms that are used to search large databases have been designed for rapid searching of large databases, rather than accuracy.
Can the sequence of an isolate be compared to an organism previously submitted for identification?
Yes. Please contact our Technical Support department and tell them which isolates you would like to compare. A sequence comparison report will be generated for your request at no additional charge.
Why do I occasionally receive a “No Match” report for a fungal sample?
The dramatic increase in our ITS2 fungal identification database makes it possible to follow taxonomic classification above the genus level with confidence and assurance, providing you with more information for tracking and trending. There will still be the occasional sample that you may receive a “No Match” report which reflects the diversity of fungal isolates still in need if classification.
Strain Typing
How many samples can be compared in one Strain Typing Report?
There is no limit to how many samples can be compared in one request, however, the report format is limited to 7 samples per page. The Comparison Report may be multiple pages as a result. Due to the time required to generate and interpret the comparison, there is not a guaranteed turnaround time for comparison requests. Please discuss your needs with Technical Support when requesting the comparison.
Does bacterial characterization by ribotyping work for all bacterial samples?
Because there are multiple copies of the target region examined for ribotyping, many species will result in banding patterns or a fingerprint that can be used for characterization. However, there are some species that will not produce usable data. For example, organisms that only have one or a couple copies of the target region may not produce enough bands for determining strain relatedness. The solution to these problems is to use single of multi-locus strain typing, because these methods are sequence-based and not fragment-based.
Single or Multi-Locus Sequence Typing
How do I initiate a strain typing project with Charles River?
Please contact Technical Support 1.800.886.9654 to discuss your project timelines.