TITLE: External Quality Control for inter-Batch comparison (English)
AUTHORS: Ricardo M. Borges

[DESCRIPTION]
External Quality Control (QCExt): The sample containing the strain code CCMR0280, cultivated in parallel with all batches, will serve as a reference for comparative studies between the strains of the CCMR (Culture Collection of Microorganisms at the Federal University of Rio de Janeiro). After drying the biomass, the new sample of the strain code CCMR0280 obtained in the current cultivation will be combined with the stock of CCMR0280-QCExt for equitable distribution of inter-batch Quality Control.

This document is based on the article: Long-Term Metabolomics Reference Material. G.J. Gouveia, A.O. Shaver, B.M. Garcia, A.M. Morse, E.C. Andersen, A.S. Edison*, and L.M. McIntyre*. Analytical Chemistry. 2021, 93, 26, 9193–9199. https://doi.org/10.1021/acs.analchem.1c01294

[STEPS]
SECTION: Material
1. 2 mL microtubes with screw cap
50 mL Falcon type centrifuge tube
Analytical balance (AUW220, Shimadzu)

SECTION: Material in Stock
2. Initially, a starting batch composed of 25 replicas of the cultivation  of the strain code CCMR0280 was (i) cultivated, (ii) collected and combined, (iii) subjected to lyophilization, and (iv) subsequently 
ground into powder.
SECTION: Material in Stock
3. This ground sample is meticulously divided into 50, if possible, 50 mg aliquots in 2 mL microtubes with screw caps.

SECTION: For each batch to be produced
4. In each batch of cyanobacteria cultivated for the comparative study between the CCMR strains, the cultivation of the strain code CCMR0280 will be added, maintaining the same number of replicas as the study.

This sample will be used for intra-batch quality control and will also be part of the composition of the CCMR0280-CQExt sample, which, in turn, will be used for inter-batch quality control.
SECTION: For each batch to be produced
5. For each batch of strains to be cultivated, after completing the collection and lyophilization stage of the biomass, a sample of CCMR0280-CQExt will be added as a quality control sample.

The goal is to minimize variance between samples at all stages of batch analysis, facilitating the process of correction between different batches.
SECTION: For each batch to be produced
6. From the samples of the strain code CCMR0280 cultivated in each batch, after biomass collection and lyophilization, an aliquot will be combined in a 50 mL Falcon-type centrifuge tube to the equivalent amount of at least 100 mg.
SECTION: For each batch to be produced
7. This combined sample will be grounded and homogenized.
SECTION: For each batch to be produced
8. A 50 mg aliquot of this sample (of the strain code CCMR0280 cultivated in each batch) will be added to the sequence of CCMR0280-CQExt samples.
 
The CCMR0280-CQExt samples will be stored in a freezer at -20°C in a properly identified box. Each new sample should be labeled with the batch number and production date.

At this point, we will have a series of 50 mg aliquots of CCMR0280-CQExt samples, including those that will be produced with each new cultivation batch.
SECTION: For each batch to be produced
9. When the time comes to carry out the extraction and preparation stage of the samples from each batch, the first 10 CCMR0280-CQExt samples in the series will be (i) gathered, (ii) homogenized, and (iii) 5 representative aliquots of 50 mg will be added to the extraction sequence.

For the first batch, samples 1-10 will be used.
For the second batch, samples 2-11 will be used.
For the third batch, samples 3-12 will be used.
For the fourth batch, samples 4-13 will be used, and so on.

 
SECTION: For each batch to be produced
10. The extraction protocol for the biomasses must be strictly followed with all the samples from each batch, including these 5 CCMR0280-CQExt samples.
SECTION: For each batch to be produced
11. All analytical data acquired from the CCMR0280-CQExt samples will be evaluated for their homogeneity.
10 of the aliquots initially produced (step 3) will be separated and submitted to the extraction and preparation stage of the samples for the acquisition of analytical data.

These analytical data will be used to prove homogeneity.