Microbe Division (JCM)

JCM commonly performs quality checks of each strain as described below at the time of its acceptance and, preservation at JCM. JCM also performs quality tests of the holdings when necessary. JCM does not verify characteristics and/or functions cited in the on-line catalogue. JCM is not responsible for any differences between the properties of the strain deposited in JCM and properties provided in the literature, database and any information sources. Users are recommended to check the properties necessary for their researches by themselves.

• Performed
• Acceptance test

Every strain deposited in JCM is subcultured at JCM, and its viability, purity, colony appearance and cell morphology are checked by microscopic and/or macroscopic observations. At the same time, a DNA sequence of a PCR-amplified fragment such as that of the rRNA gene is determined and compared with the sequence provided by a depositor of the strain to confirm the authenticity of the received culture. This DNA sequence check at the time of acceptance has been performed since 2011. Archaeal strains deposited after 2015 have been checked also if there is bacterial contamination by the PCR analysis under the same experimental condition for the bacterial 16S rRNA gene amplification.

• Preservation test

Viability and purity of each batch are confirmed after preservation by the freezing, freeze-drying and/or L-drying methods.

• Not performed

Characteristics and/or functions of each strain appearing in the on-line catalogue are based on information from the corresponding literature and not examined by JCM.

• Supplementary tests

When any other tests are performed, the results are shown in the on-line catalogue.

Method for DNA sequence check

The standard protocol for the DNA sequence check performed at JCM is as follows:

(1) Preparation of cell extract
(2) PCR
(3) DNA sequencing

Materials:

• FastPrep-24 (MP Biomedicals), heat-sterilized acid-washed glass beads (SIGMA G4649 [≤106 µm]), a 2.0 mL screw-cap tube (e.g. AS ONE C-2.0) for cell disruption.
• MultiScreen PCR96 filter plate (Merck-Millipore MSNU03010), MonoFas DNA purification kit I (GL Sciences) for DNA purification.
• TaKaRa Ex Taq^® Hot Start Version (Takara Bio), and other reagents, buffers, and instruments for PCR and DNA analysis.

(1) Preparation of cell extract

1. Add 0.3 g grass beads and 400 µL TE buffer in a 2.0 mL screw-cap tube and suspend an appropriate amount of microbial cells.
2. Disrupt cells using FastPrep-24, usually at speed 6 for 20 sec.
3. Centrifuge and precipitate the cell debris. Transfer the supernatant into a new tube.
4. The supernatant is purified or concentrated with MonoFas, if necessary.

(2) PCR

1. Crude cell extract or its purified solution is used as template for PCR after diluting appropriately.
2. Amplify the gene fragment with the reaction mixture, the PCR primers and the cycle condition described in Tables 1-4.
3. Electrophorese the PCR product and confirm its amount.

(3) DNA sequencing

1. Purify the PCR product with a DNA purification kit such as MultiScreen or MonoFas.
2. Determine DNA sequence according to the instruction of DNA sequencer. For the DNA sequencing primer, choose appropriate one among those shown in Table 2.
3. Correct DNA sequence data, if necessary, and compare with the data from the depositor. Compare also with the database sequences using the BLAST service provided by e.g. NCBI, if appropriate.

 
Table 1. PCR mixture

Reagent	Volume (µL)
Milli-Q Water	37.75
10 x Buffer	5.0
dNTPs (2.5 mM)	4.0
Forward primer (10 µM)	1.0
Reverse primer (10 µM)	1.0
TaKaRa Ex Taq® HS	0.25
Template DNA	1.0

Total reaction volume 50 µL.

 
Table 2. Primers used in PCR amplificaion and sequencing

Primer	Sequence (5’–3′)	Target Group
27F1	AGAGTTTGATCCTGGCTCAG	Bacteria
786F2	GATTAGATACCCTGGTAG	Bacteria
1492R3	GGTTACCTTGTTACGACTT	Bacteria
907R1	CCGTCAATTCMTTTRAGTTT	Bacteria
826R4	GACTACCAGGGTATCTAATCC	Bacteria
519R1	GWATTACCGCGGCKGCTG	Bacteria/Archaea
530F1	GTGCCAGCMGCCGCGG	Bacteria/Archaea
1053F2	GCATGGCYGYCGTCAG	Bacteria/Archaea
A21F5	TTCCGGTTGATCCYGCCGGA	Archaea
A348F6	TCCAGGCCCTACGGG	Archaea
A1400R7	GACGGGCGGTGTGTGC	Archaea
A1100R7	CGGGTCTCGCTCGTT	Archaea
ITS58	GGAAGTAAAAGTCGTAACAAGG	Fungi
ITS38	GCATCGATGAAGAACGCAGC	Fungi
NL19	GCATATCAATAAGCGGAGGAAAAG	Fungi
NL49	GGTCCGTGTTTCAAGACGG	Fungi
ITS48	TCCTCCGCTTATTGATATGC	Fungi
ITS28	GCTGCGTTCTTCATCGATGC	Fungi

 
Table 3. PCR primer sets

Target Group	Primer set	Target gene
Bacteria	27F + 1492R	16S rRNA
Archaea	A21F + A1100R	16S rRNA
Archaea	A21F + A1400R	16S rRNA
Fungi	ITS5 + NL4	ITS, LSU
Fungi	ITS5 + ITS4	ITS
Fungi	NL1 + NL4	LSU

 
Table 4. PCR cycling condition

Step	Temp. (°C)	Time
1	98	2 min.
2	98	10 sec.
3	53	20 sec.
4	72	60 sec.
5	Followed steps 2-4 by 30 to 35 cycles
6	72	5 min.
7	10	hold

 
Reference

1. Lane, D. J. (1991). 16S/23S rRNA sequencing, In: Stackebrandt, E. and Goodfellow, M. (eds.), Nucleic Acid Techniques in Bacterial Systematics, pp. 115–176, Jon Wiley & Sons, Chichester.
2. Baker, G. C., Smith, J. J., and Cowan, D. A. (2003). Review and re-analysis of domain-specific 16S primers. J. Microbiol. Methods 55: 541–555.
3. Turner, S., Pryer, K. M., Miao, V. P., and Palmer, J. D. (1999). Investigating deep phylogenetic relationships among cyanobacteria and plastids by small subunit rRNA sequence analysis. J. Euk. Microbiol. 46: 327–338.
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6. Achenbach, L. and Woese, C. (1995). Appendix 6. 16S and 23S rRNA-like primers, In: Robb, F. T. and Place, A. R. (eds.), Archaea: a Laboratory Manual. Thermophiles, pp. 201-203, Cold Spring Harbor Laboratory Press, New York.
7. Itoh, T., Suzuki, K., and Nakase, T. (1998). Occurrence of introns in the 16S rRNA genes of members of the genus Thermoproteus. Arch. Microbiol. 170: 155–161.
8. White, T. J., Bruns, T., Lee, S., and Taylor, J. W. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, In: Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J. (eds.), PCR Protocols: A Guide to Methods and Applications, pp. 315-322, Academic Press, New York.
9. O’Donell, K. (1993). Fusarium and its near relatives, In: Reynolds, D. R. and Taylor, J. W. (eds.), The Fungal Holomorph: Mitotic, Meiotic and Pleomorphic Speciation in Fungal Systematics, pp. 225-233, CAB International, Wallingford.