TABLE 1.

Procedures and time to perform 16S rRNA gene sequence analysis for bacterial identification in a routine clinical microbiology laboratory^a

Step	Procedure	Time (hands-on)	Waiting time (machine time)b
1	Harvest. Organism can be harvested from any plate or broth if it is a pure culture, e.g., antibiotic susceptibility plate. The age of the culture is not important. One or two 0.01 loops full is sufficient. Save for processing as a batch.	3-5 min each
2	Extraction of DNA.	0.5 h	10 min and 3 min
3	PCR amplification.	0.5 h	2.0 h
4	Analysis of the PCR product. Loading, running, and examining gel.	20 min	1 h
5	Purification of PCR products.	1 h
6	Cycle sequencing.	30 min	3.0 h
7	Purification of PCR products.	1 h
8	Sequencing of the 16S rRNA gene. Load capillary tray; allow to run during time away, e.g., overnight or while doing something else.	1 h	2.5 h
9	Analysis time. It takes 5 min or less to edit the sequence if the operator, software, and runs are good.	5-15 min/sample
10	Assignment of a name. If the organism is in the database, it takes 1 min; if it is a novel organism and several databases must be searched and sequences compared in detail, 15-30 min. At this point, correlation with phenotypic characteristics and clinical presentation is also done.	Not counted for this analysis
11	Reporting of results.	30 min
	Total labor time, based on integrating and completing three runs of 20 samples per wk.	60 samples/40 h	1 sample/40 min

^aModified from Clarridge et al., Abstr. 101st Gen. Meet. Am. Soc. Microbiol. 2001, with subsequent contributions by Kristina Hulten.

^bBased on an ABI 3100 instrument.