From normally sterile clinical samples there are few problems with isolating E.sakazakii. However, isolation from a sample of infant formula requires some degree of luck as the organism will most likely be stressed, unevenly distributed throughout the batch and also numbers will probably be fairly low, in the region of less than 1 CFU per g.

Pre-enrichment is used to resuscitate the stressed cells and this is followed by a selective enrichment, the FDA method uses Enterobacteriaceae enrichment (EE) broth which is then streaked onto VRBG agar and suspect colonies subcultured onto TSA agar where yellow pigmented colonies need to be confirmed by oxidase test and a biochemical identification panel. Other methods use modified lauryl sulphate broth with vancomycin for the selective enrichment stage and this may be done at an elevated temperature 44°C.

After enrichment, there are now newer chromogenic agars available which greatly reduce the lab workload, give a faster time to result and are generally more reliable. These use the fact that alpha-glucosidase is produced by E. sakazakii, and not by most other Enterobacteriaceae. Typical colonies will show distinct colouration but biochemical confirmation is still required.

Concentrating bacterial cells can help with raising available cell numbers. Immunomagnetic beads capture the target cells in the sample, which can then be transfered to either a chromogenic media or for an even faster result onto a molecular method.

Molecular methods, used after the enrichment steps, include PCR or gene probe assays both giving faster results that need no further confirmation.