Scott Sutton, Ph.D.
This article first appeared in the PMF Newsletter of November, 2007 and is protected by copyright to PMF. It appears here with permission.
We frequently see the Seed Lot Technique referenced in the USP, literature publications, and conference presentations. I would like to take a few minutes to explore the technique in some detail.
The goals of the seed lot technique in our industries is three-fold:
1. To provide a method for generation of a working culture no more than 5 passages removed from the original stock from the national repository
2. To provide a method for generation of a pure culture for laboratory work
3. To provide a method for generation of a traceable culture for laboratory investigations
Let’s look at each of these goals separately.
The goal of “no more than 5 passages removed” is now pretty common in the compendia, but not so commonly provided are the details on exactly how to accomplish this. This explanation will rely on Figure 1 below.
In this design, the lyophilized culture is received, resuscitated and grown in broth. This growth step is the first passage removed from the national repository. The broth culture is then prepared for cryopreservation (see “Cryopreservation of Bacteria” by Liz Kerrigan that appeared in the February 2007 issue of PMF News). The prepared culture can be (should be) split into many different vials which are frozen individually. Standard techniques for this freezing step can be found from ASTM (1997). Note that the long-term viability of these cultures is not clear for all storage conditions. Therefore, viability checks should be run on standard strains annually.
The next passage for the organism occurs when the frozen vial is removed and streaked on nutrient plates or tube slants. This second passage is acceptable for growth promotion studies, but there is evidence in the literature of the effect of inoculum preparation variables affecting results of some tests (see Boomfield 1995, Gilbert 1987, Orth 1989). If you wish to pass the cultures once (recommended for most tests) or twice more (recommended for the antimicrobial effectiveness test) you still are only 4 passages removed from the national repository strain.
An assumption of most QC microbiology assays is that we are working with a pure culture of the correct organism. We put media under incoming quarantine and release procedures, why shoulder not do the same for the challenge organisms we use (Sutton, 2007). This is, in fact, recommended by USP (USP, 2008). The best place to withdraw a sample for testing is at the first passage where you have a lab batch ready for cryopreservation. This should be tested by streaking for single colonies (see PMF Newsletter of December, 2006) and checking for purity of the culture, the identity of the microorganism, and confirmation that the new lot is the same strain as the ones previously used (genotypic methods?).
Having established the purity and identity of the batch grown and placed in cryopreservation, this state must be protected. Most of us can pull out the frozen vial, sterilize a loop and take a sample without contaminating that frozen vial. However, it is impossible to prove that we did not do that in a situation where contamination crept into the process at some point. It is therefore recommended to discard each frozen “Cryo” stock vial after sampling. Similarly, once a container along the chain (plate or slant tube) is sampled, that container should be considered contaminated and discarded. The purity of the inoculum should be confirmed from the inoculum preparation as the final check in this process.
Ideally we can trace the inoculum used in a test from that test all the way back to receipt of the shipment from the national repository and then back forward to all tests that used this particular shipment. This is very possible if all incoming microorganisms are assigned identification numbers for this purpose, and all sub-batches are traced using a reasonable system. The value of this system in eliminating the culture (clear evidence of purity, lack of problems in other tests that used the culture, etc) make the effort involved in tracing the cultures.
One method that is straightforward to execute is to assign the incoming microorganism ID number, then a second 3-digit number is added after a decimal point to identify the “Cryo” vial, then another decimal point followed by a 2-digit number to identify the working stock preparation followed (finally) by a T1 or T2 to denote the first or second transfer from that working stock. This leads to long, but precise, identification numbers. For example, the culture batch number 12345.333.22.T2 would identify the inoculum batch as the second transfer from a microorganism that had been assigned the identification code “12345” upon receipt and then stored in the “cryo” tube “333” before being streaked as the working culture preparation identified as “22”. Obviously, for this system to work quality checks (of some sort) and records of each step of the seed lot procedure would be required. Thi This method of tracking the cultures is only one of many that would be suitable for the purpose.
The use of a well-designed seed lot culture technique can provide many benefits to a regulated lab (or for that matter a non-regulated lab). If implemented and executed with an eye to making it part of the overall Quality system for the lab, this seed lot technique and its associated records can help in assuring pure cultures for inocula that are traceable if need be for investigations. In addition, the system can generate a large supply of fresh cultures, none of which exceed 5 passages from the original shipment from the national repository.
ASTM 1997 E1342-97 Standard Practice for Preservation by Freezing, Freeze-Drying, and Low Temperature Maintenance of Bacteria, Fungi, Protista, Viruses, Genetic Elements, and Animal and Plant Tissues
Bloomfield, S et al. 1995. Development of Reproducible Test Inocula for Disinfectant Testing. Intl Biodeter Biodegrad. pp. 311-331
Gilbert, P et al. 1987. Inocula for Antimicrobial Sensitivity Testing: a Critical Review. J Antimicrob Chemother. 20:147-154
Orth, D.S. et al. 1989. Effect of Culture Conditions and Method of Inoculum Preparation On the Kinetics of Bacterial Death During Preservation Efficacy Testing. J Soc Cosmet Chem. 40:193-204
Sutton, S. 2007. “Microbiology Laboratory Quality Control Practices” IN Pharmaceutical Quality Control Microbiology: A Guidebook to the Basics DHI Publishers, Inc. 2007.
USP. 2008. <1117> Microbiological Best Laboratory Practices. USP 31 Volume 1 United States Pharmacopeial Convention. pp 589-593.
Experts at the Microbiology Network are ready to assist with consulting or training to meet your needs. Have customized, in-house assistance with your questions from our recognized experts at your schedule either through consulting agreements, in-house training or customized webinars – contact us using the “Let’s Talk” communication found in the upper right of this page.