Worsham (Worsham and Sowers, 1999). These included point mutations as well as deletions and insertions. Sastalla and colleagues further showed that the spo0A mutants exhibited a shorter lag time than did the wild type when inoculated on rich medium. Thus, one factor contributing to the enrichment of sporulation-deficient mutants upon repeated passage is that at least some sporulation mutants resume rapid (exponential phase) growth more quickly than does the wild type.
Part of the analysis of the anthrax letter evidence samples involved the preparation of relatively low-density liquid suspensions of spores recovered from the letters and the spreading of these suspensions on the surface of a solid growth medium. This procedure allowed each viable spore to germinate, grow, and divide in isolation, producing a clonal population of millions of cells—all derived from a single cell—in the form of a colony. This is a standard microbiological practice used for the enumeration of viable spores in a sample. The method also allows the visual detection of variants in the population that may carry genetically heritable traits responsible for altered phenotypic properties, although the sensitivity and discriminatory capacity of this method are low. Relatively early in the investigation (fall 2001), Terry Abshire, a microbiologist at USAMRIID, observed the presence of multiple phenotypic variants (morphotypes) among the colonies generated from the anthrax letter spore samples. The analysis of these variants eventually became a major feature in the FBI’s effort to identify the most likely source of the specific culture used to produce the letter samples. To the advantage of the FBI, Patricia Worsham was one of the USAMRIID scientists that assisted with the analysis of the letter spore samples. Due to her extensive experience with B. anthracis and her specific experience studying B. anthracis phenotypic variants, Worsham and her colleagues were readily able to apply methods for the study and characterization of phenotypic variants in the letter samples.
In early analysis of the Leahy letter sample, spores were plated on sheep blood agar and incubated for three days. On these plates, Abshire observed the presence of morphotypes with more defined colony edges and a yellow tint relative to the majority of the colonies. Based on this observation, Worsham was asked to develop a protocol to characterize morphotypes in the multiple evidentiary samples. The goal of this analysis was the identification of phenotypic and eventually genetic signatures of the letter samples that might contribute to determining the source of the letter material.
Spore samples from the Daschle, Leahy, and New York Post letters were suspended in water at a low density, such that they could be spread to give rise to between 10 and 50 colonies per plate, allowing the observation of individual