The study by Anagnostopoulos and Spizizen investigates the requirements for transformation in *Bacillus subtilis* strain 168, a highly transformable strain. The research focuses on the optimal conditions for transforming auxotrophic mutants to prototrophy, using simple chemically defined media. Key findings include: 1. **Optimal Competence Conditions**: Vegetative cells of *B. subtilis* strain 168 become optimally competent for transformation when grown in minimal media containing glucose and an ammonium salt as carbon and nitrogen sources. The presence of indole or L-tryptophan and a chelator for cupric ions is essential. 2. **Growth Phases**: The transformation process involves two growth phases. In the first 4-hour phase, the medium contains yeast extract, which can be replaced by indole or L-tryptophan and acid casein hydrolyzate. High concentrations of casein hydrolyzate inhibit competence. In the second 90-minute phase, the medium can be replaced by L-histidine or metal chelating compounds like EDTA. 3. **Chelating Compounds**: Chelators that form stable complexes with heavy metals, such as 2,2′-bipyridyl, β-mercaptoethanol, ethylenediamine, and EDTA, enhance competence. EDTA-Cu++ is inactive, while EDTA-Na-Fe+++ is active, possibly due to modified iron state. 4. **Amino Acid Levels**: Limited amino acid content in the medium is crucial for transformability. Higher concentrations of amino acids, such as those in acid hydrolyzed casein, reduce sensitivity to transformation by allowing the synthesis of excess cell wall components. 5. **DNA Concentration**: Transformation efficiency is linear up to 10−1 μg of DNA, similar to pneumococcal transformation. The quality of DNA preparation affects the transformation response. 6. **Physiological and Genetic Factors**: The development of competence may involve structural changes in the cell wall and enzymatic mechanisms for DNA uptake. Sensitivity to transformation occurs during presporulating events, suggesting that alterations in cell wall structure are key. The study provides a detailed framework for understanding the physiological and genetic factors that enable *B. subtilis* to become competent for transformation, offering insights into the broader mechanisms of bacterial transformation.The study by Anagnostopoulos and Spizizen investigates the requirements for transformation in *Bacillus subtilis* strain 168, a highly transformable strain. The research focuses on the optimal conditions for transforming auxotrophic mutants to prototrophy, using simple chemically defined media. Key findings include: 1. **Optimal Competence Conditions**: Vegetative cells of *B. subtilis* strain 168 become optimally competent for transformation when grown in minimal media containing glucose and an ammonium salt as carbon and nitrogen sources. The presence of indole or L-tryptophan and a chelator for cupric ions is essential. 2. **Growth Phases**: The transformation process involves two growth phases. In the first 4-hour phase, the medium contains yeast extract, which can be replaced by indole or L-tryptophan and acid casein hydrolyzate. High concentrations of casein hydrolyzate inhibit competence. In the second 90-minute phase, the medium can be replaced by L-histidine or metal chelating compounds like EDTA. 3. **Chelating Compounds**: Chelators that form stable complexes with heavy metals, such as 2,2′-bipyridyl, β-mercaptoethanol, ethylenediamine, and EDTA, enhance competence. EDTA-Cu++ is inactive, while EDTA-Na-Fe+++ is active, possibly due to modified iron state. 4. **Amino Acid Levels**: Limited amino acid content in the medium is crucial for transformability. Higher concentrations of amino acids, such as those in acid hydrolyzed casein, reduce sensitivity to transformation by allowing the synthesis of excess cell wall components. 5. **DNA Concentration**: Transformation efficiency is linear up to 10−1 μg of DNA, similar to pneumococcal transformation. The quality of DNA preparation affects the transformation response. 6. **Physiological and Genetic Factors**: The development of competence may involve structural changes in the cell wall and enzymatic mechanisms for DNA uptake. Sensitivity to transformation occurs during presporulating events, suggesting that alterations in cell wall structure are key. The study provides a detailed framework for understanding the physiological and genetic factors that enable *B. subtilis* to become competent for transformation, offering insights into the broader mechanisms of bacterial transformation.