Giant yeast colonies develop a low redox potential, which mimics the electrophilic milieu of both the mitochondrial drug metabolizing compartment and the hypoxic core of many tumors. The major metabolic mediators of low redox potential include: ATP, glutathione, NAD+/NADH, and NADP+/NADPH. Ammonia signaling is the critical mechanism that induces stratification of the giant yeast colonies to allow a low redox potential. A comparison of two powerful investigative models for drug pathways using Saccharomyces cerevisiae have been compounded by the use of different growth media and stimuli to the system. Chemogenetic profiling, which uses a pool of yeast deletion mutants to determine survival changes, is heavily slanted to the use of rich media. Giant yeast colonies studies are heavily slanted to the use of poor media. The current study answers the question “what is the difference over time in redox potential, and its major metabolic mediators, between giant yeast colonies grown on rich and poor media?” Using gene deletion tools, we show that cell death in giant yeast colonies is ammonia-dependent. In poor nutrient, ammonia-depleted (Sok2 deletion mutants) giant yeast cultures, rotation can allow manipulation of reactive oxygen species, providing a model to compare high and low redox states without chemical administration. Mechanistically, these changes are not due to detectable NAD/NAPH or NADP/NADPH changes, but are related in changes in glutathione and ATP concentration.