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Teachers foster students’ scientific literacy by integrating the nature of science (NOS) into instruction. Sixty-one middle school teachers completed VNOS and CoRe that measured their content knowledge (NOS-CK) and pedagogical content knowledge (NOS-PCK) of NOS. Teachers demonstrated strengths in understanding some aspects of NOS (e.g., empirical, creative) but weaknesses in others (e.g., myth of “the scientific method” and social-cultural dimension). Regression analysis indicates that, despite a slight positive relationship, NOS-CK was not a significant predictor of NOS-PCK. Among gender, certification area, degree level and certification status, only certification status was a significant predictor of NOS-CK. Classroom observations and follow-up interviews of 12 teachers showed that they struggled to translate their knowledge into practice. Implications for professional development were discussed.