<p><strong>BACKGROUND: </strong>Clinicians often express concerns about poor sensitivity of blood cultures in neonates resulting from inadequate inoculant volumes. Our objective was to determine the inoculant volume sent for neonatal sepsis evaluations and identify areas of improvement.</p>

<p><strong>METHODS: </strong>Single-center prospective observational study of infants undergoing sepsis evaluation. Blood volume was determined by clinician documentation over 21 months, and additionally by weighing culture bottles during 12 months. Adequate volume was defined as ≥1 mL total inoculant per evaluation. For first-time evaluations, local guidelines recommend sending an aerobic-anaerobic pair with 1 mL inoculant in each.</p>

<p><strong>RESULTS: </strong>There were 987 evaluations in 788 infants. Clinicians reported ≥1 mL total inoculant in 96.9% evaluations. Among 544 evaluations where bottles were weighed, 93.4% had ≥1 mL total inoculant. Very low birth weight infants undergoing evaluations &gt;7 days after birth had the highest proportion of inadequate inoculants (14.4%). Only 3/544 evaluations and 26/1011 bottles had total inoculant &lt;0.5 mL. Ninety evaluations had &lt;1 mL in both aerobic and anaerobic bottles despite a total inoculant volume that allowed inoculation of ≥1 mL in one of the bottles.</p>

<p><strong>CONCLUSIONS: </strong>Obtaining recommended inoculant volumes is feasible in majority of neonates. Measuring inoculant volumes can focus improvement efforts and improve test reliability.</p>

<p><strong>IMPACT: </strong>Clinicians express concern about the unreliability of neonatal blood cultures because of inadequate inoculant volume. We investigated over 900 evaluations and found &gt;90% of evaluations have ≥1 mL inoculant. Monitoring adequacy of blood culture technique can identify areas of improvement and may allay concerns about blood culture reliability. Current recommendations for adequate inoculant volume for blood cultures are met in a majority of neonates. Areas of improvement include preterm late-onset sepsis evaluations and distribution techniques during inoculation.</p>