The Isolation of Nitrosomonas europaea in Pure Culture

Jane Meiklejohn successfully isolated Nitrosomonas europaea in pure culture from Rothamsted soil using a modified version of Winogradsky's method. The isolation procedure involved three key steps: building up the nitrifier population in enrichment cultures using mineral medium with ammonium sulfate and calcium carbonate, removing cells from carbonate debris using a carbon dioxide stream, and picking colonies from poured silica gel plates. The enrichment culture was maintained for five months with periodic additions of ammonium sulphate and calcium carbonate. After multiple liquid transfers, a modified medium (Medium B) was developed to maintain nitrifying activity over 24 successive transfers without loss of function. The isolated organism consisted of small oval, non-motile, Gram-negative cells measuring 1.2-1.7 micrometers in length and 1.0-1.2 micrometers in width. Colonies on silica gel were tiny (80-140 micrometers diameter), compact, glassy, and initially colorless becoming brown, sometimes displaying a distinctive starfish shape. The pure cultures oxidized ammonia to nitrite but did not further oxidize nitrite to nitrate, confirming their identity as ammonia-oxidizing bacteria matching Winogradsky's original description of N. europaea.

Key findings

• Meiklejohn developed a practical three-step isolation method using enrichment cultures, CO2 treatment to separate cells from carbonate, and silica gel plating to obtain pure N. europaea cultures
• Modified mineral medium (Medium B) maintained nitrifying activity over 24 successive liquid transfers without contamination loss, though contaminants persisted as detected on nutrient agar
• The isolated N. europaea cells were small oval, non-motile, Gram-negative bacteria forming tiny glassy colonies on silica gel with characteristic starfish-shaped morphology
• Pure cultures specifically oxidized ammonia to nitrite without further conversion to nitrate, distinguishing them from the complete nitrification pathway
• The carbon dioxide treatment step proved effective for removing bacterial cells from calcium carbonate particles, overcoming limitations of Winogradsky's original motility-dependent method