Methods for intense aeration, growth, storage, and replication of bacterial strains in microtiter plates

Abstract

Miniaturized growth systems for heterogeneous culture collections are not only attractive in reducing demands for incubation space and medium but also in making the parallel handling of large numbers of strains more practicable. We report here on the optimization of oxygen transfer rates in deep-well microtiter plates and the development of a replication system allowing the simultaneous and reproducible sampling of 96 frozen glycerol stock cultures while the remaining culture volume remains frozen. Oxygen transfer rates were derived from growth curves of Pseudomonas putida and from rates of oxygen disappearance due to the cobalt-catalyzed oxidation of sulfite. Maximum oxygen transfer rates (38 mmol liter(-1) h(-1), corresponding to a mass transfer coefficient of 188 h(-1)) were measured during orbital shaking at 300 rpm at a shaking diameter of 5 cm and a culture volume of 0.5 ml. A shaking diameter of 2.5 cm resulted in threefold-lower values. These high oxygen transfer rates allowed P. putida to reach a cell density of approximately 9 g (dry weight) liter(-1) during growth on a glucose mineral medium at culture volumes of up to 1 ml. The growth-and-replication system was evaluated for a culture collection consisting of aerobic strains, mainly from the genera Pseudomonas, Rhodococcus, and Alcaligenes, using mineral media and rich media. Cross-contamination and excessive evaporation during vigorous aeration were adequately prevented by the use of a sandwich cover of spongy silicone and cotton wool on top of the microtiter plates.

FIG. 1

Methods of aeration that were assessed for maximum OTRs as shown in Table 1 and Fig. 2. The solid lines inside the wells in the upper two panels represent the culture surface when the orbital shaker is at the outer right side of its rotating movement (as determined by photographic analysis). The dashed lines represent the culture surface when the orbital shaker is at the outer left side of its rotating movement.

FIG. 2

Growth curves of P. putida CA-3 on a glucose mineral medium (750 μl) in deep-well microtiter plates incubated under various conditions: orbital shaking, 2.5-cm diameter, 300 rpm (⧫ and ◊); orbital shaking, 2.5-cm diameter, 375 rpm (■ and □); and orbital shaking, 5-cm diameter, 300 rpm (▴ and ▵). The dashed lines denote the slopes resulting from linear regression of the data points represented with solid symbols in the linear, oxygen-limited part of the growth curve. These slopes were used to calculate the OTRs given in Table 1.

FIG. 3

System for the retrieval and growth of a bacterial strain collection in microtiter plate format. The spring-loaded replicator is used for the parallel sampling of up to 96 stock cultures frozen in glycerol; the surfaces of all stock cultures are contacted by the pins even though the surfaces are not exactly at the same level. The sampled cells are allowed to grow on an agar plate for several days. The colonies formed are then transferred to a square-well microtiter plate filled with liquid medium, covered as shown, and incubated on an orbital shaker.