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An Enrichment Technique for Isolation of Marine Chemotactic Bacteria I. CHET* AND R. MITCHELL Laboratory of Applied Microbiology, Division of Engineering and Applied Physics, Harvard University, Cambridge, Massachusetts 02138

Chemotaxis is the movement of an organism toward or away from a chemical source [1, 2, 5, 8]. Bacteria are attracted to many kinds of chemicals, some of which can serve as nutrients. However, there is no correlation between the metabolism of a substance, energy production from it and its ability to attract bacteria [3]. Mesibov and Adler [7] found that extensive metabolism of amino acids is neither required nor sufficient for attraction. We have developed an enrichment technique for isolating active motile bacteria from aquatic habitats, which are able to degrade and metabolize the specific s u b s t a n c e t o which they are attracted. Marine bacteria were grown in enrichment liquid media containing seawater taken from the Atlantic Ocean near Boston, Mass., amended with 1% of either albumin, casein, or L-alanine as carbon and nitrogen sources. When glucose was used as a carbon source, 0.5% NH4NO3 was added as the nitrogen source. Seawater analysis indicated the presence of 210 /zg]liter phosphate which enabled bacterial growth without further addition of phosphorus. The bacteria were grown at 30~ for 2 - 4 days until the culture reached a density of 101~ bacteria/ml. These bacteria were tested for chemotaetic activity toward the substances which previously served as a carbon or carbon + nitrogen source, respectively. The design of the experiment was modeled after the method of Adler [ 4 ] . Test materials were suspended in artificial seawater [6] and were placed in 1 /~1 capillaries. Controls consisted of artificial seawater in the

*Permanent address: The Hebrew University, Department of Plant Pathology and Microbiology. Faculty of Agriculture, Rehovot, Israel. 75 MICROBIAL ECOLOGY 3:75-78 (1976) 1976 by Springer-Verlag New York Inc.

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microcapillary. Numbers were determined by the dilution plate method. Each number represents data from at least four experiments. The process described in Fig. 1 shows the successive stages involved in the enrichment technique. In each experiment the enrichment involved growth of a mixed culture o f marine bacteria in seawater amended with a specific substrate. Samples of water containing large numbers o f bacteria were transferred to chemotaxis test cells [ 4 ] . The appropriate attractant was inserted in capillaries which were placed in the bacterial suspension for l0 min. The bacteria entering the capillary (first chemotactic response in Fig. 1) were transferred to a medium containing the same substrate and incubated for 72 hr. The chemotactic response of the resultant culture was tested for ability of the bacteria to be attracted to the substrate in the enrichment (second chemotactic response in Fig. 1). The technique yielded large populations of bacteria capable of being attracted to and metabolizing a substrate. In most

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marinebacteria

non-attracted bacteria

enriched cultures on specific C or C + N sources

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1 E first chemotactic response [

J

1

[ attractedbacte"a ]

l

isolation on the corresponding substrates

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sec~ chem~

resp~

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1 isolation of very active bacteria

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Fig. 1. Flow diagram of enrichment technique for bacteria capable of both metabolizing and attraction to specific substrates.

Isolation of Marine Chemotactic Bacteria

77

cases we obtained pure cultures. The bacteria were highly motile and strongly attracted to the test chemical. The quantitative data in Table 1 show a comparison of chemotactic activity of the bacteria following initial enrichment (first chemotactic response) and the chemotactic response following subculture on a solidified medium containing the test substrate (second chemotactic response).

Table 1

Enrichment of Chemotactic Marine Bacteria Attracted to Specific Metabolizable Substrates Number of attracted bacteria a Substrate

Nutrient source

First chemotactie response

Second chemotactic response

Albumin Casein Glucose L-Alanine

N + Cb N + C C N + C

32,000 25,000 12,000 18,000

520,000 275,000 160,000 95,000

aAttraction to artificial seawater alone: 6000 bacteria//~l. bN, nitrogen; C, carbon. In all the attractants which we tested, the number of the attracted bacteria did not exceed 32,000 within 10 min in the first enrichment phase. However, the chemotactic response was significantly higher in the second phase when albumin attracted 520,000 bacteria and L-alanine, which was the poorest attractant, attracted 95,000 bacteria. The technique which we describe may serve as a very useful tool both for ecological and physiological studies. The process enables isolation of bacteria from natural ecosystems which are capable of metabolizing and are attracted to specific substrates. This research was supported in part by Contract No. N00014-67-A0298-0037 between Harvard University and the U.S. Office of Naval Research.

References

1.

Adler,J. 1966. Chemotaxis in bacteria. Science 153: 708-716.

2.

Adler,J. 1969. Chemoreceptors in bacteria. Science 166: 1588-1597.

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3.

Adler, J, 1973. Chemotaxis and chemotropism. In: Behavior of Microorganisms. A. Perez-Miravete, (editors.) Plenum Press, New York.

4.

Adler, J. 1973. A method for measuring chemotaxis and use of the method to determine optimal conditions for chemotaxis for Escherichia coli. J. Gen. Microbiol. 74:77-91.

5.

Chet, I., Fogel, S., and Mitchell, R. 1971. Chemical detection of microbial prey by bacterial predator. J. Bacteriol. 106: 863-867.

6.

Marshall, K. C., Stout, R,, and Mitchell, R. 1971. Mechanism of the initial events in the sorption of marine bacteria to surfaces. J. Gen. Microbiol. 68: 337-348.

7.

Mesibov, R., and Adler, J. 1972. Chemotaxis toward amino acids in Escherichia coli. J. Bacteriol. 112: 315-326.

8.

Weibull, C. 1960, Movement. p, 153-205 In: The Bacteria. I. C. Gunsalus and R. Y. Stanier, (editors.) Pp. 153-205, Vol. 1. Academic, New York.

An enrichment technique for isolation of marine chemotactic bacteria.

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