The fluid was transferred to a tube of BHI broth, streaked to plates of TCBS and TS A and
prepared for AODC, FAB and DVC counts. Results showed that CA 401 cells from all
microcosms injected into the rabbit ileum, were recoverable in BHI broth and on TSA
and TCBS plates. Surprisingly, all plate cultures grew as pure cultures. Slide agglutination tests were 4
+ positive with V. cholerae 01 antisera. DVC counts increased to 10 8 to
10
9 cells/ml. AODC counts increased from 10 8 to 10
10 cells/ml. Transfers from the T
1
N
1
loop revealed no growth on TSA, TCBS, and EMB plates and in BHI broth. Microscopic
examination of the T1N1 loop revealed large rods in chains, consistent with the morphology of gut anaerobes.
A similar ileal loop assay was performed using the viable, but non-culturable cells
recovered from membrane chambers. Aspirated fluid from the loops were streaked to
EMB and BHI-Instant Ocean plates. E. coli grew on all the media tested.
In conclusion, immunofluorescent-epifluorescent techniques have provided information
concerning the survival patterns of selected bacteria in the aquatic environment.
Although “die-off’ is indicated by conventional plating techniques, fluorescent microscopy reveals that viable pathogens continue to be viable and to remain present in far
greater numbers than reported. Thus, bacteria possess a “starvation-survival” mechanism
(Baker et al, 1983; Hoppe, 1976; Jones et al., 1981; Mitchell, 1968; Morita, 1983;
Novitsky et al., 1977 and 1978; Stevenson, 1978; and Tabor et al, 1981. ) during the
absence of suitable energy-yielding substrates, and prevail in a dormant phase until
suitable environmental conditions return. The latter may occur in the water column or in
animal hosts. The results of the animal studies dramaticalllion-survival” mechanism
(Baker et al, 1983; Hoppe, 1976; Jones et ai, 1981 ; Mitchell, 1968; Morita, 1983;
Novitsky et al, 1977 and 1978; Stevenson, 1978; and Tabor et al, 1981. ) during the
absence of suitable energy-yielding substrates, and prevail in a dormant phase until
suitable environmental conditions return. The latter may occur in the water column or in
animal hosts. The results of the animal studies dramatically demonstrate that nonrecoverable cells continue to harbor the potential for virulence, the public health implications of which cannot be ignored. Reevaluation of the traditional enumeration procedures clearly are needed.
BAKER R.M., F.L. SINGLETON, and M.A. HOOD, 1983. The effects of nutrient deprivation on Vibrio cholerae.
Appl Environ. Microbiol. 46 : 930-941.
COLWELL R.R., H.Xu, N. ROBERTS, R. SIEBELING, A. HUQ P. WEST, I. HUQ. K M. S. AZIZ, D. MANEVAL, P.
BRAYTON, D. ROSZAK, M. MACDONELL, 1984. Ecology and systematics of Vibrio cholerae in endemic and
non-endemic areas. US Japan cholerae conference.
GRIMES D.J. and R.R. COLWELL, 1984. Viability and virulence of Escherichia coli suspended by membrane
chamber in semitropical ocean water. J.C. Clin. Microbiol. (Submitted).
HOBBIE J.E., R.J. DALEY and S. JASPER. 1977. Use of nuclepore filters for counting bacteria by fluorescence
microscopy. Appl. Environ. Microbiol. 33 : 1225-1228.
HOPPE H.G., 1976. Determination and properties of actively metabolizing heterotrophic bacteria in the sea,
investigated by means of autoradiography. Mar. Biol. (Berlin). 36: 291-302.
JONES J.L. and M.E. RHODES-ROBERTS. 1981. The survival of marine bacteria under starvation conditions. J.
Appl. Bacteriol. 50 : 246-258.
KOGURE K., U. SIMDU and N.TAGA, 1979. A tentative direct microscopic method for converting living bacteria.
Can. J. Microbiol. 25 : 415-420.
513
prepared for AODC, FAB and DVC counts. Results showed that CA 401 cells from all
microcosms injected into the rabbit ileum, were recoverable in BHI broth and on TSA
and TCBS plates. Surprisingly, all plate cultures grew as pure cultures. Slide agglutination tests were 4
+ positive with V. cholerae 01 antisera. DVC counts increased to 10 8 to
10
9 cells/ml. AODC counts increased from 10 8 to 10
10 cells/ml. Transfers from the T
1
N
1
loop revealed no growth on TSA, TCBS, and EMB plates and in BHI broth. Microscopic
examination of the T1N1 loop revealed large rods in chains, consistent with the morphology of gut anaerobes.
A similar ileal loop assay was performed using the viable, but non-culturable cells
recovered from membrane chambers. Aspirated fluid from the loops were streaked to
EMB and BHI-Instant Ocean plates. E. coli grew on all the media tested.
In conclusion, immunofluorescent-epifluorescent techniques have provided information
concerning the survival patterns of selected bacteria in the aquatic environment.
Although “die-off’ is indicated by conventional plating techniques, fluorescent microscopy reveals that viable pathogens continue to be viable and to remain present in far
greater numbers than reported. Thus, bacteria possess a “starvation-survival” mechanism
(Baker et al, 1983; Hoppe, 1976; Jones et al., 1981; Mitchell, 1968; Morita, 1983;
Novitsky et al., 1977 and 1978; Stevenson, 1978; and Tabor et al, 1981. ) during the
absence of suitable energy-yielding substrates, and prevail in a dormant phase until
suitable environmental conditions return. The latter may occur in the water column or in
animal hosts. The results of the animal studies dramaticalllion-survival” mechanism
(Baker et al, 1983; Hoppe, 1976; Jones et ai, 1981 ; Mitchell, 1968; Morita, 1983;
Novitsky et al, 1977 and 1978; Stevenson, 1978; and Tabor et al, 1981. ) during the
absence of suitable energy-yielding substrates, and prevail in a dormant phase until
suitable environmental conditions return. The latter may occur in the water column or in
animal hosts. The results of the animal studies dramatically demonstrate that nonrecoverable cells continue to harbor the potential for virulence, the public health implications of which cannot be ignored. Reevaluation of the traditional enumeration procedures clearly are needed.
BAKER R.M., F.L. SINGLETON, and M.A. HOOD, 1983. The effects of nutrient deprivation on Vibrio cholerae.
Appl Environ. Microbiol. 46 : 930-941.
COLWELL R.R., H.Xu, N. ROBERTS, R. SIEBELING, A. HUQ P. WEST, I. HUQ. K M. S. AZIZ, D. MANEVAL, P.
BRAYTON, D. ROSZAK, M. MACDONELL, 1984. Ecology and systematics of Vibrio cholerae in endemic and
non-endemic areas. US Japan cholerae conference.
GRIMES D.J. and R.R. COLWELL, 1984. Viability and virulence of Escherichia coli suspended by membrane
chamber in semitropical ocean water. J.C. Clin. Microbiol. (Submitted).
HOBBIE J.E., R.J. DALEY and S. JASPER. 1977. Use of nuclepore filters for counting bacteria by fluorescence
microscopy. Appl. Environ. Microbiol. 33 : 1225-1228.
HOPPE H.G., 1976. Determination and properties of actively metabolizing heterotrophic bacteria in the sea,
investigated by means of autoradiography. Mar. Biol. (Berlin). 36: 291-302.
JONES J.L. and M.E. RHODES-ROBERTS. 1981. The survival of marine bacteria under starvation conditions. J.
Appl. Bacteriol. 50 : 246-258.
KOGURE K., U. SIMDU and N.TAGA, 1979. A tentative direct microscopic method for converting living bacteria.
Can. J. Microbiol. 25 : 415-420.
513