Making use of the streak plate technique and low temperature incubation, 28 cultures belonging to six genera namely, Achromobacter, Flavobacterium, Pseudomonas, Micrococcus, Vibrio and Alcaligenes were isolated from different varieties of marine fish. The growth studies indicated that all of them were able to grow between -5 and +5°C within a week's time and none of them showed growth at 37°C. The optimum temperature of growth for all these cultures was in the range 25-28°C. Among these only one, i.e., a Vibrio sp., was found to be an obligate psychrophile

Anand, C.P.

Rudra Setty, T.M.

Aquatic Commons

E 
10 
A 
c 
OF PSYC ROP ILIC 
E FISH 
C. P. ANAND AND T. M. RUDRA SETTY 
College of Fisheries, University of Agricultural Sciences, Mango/ore 
Making use of the streak plate technique and low tempe-
rature incubation, 28 cultures belonging to six genera namely, 
Achromobacter, Flavobacterium, Pseudomonas, Micrococcus, Vibrio 
and Alcaligenes were isolated from different varieties of marine 
fish. The growth studies indicated that all of them were able 
to grow between -5 and +SoC within a week's time and none 
of them showed growth aL 3TC. The optimum temperature 
of growth for aH these cultures was in the range 25-28°C. 
Among these only one, i.e., a Vibrio sp., was found to be an 
obligate psychrophile. 
INTRODUCTION 
Many attempts have been made in 
the past to isolate and identify the psy-
chrophilic bacteria in fish. The recent 
revievvs on this aspect by Ingraham 
and Stokes (1959), Witter (1961) and 
Morita (1975) give a full account of the 
various attempts made in the past and 
of the psychrophilic groups identified. 
The attempts made so far to check the 
growth of psychrophilic bacteria in ice 
stored fish by the use of various anti-
biotics and chemical preservatives have 
not given satisfactory results for commer-
cial application though chlorotetracydine 
has been found to be of some use com-
98 
mercially. While most of the earlier 
studies were directed towards treating the 
fish with preservative added to water or 
ice, only recently a few attempts have 
been made to study the effect of some 
of these preservatives on isolated cultures 
(Velankar, 1958; Heather and Vander 
Zant, 1958; Surendran and Mahadeva 
Iyer, 1971; Tunstal and Gowland, 1974). 
Even in these cases the studies were not 
quite comprehensive in the sense that 
either only a few isolates from fish have 
been included for the ~tudy or preserva-
tives have been tried at arbitrary conce-
ntrations. As seen from the current 
literature, it appears that this problem 
could be solved not by any one preser-
Fish Techno!. 
Jlnand & (Setty: Cfiemical control of psycfiropfiilic ()acterial spoilage of fist! 
vative but by a combination of two or 
three preservatives as pointed out by 
Partmann (19S2). Therefore, the objective 
of this investigation was to try these 
preservatives on the various groups of 
psychrophilic bacteria that normally exist 
in ice stored fish and to see which of 
these groups are inhibited by the particular 
preservative and then choose that preser-
vative or a combination of preservatives 
which can effectively check the growth of 
all these groups. In the present study, 
attempts have been made to isolate and 
identify the various groups of psychro-
philic bacteria that normally exist in fishes 
of Man galore coast .. 
MATERIALS AND METHODS 
The fish used m the study were 
collected from the sea at a depth of 
20-2S fathoms using the college's boat. 
The hauled fish were iced immediately, 
brought to the labo~atory and kept in the 
ante-room of cold storage (0 to + 2°C) 
prior to sampling. The samples were 
drawn over a period of IS days at inter-
vals of 2 days. The types of marine fish 
used were Rastrelliger kanagurta, Sardi-
nella spp., Johnius sp., Nemipterus japo-
nicus, Lactarius lactarius and Tachysu-
rus sp. 
Both sea water agar and nutrient agar 
were employf'd for isolation of bacterial cul-
tures from fish samples. The composition of 
the two media are shown tn Table I. 
While both the media were used for 
isolation purpcses in order to isolate as 
many types as possible, sea water agar 
was used for maintenance of isolated 
cultures. 
isolation of cultures 
Streak plate technique was adopted 
VoL No. 2 1977 
for the isolation of organisms. The 
streaking was done by means of a glass 
hockey stick. Whole fish samples were 
homogenised m sterilized and cooled 
homogenising cups from which lOg. ma-
terial was transferred to 90 mL of 
precooled 0.8S% saline. The serial dilut-
ions were then prepared from this for 
inoculating the medium. The diluents 
and the petriplates containing the medium 
we1 e precooled (O-S°C) before they were 
inoculated. The streaked plates were in-
cubated at -3 to -S°C, 0°C ( ± 2) and 3 
. to S°C. The morphologically different 
colonies were picked and transferred to 
sea water agar slants and pure cultures 
obtained. The pure cultures were stored 
in a refrigerator at S-TC and were 
subcultured periodically. 
Growth studies 
All the 28 pure cultures were studied 
for their optimum temperature require-
ments for growth. The incubation tem-
peratures tried were 0°C ( ± 2), 3 to 5°C, 
1S-20°C, 2S-28°C, 30-32°C and 37°C, 
The studies were carried out on slants 
using both sea water agar and nutrient 
agar media and the time of appearance 
of growth over a period of time were 
noted for each. 
Identification of cultures 
Attempts were made to identify the 
cultures upto their generic level and 
wherever possible upto their species leveL 
The procedures followed were those des-
cribed in the Manual of Microbiological 
Methods (Pelczer eta!., l9S7) using Ber-
gey's Manu3.l of Determinative Bacteriology 
(Breed eta!., 1957) as the guideline for 
identification of cultures. However, for 
identification and confirmation of Pseudo-
monas cultures Kovac's oxidase test 
99 
Anand & 8ettw Cftemical control of psycfiropf'tilic 6acteria1 spoilage of fisf1 
TABLE I 
COMPOSITION OF MEDIA 
Components of the 
medium 
Sea water agar 
medium 
Nutrient agar 
medium 
-------·--------------------------------------------------------------------
Bacto-peptone (Difco) 
Beef extract (Difco) 
Glucose (BDH) 
Agar (Japan make) 
Sodium chloride (BDH) 
Distilled water 
Aged sea water 
pH 
as described by Shewan, Hobbs and 
Hodgkiss (1960) was also made use of. 
RESULTS AND DISCUSSION 
Making use of the streak plate tech-
nique and low temperature incubation 
158 cultures were picked out based on 
their colony morphology. They were fur-
ther subjected to primary and secondary 
screening to eliminate the duplicates 
yielding about 70 isolates. Out of these, 
28 isolates which showed distinctly diff-
erent morphological and cultural chara-
cteristics were finally chosen for purification 
and identification. 
The enrichment culture technique recom-
mended by Morita (1975) was tried for 
isolation of psychrophiles by incorporat-
ing a small quantity of fish extract into 
the sea water agar medium. However, 
it was observed that this medium was 
not in any way superior to sea water 
agar or nutrient agar media, since the 
number and types of C.)lonies that grew 
up on these media were almost the same 
and hence for an further isolations only 
100 
5 g. 
3 g. 
1 g. 
15 g. 
1000 ml. 
7 
5 g. 
3 g. 
1 g. 
15 g. 
30 g. 
1000 ml. 
7 
sea water agar and nutrient agar media 
were used. 
Though the low temperature incu ba-
tion technique was followed right throuah 
their isolation, purification and mainte~1 -
ance, these 28 cultures were again subje-
cted to growth studies to determine their 
optimum growth temperatures and also 
to confirm their psychrophilic nature. 
For this purpose they were incubated at 
ooc ( ± 2), 3 - soc, 15 - 20°C, 25-28°C, 
30-32oC and 37°C. It was observed that 
all these cultures took 5-7 days to show 
visible growth at ooc ( ± 2), 3-4 days at 
3 to soc, 2 days at 15-20°C, 24 hrs. at 
25-28°C and 30-32°C and showed no 
growth at 3rC. While the growth of all 
these cultures was found to be abundant 
at 25-28°C within 24 hours, their growth 
at 30-32°C was moderate and quite a 
few of the cultures showed very slight 
growth at this range. While the growth 
of these isolates was good on sea water 
agar and nutrient agar with 3% sodium 
chloride, the growth in nutrient 
agar without sodium chloride was, 
Fish. Techno!. 
}\nand & 8etty: Cftemica! contra! of psyciiropftilic IJacteria! spoilage of fisft 
TABLE H 
IDENTIFICATION OF CULTURES 
Isolate No. 
I. 
2. 
3. 
4. 
5. 
6. 
7. 
8. 
9. 
10. 
11. 
12. 
13. 
]4. 
15. 
16. 
17. 
18. 
19. 
20. 
21. 
22. 
23. 
24. 
25. 
26. 
27. 
28. 
Name of the organisms 
Flavobacterium halmephilum 
Vibrio sputigenus 
Achromobacter sp. 
Vibrio costicolus 
Achromobacter sp. 
Micrococcus conglomeratus 
Achromobacter superficialis 
Achromobacter liquefacienes 
Micrococcus varians 
Pseudomonas striata 
Pseudomonas fragi 
Vibrio sp. 
Achromobacter guttatus 
Micrococcus agilis 
Achromobacter sp. 
Achromobacter sp. 
Achromobacter delmarvac 
F lavobacterivm. diffusum 
Achromobacter sp. 
Alcaligenes bucheri 
Achromobacter aqcwmarinus 
Achrornobacter sp. 
Alcaligenes faecalis 
Pseudomonas sp. 
AcoromobaCter delicatulus 
Flavobacterium sp. 
Flavobacterium sp. 
Achromobacter sp. 
very poor even at optimum growth te-
mperature. 
It was c0ncluded from this study that 
the optimum temperature for all these 
cultures lies between 25-28°C, except the 
isolate number 12 (Vihrio sp.) which could 
not grow at temperatures above 20°C but 
grew well between 0 to 5°C. 
VoL 14 No. 2 1977 
The 28 cultures thus screened were 
subjected to various morphological, cul-
tural and physiological tests after ensuring 
their purity. These cultures were identi-
fied as given in Table IL 
Of the 28 cultures, 13 were identified 
as species of Achromobacter, 4 as Flavo-
bacterium, 3 each as A1icrococcus, Pseudo-
monas and Vibrio and 2 as Alcaligenes. 
Efforts were made to identify these 
28 cultures upto their species level making 
use of various tests. However, some of 
the cultures could not be identified upto 
their species level by the above tests alone 
as they were not agreeing with any of 
the type cultures described under their 
respective genera. H was concluded that 
either they were new species or further 
work was needed for proper identification 
of these cultures. 
Though it has been possible to obtain 
psychrophilic bacteria belonging to the 
genera Achromobacter, Flavobacterium, Pse-
udomonas, Alcaligenes, Micrococcus and 
Vibrio, the psychrophiles belonging to 
other genera like Bacillus, Serratia, Aero-
monas, Corynebacterium, Photobacterium, 
Cytophaga, Moraxella, Mycoplana. Ente-
robacters etc. were not encountered in 
the present study. However, the most 
commonly occurring psychrophilic genera 
on fish have been isolated in this study. 
The facts that all these cultures grow 
well at ooc within a week's time, grow 
best between 25-28°C, show only a scanty 
growth above 30oC and no growth at 
3rC, go to show that they are definitely 
psychrophiles as per the definitiJns of 
Ingraham and Stokes (1957) and Stokes 
(1963). Among these only the Vibrio sp. 
101 
Anand & 8etty: Cfie-m'icat conirot o-f ps.ycf'irop/Wic tJacterial spoitoge of fisf'i 
could be called an obligate psychrophile 
since it could not grow at temperatures 
above 20°C but could grow well between 
0- 5°C. 
While there was no difficulty in 
identifying these cultures upto their generic 
level, differentiation of Pseudomonas 
from Achromobacter was slightly diffi-
cult since both were motile. Kovack's 
oxidase and fermentative test could how-
ever differentiate these two according to 
the determinative scheme suggested by 
Shewan et aL, (1970). 
ACKNOWLEDGEMENT 
The authors wish to express their 
sincere thanks to Prof. H. P. C. Shetty, 
Director of Instruction, College of Fish-
eries, Mangalore, for providing opportunity 
and facilities and for the encouragement 
given throughout this study. 
REFERENCES 
Breed, R. S., E. G. D. Murray and N. R. 
Smith. 1957. Bergey's Manual of 
Determinative Bacteriology., 7th Edn., 
Bailliere, Tindall and Cox, Baltimore. 
Heather, C. D. and C. Vanderzant. 1958. 
Fd. Techno!., : 263. 
102 
Ingraham, J. L. 1958. J. Bacterial., 
16:75. 
Ingraham, J. L. and J. L. Stokes. 1959. 
Bacterial Rev., 23 : 99. 
Morita, R. Y. 1975. Bactertol Rev., 
: 144. 
Partmann, W. 1952. z. Levertsm. - Unter-
such. U. Forsch., 94 : 246. 
Pelczar, M. J. Jr. 1957. Manual of Mic~ 
robiological Methods,: McGraw Hm 
Book Co., New York. 
Shewan, J. M., G. Hobbs and W. Ho-
dgkiss. 1960. J. Appl. Bacterial:, 
23 : 379. 
Stokes, J. L. 1963. Recent Progress iir, 
Mfcrobiology; Ed. N. E. Gibbons : 
187. University of Toronto Press. 
Surendran, P. K. and K. Mahadeva Iyer. 
1971. Fish. Techno!., 3, 2 : 128. 
Tunstall, A. M. and G. Gowland. 1974. 
J. Appl. Bacterial., 31 : 455. 
Velankar, N. K. 1958. Proc. Indian Acari. 
Sci., 47B : 87. 
Witter, L. D. 1961. J. Dairy Sci., 44 : 983. 
Fish. Techhol. 


Chemical control of psychrophilic bacterial spoilage of fish 1. Isolation and identification of psychrophilic bacteria from marine fish

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Chemical control of psychrophilic bacterial spoilage of fish 1. Isolation and identification of psychrophilic bacteria from marine fish

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