Thalassas, 2007, 23 (2): 17-21
An International Journal of Marine Sciences
A SIMPLE SCHEME FOR THE IDENTIFICATION OF MARINE
HETEROTROPHIC BACTERIA
SURAJIT DAS*, P. S. LYLA & S. AJMAL KHAN
Keywords: Marine heterotrophic bacteria; biochemical tests; identification scheme.
ABSTRACT
INTRODUCTION
A scheme has been proposed to identify the marine
heterotrophic bacterial genera based on the minimum
biochemical tests. Fifteen gram negative and six gram
positive marine bacterial genera could be identified
with this proposed scheme which involves staining,
morphology, motility and some major biochemical
tests
A number of reports are available on key
characters for the identification of heterotrophic
bacteria (Simidu and Aiso, 1962; Baumann et al.,1972;
Buchanan and Gibbons, 1974; Starr et al.,1981;
Sneath, 1986), and in particular for the marine
heterotrophic bacteria (Baumann et al.,1972; Oliver,
1982). But, most of the schemes are based on several
time consuming biochemical tests. In spite of
performing all the laborious biochemical tests,
sometimes, the isolates are misidentified. Oliver
(1982) proposed a key for the identification of gramnegative bacteria and Jensen and Fenical (1995) for
gram-positive bacteria and these are widely followed.
More bacterial genera have been reported from the
marine environment, and these are not included in
those schemes. Hence, in the present study an attempt
has been made to simplify and modify these schemes
to include the recently reported marine heterotrophic
bacteria and to propose a collated scheme for the
identification of marine bacterial genera (both gram
negative and gram positive) based on minimum
biochemical tests. The scheme relies on the
information given by Shewan (1963), Okami and
Okazaki (1972), Buchanan and Gibbons (1974), Starr
et al. (1981), Oliver (1982), Jensen and Fenical (1995)
and Cappuccino and Sherman (2001).
Centre of Advanced Study in Marine Biology, Annamalai
University
Parangipettai- 608 502, Tamil Nadu, India
*E-mail: [email protected]
Tel: +91-4144-243223 ext. 220; Fax: +91-4144-243555
17
Surajit Das, P. S. Lyla & S. Ajmal Khan
Figure 1.
Diagrammatic scheme for the identification of gram-negative marine bacteria
MATERIALS AND METHODS/ RESULTS
AND DISCUSSION
The scheme starts with the gram staining,
developed by Hans Chirstian Gram to separate the
bacteria into two groups: gram +ve and gram –ve;
based on the cell-wall structure. But commercially
available kit for the gram staining requires time and
involves morphological changes of bacteria during
heat fixation. The non-staining KOH method
originally proposed by Fluharty and Packard (1967)
and later modified by Buck (1982), is a simple and
rapid method to determine gram reaction where small
colonies could also be identified (Jensen and Fenical,
1995). The advantage with respect to time, ease and
cost of this technique is obvious. To perform this test,
a drop (10µl) of 3% aqueous KOH (Potassium
hydroxide) is to be placed on a slide. As many as 8 to
10 tests per slide can be done conveniently. Using a
sterile loop, visible amount of bacterial growth from an
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agar culture is to be transferred to the drop of KOH.
The cells and KOH are to be mixed thoroughly on the
slide, constantly stirred over an area about 1.5 cm in
diameter. If the bacterium-KOH suspension becomes
markedly viscid or gels within 50-60 seconds, the
isolate is gram negative. If no gelling is observed, the
isolate is gram positive. The best way to determine
viscosity is to raise the loop about 1 cm from the slide.
If an obvious stringiness is present, then the culture is
gram negative.
The scheme for gram-negative isolates is presented
in Figure 1. The gram-negative rods which are without
spores (rods only have spores) are to be tested for
motility in the semi-solid nutrient agar medium (1.5%
agar) by stabbing the bacterial culture. Gram negative,
heterotrophic bacteria, which are motile by means of
flagella, are readily isolated from the ocean and appear
to comprise a major component of the bacterial flora of
the sea (Baumann et al.,1972). The non-motile isolates
A Simple Scheme For The Identification Of Marine Heterotrophic Bacteria
are then to be examined for pigment production in
Seawater Yeast Extract Peptone agar media (Peptone5g; Yeast extract- 3g; Aged seawater- 750ml; Distilled
water- 250 ml; pH- 8; Agar- 15g) (Schneider and
Rheinheimer, 1988). The pigment producing bacteria
show yellow or orange pigmentations and these
pigmented bacteria are subjected to Catalase test
further with 3% H2O2. The catalase negative isolates
are tentatively identified as Flexibacter and the
catalase positive isolates are tested for polymyxin B
sensitivity and the sensitive isolates belong to
Flavobacterium and non-sensitive isolates belong to
Cytophaga genus.
The fermentative strains are then subjected to
Kovac’s oxidase test. A filter paper is to be soaked in
1% aqueous tetramethyl-p-phenylene diamine
dihydrochloride solution and dried. Young cultures
(less than 18 hrs) of the isolates are to be streaked on
this filter paper and the results are read as follows:
Pink or purple- Positive; No colour- Negative. The
oxidase negative luminescent strains are Photobacter
and non- luminescent strains are Enterobacteriaceae
(Klebsiella). The oxidase positive strains are then
subjected to pteridine test with 0/129 and the sensitive
strains are Photobacterium and the resistant strains are
Aeromonas.
The non-pigmented isolates are to be tested for
glucose dissimilation in Hugh-Leifson’s basal medium
(Hugh and Leifson, 1953) with glucose as substrate.
The basal media is prepared with the composition of
Peptone-2g; Sodium chloride- 5g; Dipotassium
orthophosphate- 0.3g; Bromothymol blue (1% aqueous
solution)- 3 ml; Distilled water- 1000ml; pH- 7.1 and
Agar- 3g with filter sterilized 100ml glucose (10%
aqueous solution). The required amount of medium is
to be disposed in to the test tubes into which Durham’s
tubes are placed to trap the gas bubbles. The result may
be read as: Yellow (aerobic and anaerobic) Fermentative; Yellow (aerobic) - Oxidative; Green/ No
change- No reaction/Alkaline.
The oxidative/alkaline/no reaction strains are also
to be tested for oxidase production. The negative
oxidase isolates belong to Acinetobacter and the
positive oxidase isolates are checked for penicillin
sensitivity (1.5 IU). The sensitive strains are Moraxella
and the resistant strains are Pseudomonas.
The other parts of motility test, i.e. motile isolates
are subjected to Kovac’s Oxidase test and the isolates
showing negative results are Enterobacteriaceae. The
positive oxidase isolates are then examined for glucose
dissimilation as described earlier. The results may be
recorded as follows: Fermentative (F), Oxidative
(OX), oxidative/Negative (OX/N) and No growth/
Figure 2.
Diagrammatic scheme for the identification of gram-positive marine bacteria.
19
Surajit Das, P. S. Lyla & S. Ajmal Khan
Negative (NG/N). The isolates showing the last
fraction of the result are considered as Alcaligenes
(Kakimoto et al., 1974). The isolates showing OX and
OX/N belong to Pseudomonas and Alteromonas
respectively. But to have confirmed identification of
these two group’s, isolates are to be examined for
penicillin sensitivity and the sensitive strains are
Pseudomonas and the resistant strains are Alteromonas
(Lee et al., 1977). The fermentative (F) strains are then
treated in Kovac’s Oxidase test and the negative strains
are Enterobacteriaceae. If gas is evolved from the
positive isolates the isolates are Photobacterium. The
negative gas producing strains are then treated with
vibriostatic pteridine. 0/129 (2, 4- diamino-6, 7
diisopropyl pteridine) can be used as saturated solution
on filter paper disks to test for vibriostatic action
according to the method of Collier et al. (1950). The
sensitive strains are Vibrio and the resistant strains are
Aeromonas.
The scheme for gram-positive isolates is presented
in Figure 2. The gram-positive strains are looked under
light microscope (400x) for the morphology. The
gram-positive bacilli (rod shaped) spores are to be
stained then. After fixing the smear in a glass slide,
malachite green may be added and steamed. After
draining, safranin should be added as counterstain and
the results are: spores- green colour and without
spores- red colour. The endospore forming isolates are
Bacillus and without spores are Corynebacterium.
The gram-positive cocci (round shaped) are tested
for catalase production and the negative catalase
isolates are Streptococcus. The catalase positive
spherical isolates are Arthrobacter and the clumped
isolates are tested for glucose dissimilation. The
oxidative strains are Micrococcus and the fermentative
strains are Staphylococcus.
In any ecological study on bacteria, identification
of the isolates gives an exact picture about the
community structure, thereby, enabling to understand
the role of those genera in the biogeochemical process
in the marine environment. Although, almost all
contemporary marine microbiologists have been
flipped over to molecular approaches for the
identification, the present key would be useful for
tentative identification of bulk samples where the
molecular approaches would not be cost effective.
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With this proposed key, the generic identification of
heterotrophic bacteria would be rather easier and faster
and would be a boon for the marine microbial
ecologists.
ACKNOWLEDGEMENTS
The authors are thankful to the Director, Centre of
Advanced Study in Marine Biology and the authorities
of Annamalai University for the facilities. One of the
authors (S.D) is thankful to the Centre for Marine
Living Resources and Ecology, Ministry of Ocean
Development, Government of India for Research
Fellowship.
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