Fish contributes about 60% of the worlds protein supply and 60% of the
developing world derives more than 30% of their annual protein from fish (FAO,
2007; Kumolu-Johnson and Ndimele, 2011). In Malaysia,
fish is an important part of human diet as it is one of the major cheap protein
sources. Malaysia is a country that surrounded by the sea and fisheries is one
of the countrys major industry. Among various fishes, Asian seabass (Lates
calcarifer) is one of the most popular fish in Malaysia. It is an economically
important food fish in Malaysia. Malaysian people prefer Asian seabass due to
its delicately flavoured flesh. However, total production of Asian seabass in
Malaysia is increasing day by day. In 2004, in Malaysia, total production of
Asian seabass was 5.7 thousand MT while in 2009 it was nearly 16 thousand MT
(FAO, 2011). In Malaysia, the production of Asian seabass
is mainly from aquaculture farms in east Malaysia, where Asian seabass is generally
sold to the wet market and are also distributed to the supermarket.
Fish is a very perishable product therefore, the quality and freshness of fish
decline rapidly upon dying. Microbial activity starts in the different organs
of fish after dead and the quality of fish is gradually deteriorating. However,
the rate of deterioration or bacterial activity depends directly on storage
and processing conditions of fish. A good storage condition can stop microbial
activity and spoilage. Storage conditions may also influence the sensory profile
(Aaraas et al., 2004). Therefore, storage conditions
play a very important role in maintaining the quality of fish. To maintain the
quality, fish and fishery products are stored or preserved for years using a
wide range of technological processes. These include curing, freezing, chilling,
canning, etc. Curing method includes salting drying, smooking and pickling.
However, in many cases, applied preservation technology is greatly dependent
on types of fish. Selection of proper storage and preservation methods are also
based on duration of storage and the quantity of fish to be stored (Burt
et al., 1992).
In practice, Asian seabass is generally kept in chilled condition using crushed
ice for a short-term storage. However, chilling does not kill the microorganisms
but reduces microbial metabolism which is responsible for spoilage. Other alternative
might be using sea salt (sodium chloride). Sea salt has been using for centuries
as a preservative agent for many fishes (Takiguchi, 1989).
Salting lengthens the shelf life of fish and fishery products. Besides preservation,
it also acts as enhancer attractive flavor of many fishes. There are many studies
which discuss the bacteriological quality at different storage conditions of
different fishes (Aaraas et al., 2004; Tejada
et al., 2007; Zambuchini et al., 2008).
Unfortunately, there is no study that discuss about bacteriological quality
of Asian seabass when stored with crushed ice and sea salt. Therefore, the proposed
study was conducted to evaluate the bacteriological quality of Asian seabass
when preserve with crushed ice and sea salt. The main objectives of the study
were to identify different bacteria in Asian seabass when preserve with ice
and sea salt, and to compare their quantitative bacterial load.
MATERIALS AND METHODS
Sample collection and storage: A total of six live seabass was collected
from a marine fish cage at Kuala Pahang and transferred to the laboratory and
allowed to die in container with ice. All fishes were mixed and divided into
two groups based on random selection. Each group contained three fish, each
of which were considered as a replication in this study. The first group was
stored into a plastic container with sea salt while the second group was stored
with crushed ice. Both of the storage conditions were maintained for two days.
Examination of morphological characteristics of fish: The basic morphological
characteristics of fish were examined in the laboratory before and after storage
conditions. The basic morphological test of fish included appearance of skin,
eyes and gill. Skin was examined for their firmness and shiny characteristic
while cornea of eyes was examined for its clarity and the red colour criteria
for the gills.
Microbiological analysis: Microbiological analysis was done according
to Buller (2004). It involved the isolation and inoculation
of the bacteria from selected parts of the fish samples followed by subsequent
incubation and subcultures. Nutrient agar was used specifically as the general
media and nutrient broth was used as the media for subculturing pure bacteria
The fish samples were swabbed using sterile cotton tips at two different parts:
skin and flesh. A special care was taken to maintain an equal swabbing at each
time. Then, the swabs were directly inoculated into nutrient agar plates. For
the skin, swabbing was made at the surface of each of the fish using sterile
cotton tips. In order to swab the flesh part, a fillet cut was made at the posterior
part of the fish using a sterile blade. Then swabbing was made using sterile
cotton tips. Direct inoculation to the nutrient agar plate was made upon the
cotton tip was swabbed on the appropriated part by a single streaking then followed
by several subsequent streaks over the first streak using sterile inoculating
The primary isolation plate media were incubated at 30°C for 24 h. After
incubation, each culture plate was examined with the dissecting microscope for
any appearance of discrete bacterial colony growth. To obtain pure growth, a
representative of each colonial type has been picked and subcultured to nutrient
agar plates which served as secondary plates. In this step, the primary culture
of plate was examined and identified how many colonies had similar size, morphology,
color, wrinkled, mucoid, spreading, clear, etc. and then was selected, circle
and number them with a marker or wax pencil. After that, colonies were picked
to be isolated. The subculturing of single colonies was performed by using dilution-streaking
technique and then was incubated at 30°C for 24 h. After that, each pure
colony was inoculated into Falcon tube containing 30 mL nutrient broth and further
incubated at 30°C for 24 h. This was served as a stock culture for each
pure bacterial colony.
Gram-positive bacteria and Gram-negative bacteria were identified using Gram
staining which was done based on American Society of Clinical Pathology (ASCP,
2004) method with some modification. Firstly a bacterial smear was prepared
from a freshly cultured bacterial colony (18-24 h old). Then, a drop of water
was placed onto the glass slide. A minute amount of a bacterial colony was spread
evenly after aseptically transferred onto the water drop and allowed to fully
dry. The procedure was continued with the fixation of the specimen onto the
glass slide by gently passed the underside of the slide through a flame 2 to
3 times and the slide were let cool.
The smear was flooded with crystal violet solution and kept for 30 sec before
being rinsed with water. After that, the slide was flooded again with iodine
for 30 and 60 sec before being rinsed with water. An iodine solution was used
as a mordant. A mixture of acetone and alcohol was added to the slanted slide
drop-wise until no colour appears in the drippings or for about 5 seconds. Then,
the slide was rinsed with water immediately. Lastly, the slide was flooded with
safranin for 30 to 60 sec before being rinse gently with water until no colour
appear in the effluent. The preparation of smear was completed by blotting the
smear dry or letting the smear air-dried. After that it was examined under the
light microscope to observe the cell shape and differentiate whether it is gram
negative or positive bacteria.
Identification of fish pathogenic bacteria was done using API®
20E test, which uniquely used for identifying Enterobacteriaceae and other non-fastidious
Gram-negative rod. The API®
20E kit is not for direct use with the specimens thus the bacterial samples
were allowed to grow on fresh nutrient agar plates first and then incubated
for 18 to 24 h at 30°C prior to use with the kit. API®
test uses 21 standardized and miniature biochemical tests. The reactions were
read according to the table provided with the kit. For this, an incubation box
that consisted of a tray and a lid was prepared. Then, a humid condition was
formed in the honey-combed wells of the tray by distributed about 5 mL of distilled
water. The reference of the bacterial isolate was written on the elongated flap
at the end of the tray for recording purpose. After that, the strip that contains
21 micro- tubes was placed on the tray.
A single well-isolated colony from nutrient agar plate was taken and transferred
into a universal bottle containing 5 mL of 0.85% sterile normal saline solution.
Different bottle containing sterile normal saline solution was use for each
different bacterial isolates. Then, the solution was homogenized and distributed
into the 21 microtubes using micropipette. For CIT, VP and GEL tubes were filled
with suspension into both tubes and cupules. Then, ADH, LDC, ODC, H2S
and URE tubes were overlaid with sterile mineral oil, filling the cupules to
create anaerobic atmosphere. After that, the strip was put into the incubation
box and incubated at 37°C for 18 to 24 h. After the incubation, the results
were read by the Reading Table in the manual of the API®
20E kit. In the case where the number of positive test (including the GLU test)
was less than 3, the strip was reincubated for a further 24 h without any addition
of any reagent. The strip was analyzed after the incubation if more than three
tests were positive (including GLU test). If not, the strip was further incubated
for another 24 h without adding any of reagents.
For TDA test, 1 drop of TDA reagent was added into the TDA tube. A positive
reaction was indicated by a reddish brown color while negative reaction indicated
by yellow color. For IND test, 1 drop of JAMES reagent was dropped into the
IND tube. A positive result was shown by a development of a pink color. A negative
result was indicated by yellow color. IND test was performed last because gaseous
product was released in the reaction which could interfere other tests interpretation.
For VP test, 1 drop of each VP 1 and VP 2 reagents were dropped into VP tube
and in 10 minutes, positive reaction occurred with a pink or red color was observed.
In contrast, if there was formation of slightly pinkish color after 10 min,
the test was considered negative. All the results were recorded on the result
sheet and analyzed by using the apiwebTM identification system.
Several supplementary tests (nitrate reduction test, motility test, growth
on MacConkey agar, oxidation of glucose and fermentation of glucose) were carried
out to confirm the characteristic and identification of the bacteria. Nitrate
Reduction Test was performed after completely adding reagent to TDA, VP and
IND microtubes after incubation of API 20E strip. 1 drop each of NIT 1 and NIT
2 reagent were added to the GLU microtube of the API 20E strip. After 2-5 min,
a red colour indicated a positive reaction (reduction of nitrate to nitrite)
while yellow colour indicated a negative reaction. When negative reaction happened,
2-3 mg of zinc dust was added to the GLU microtube. If the tubes remained yellow
after 5 min, it indicated a positive reaction to be recorded on the results
sheet. If the test turned orange-red, it was a negative reaction.
Motility Test was performed on SIM medium. An inoculating needle was used in
this test to pick a colony from a fresh bacterial culture. The inoculating needle
was then stabbed into the centre of SIM medium contained in a capped bottle.
This bottle was incubated for 24 h at 30°C. Positive reaction was indicated
by fuzzy streak, fan shape pattern, nodular growth of the bacteria and also
turbidity in the medium while negative reaction was indicated by accentuated
bacterial growth along the stab line. For the test of bacterial growth on MacConkey
agar, fresh inoculums were streaked on MacConkey agar plate by using an inoculating
loop. This was followed by incubation at 30°C at 24 h. Presence of bacterial
growth on the agar was considered as positive reaction while the absence of
bacterial growth was recorded as a negative reaction. OF basal medium was used
to observe the activity of glucose oxidation by the bacteria. With an inoculating
needle, a fresh single was picked and stabbed into the OF media contained in
a capped bottle. Then, it was incubated at 30°C for 24 h. The same procedure
was repeated to test the fermentation activity of bacteria. The different was,
a layer of sterile mineral oil about 1.5 mL was added to the top of media after
the colony as stabbed into the OF media. For both oxidation and fermentation
reaction, positive reaction was indicated by yellow colouration while green
colour indicated negative reaction.
The basic morphological condition of fish before and after storage conditions
are presented in Table 1. Before storage, skins of all seabass
were firm and shining, corneas of eyes were very clear and gills of all seabass
were red colour. Asian seabass was not different after two days of storage with
crushed ice except appearing some trans parent slime on the skin.
|| Morphological changes of Asian seabass after two days of
ice and salt storage
|| Result of Gram Staining of bacteria sample
|CF: Flesh of ice-Chilled storing fish, CS: Skin of ice-Chilled
storing fish, SF: Flesh of sea salt storage fish; SS: Skin of sea salt storage
fish, Numeric value under isolate column indicates replication number. a,
b, and c indicate different isolate in the same plate. G+ and G-:Gram positive
and Gram negative bacteria, respectively
However, some changes were observed in sea salt storage seabass. After two
days, skin of salt storage seabass was coarse and dried. Although their eyes
were also dry but showed clear after storing two days with sea salt.
The effect of ice-chilling and salting on bacteria number in flesh and skin
are shown in Fig. 1. In case of bacteria number on skin, sea
salt storage fish was statistically different that chilled fish. Sea salt storage
seabass had more bacteria than ice-chilled seabass. However, in case of bacteria
number in flesh, there was no significant different (p>0.05) between ice-chilled
and salt storage seabasses. There was a significant difference on the bacteria
number in skin and flesh (p<0.05). Overall more bacteria were observed at
the skin than in flesh (Fig. 2).
A total of 16 different isolates were distinguished in the present study. Out
of 16 isolates, only 3 (19%) isolates were identified as gram negative while
the rest 13 (81%) were gram positive (Table 2, Fig.
3). Among 16 isolates, only 2 isolates were identified as Bacilli and other
14 isolates were identified as cocci. API® test could only identified
gram negative bacteria. In the present study API® test successfully
identified 2 types of bacteria to its species level (Table 3).
The bacteria were Acinetobacter baumannii and Pseudomonas fluorescens.
However, both bacteria were identified in the skin sample ice-chilled, storage
fish. The identified bacteria were classified into either fish spoilage bacteria
or human pathogenic bacteria.
|| Effect of chilling and salting on bacteria number in (a)
Skin and (b) Flesh. A *Significantly different at p<0.01 and ns: Not
significantly different, Data are Mean±SD
||Bacteria number in skin and flesh after two days of storing.
*Significantly difference (p<0.05) between the bacteria number. in skin
and flesh. Data are Mean±SD
However, A. baumannii was classified into human pathogenic bacteria
while P. fluorescens was classified as fish spoilage bacteria. Besides
Pseudomonas fluorescens and Acinetobacter baumannii many isolates
were identified up to genus level. These included Vibrio and Myxobacteria.
||The most probable genus and species of isolates from Asian
seabass identified by the API® identification system
|CS: Skin of ice-Chilled storing fish, 1,2Replication
No. aDifferent isolate in the same plate
||Distribution of Gram-positive and Gram negative bacteria
This study focuses on bacteriological quality between sea salt and ice storages
Asian sea bass to identify the better storage method. In the present study,
result of morphological characteristics of fish indicated that all fish were
fresh even after two days of storing. However, ice-chilled storage fish showed
better morphological characteristics than that of sea salt storage fish. Before
storage skin of Asian sea bass was firm, shining but after two days, skins of
salt stored Asian seabass were coarse and dried. This indicated some chemical/bacteriological
changes in the skin skins of salt storage seabass. There is no previous study
comparing morphological characteristics between the ice and salt storage Asian
seabass. However, this result concurs with the result of bacteriological study.
In the present study higher number of bacteria was observed on the skin of
salt preserved fish than on the skin of ice preserved fish. Higher number of
bacteria might cause more deterioration of the skin of sea salt preserved fish.
Our result in a way agree with the result of Slabyj et
al. (2003), who observed that chilled fishes samples had succeed preventing
invasion and stopped bacteria from growing. The reason might be due to flash
out effect of melted ice. In the case of ice preserved fish, water produced
from melted ice removed bacteria from the skin. This reduced the number of bacteria
on the skin of ice preserved fish. Another reason might be short time preservation.
After 2 days, salted fish was not fully dried that bacteria might be survived
in that condition. Therefore, storage time is another important factor for selecting
appropriate storage method (Burt et al., 1992).
In this study, comparatively higher quantity of bacteria was observed on the
skin than in the flesh. This pattern of bacterial population distribution in
fish body was also demonstrated by many previous researches (Huss
et al. 1995), Slabyj et al. (2003)
who reported microbial growth mostly takes places at the surface of the fish
after the deterioration the quality of fish while only a limited number of microorganisms
actually invade in the fish flesh. According to Slabyj et
al. (2003), this is probably due to a consequence of bacterial enzymes,
which starts working in the flesh resulting in a lot nutrients diffuse out.
This diffused nutrient rapidly accelerates bacterial growth on the skin of fish.
In the present study, overall a few bacteria (16) were observed in both salt
and ice storage conditions. These indicate that the fishes were very fresh even
after two days of storing. The reason of freshness might be storing immediately
after dying. In this study, all fished were transported in the laboratory as
live condition and they were stored immediately after dying by cold shock. Bacteria
did not get enough time to increase their number before storing. Therefore,
storage time (time between dying and storage) is a very important factor in
maintaining quality of fish. Just after death, fish can be soft for a few hours
(pre-rigor condition) but then it starts to become stiff. This phenomenon is
called rigor mortis. The fish stays in the rigor mortis
condition for a while, but then its flesh muscles become relaxed again (NZIC,
2008). At this stage, the quality of fish starts to deteriorate very rapidly
(Aaraas et al., 2004).
Among all identified bacteria, most of the bacteria Gram positive. According
to Huss et al. (1995), Gram negative bacteria
is more dominant over gram positive bacteria in fish. However, this depends
on many factors such as fish species, location of fish body from where sample
is taken, storage time after dying, habitat of fish, etc. In this study, A.
baumannii and P. fluorescens bacteria were identified from chilled
fishes. This result in a way agrees with the result of Huss
et al. (1995), who reported that P. fluorescens generally
known as fish spoilage bacteria and observed in ice-chilled fish. However, according
to Gerischer (2009), A. baumannii is a human pathogenic
bacteria within the Acinetobacter genus or also known as opportunistic
human pathogen. Besides P. fluorescens and A. baumannii many
isolates were identified up to genus level. These included Vibrio and
In term of bacteria number and morphological characteristics, Ice-chilled preserved
fish was better than salt preserved fish. More research is needed to compare
between salt and ice storages when fish are stored for a long period. Overall,
less number of bacteria was observed in both Ice-chilled and sea salt preserved
fish. The result of the present study indicated that the quick preservation
is a very important factor to control bacterial load in the preserved fish.
However, this research cannot give any conclusion in the case of long term preservation.
Therefore, more research is recommended on the effects of chilling and salting
on bacteriological quality if fish is preserved for a long term basis.