Determining the safety and suitability of fluorescein dye for characterization of skin ulcerations in cultured Nile tilapia (Oreochromis niloticus) and African sharptooth catfish (Clarias

Journal of Advanced Research (2010) 1, 361–366

Cairo University

Journal of Advanced Research

ORIGINAL ARTICLE

Determining the safety and suitability of fluorescein dye
for characterization of skin ulcerations in cultured
Nile tilapia (Oreochromis niloticus) and African
sharptooth catfish (Clarias gariepinus)
Mai D. Ibrahem
a
b

a,*

, Salah Mesalhy

b

Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt
Department of Pathology, Faulty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt

Received 29 December 2009; revised 7 February 2010; accepted 19 April 2010
Available online 17 September 2010

KEYWORDS
Fluorescein dye;
Abrasions;

Ulcers;
Nile tilapia;
Catfish;
Detection

Abstract There is a need to identify the presence of lesions in fish skin as soon as they erupt. Fish
skin lesions are either macroscopic (can be visualized by the naked eye) or microscopic (difficult to
detect with the naked eye). Skin wounds resulting in loss of the epithelium (superficial or deep
ulcers) are serious as they may interfere with osmoregulation and open portals for opportunistic
pathogens. Herein, we report on the use of a fluorescein dye for the detection of skin ulcers that
cannot be seen by the naked eye. Due to their importance in aquaculture endeavors in Egypt, this
study focused on two indigenous species, the Nile tilapia (Oreochromis niloticus) and the scale-less
African sharptooth catfish (Clarias gariepinus). Fluorescein dye was tested for safety to fish without
interfering with microbiological analysis. Parallel to the use of the flourescein dye, the detected
ulcers were examined for the presence of bacteria or tissue alterations. Further, we experimentally
induced the formation of skin ulcers in O. niloticus physically or by injecting Aeromons hydrophila,
and then assessed the utility of fluorescein dye in detecting the induced skin lesions. Results
obtained in this study demonstrated that fluorescein dye application is harmless to Nile tilapia at

* Corresponding author. Tel.: +202 33800575; fax: 202 35725240.
E-mail addresses: , ibrahemmai20@yahoo.
com (M.D. Ibrahem).
2090-1232 ª 2010 Cairo University. Production and hosting by
Elsevier B.V. All rights reserved.
Peer review under responsibility of Cairo University.
doi:10.1016/j.jare.2010.04.002

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362

M.D. Ibrahem and S. Mesalhy
concentrations up to 0.5 mg fluorescein/ml water for up to 15 min. Indeed, a low dose of fluorescein
(0.10 mg/ml for 5 min) could identify very minute skin abrasions. We highly recommend the use of
fluorescein dye for the evaluation of skin health in farmed fish species and the visualization of minute skin abrasions.
ª 2010 Cairo University. Production and hosting by Elsevier B.V. All rights reserved.

Introduction
Loss of skin and underlying tissue is considered one of the
most common lesions affecting fish worldwide and is considered to be a reflection of the surrounding environment. There
are a number of causes for skin ulceration including pathogens
[1] and chemical and physical factors [2]. Skin loss, even if minute, can bring serious complications such as osmoregulatory
failure, swimming imbalance, respiratory distress dehydration
[1] and can predispose affected fish to opportunistic bacteria
and fungi. Skin ulcers vary in both depth and size from nonvisible microscopical erosions to grossly visible ulcers [3].
Fluoresceinating sodium salt (which will be referred to as
‘‘fluorescein’’) is a non-toxic dye that produces an intense
green fluorescence colour when dissolved in water. It has been
safely used to detect ophthalmic lesions including ulcers and
degeneration of the cornea in humans [4–7]. Fluorescein neither penetrates intact epithelium nor forms a firm bond with
vital tissue. However, when there is a break in the skin epithelium, fluorescein can rapidly penetrate [8], thereby staining exposed underlying skin layers [9]. The present study builds upon
the study of Noga and Udomkusonsri [10] and examines their
findings for the utility, safety and efficiency of the fluorescein
dye as a tool for the detection of natural and experimentally
induced skin ulcers in native fish species, and compares the results of histopathology and the use of fluorescein dye for early
detection of skin lesions.

static dechlorinated water [11]. Acclimatisation was performed
in temperature-controlled aquaria at 23 ± 1 °C. The fish were

fed twice a day with a balanced commercial fish pelleted diet
(Zoocontrol Company, Cairo, Egypt) that contained 30% protein [12]. Water quality in all aquaria was monitored regularly
for ammonia, and pH, kept within the acceptable limits during
the course of the study [13]. All fish were subjected to clinical
and bacteriological examination to prove that they were free
from Aeromons hydrophila infection.
Safety of fluorescein dye to fish
Eighty of the pre-acclimatised Nile tilapia used for the safety
test were transferred to glass aquaria (30 · 40 · 80 cm3), each
containing 40 l of 23 °C freshwater (10 fish/ aquarium). The
fish were first placed in a high concentration of dye, up to
0.5 mg fluorescein/ml for 15 min, and then observed for seven
days. Control fish (10 fish/aquarium) were similarly treated.
All tests were performed in duplicate.
In vitro antimicrobial assay (agar spot assay)

Field studies

A reference culture pathogenic, A. hydrophila (ATCC 7966
strain), was cultured in Trypticase Soya broth (Biomerieux,
France) for 18 h at 25 °C. Soft agar (composed of Trypticase
Soya broth + 0.7% bacteriological agar) containing 5% of
overnight culture of A. hydrophila in Trypticase Soya broth
was prepared. Spots were made by pipetting 10 ll of fluorescein dye dilutions (from 0.1 to 0.7 mg fluorescein/ml, each concentration on a separate plate) [14]. The plates were incubated
for 24 h. Inhibition was recorded by measuring the clearing
zone around the spot. All tests were performed in duplicate
[14].

Naturally collected fish

Fish anesthesia

African sharptooth catfish (Clarias gariepinus) of 50–60 mm
total length [TL] and Nile tilapia (Orecochromis niloticus) of
120–150 mm TL (10 fish/species) were obtained from a semiintensive aquaculture facility and transported alive in well-aerated containers to the laboratory of the Department of Fish
Disease and Management (FDML), Faculty of Veterinary
Medicine, Cairo University. Upon arrival, the fish were immediately screened for the presence of exposed skin areas using
the fluorescein dye.

Tricaine methane sulfonate (MS222) obtained from Argent
Chemical Laboratories, Redmond, WA, was used to anesthetise fish in a dose of 50 mg/l MS222 with 100 mg/l sodium
bicarbonate as a buffering reagent to adjust water alkalinity
to exceed 50 mg/l as CaCO3 as recommended by [15].

Experimental studies

The fish (both African sharptooth catfish and Nile tilapia)
were placed (one at a time) in a solution of 0.10 mg fluorescein
(FluorÒ, 10% fluorescein sodium injection, 100 mg/ml, Sigma,
USA) per ml of water for 5 min. The fish were then removed
from the fluorescein solution and immediately rinsed by placing them in clean water for 1–2 min. The fish were then euthanatized with MS222 and immediately examined for skin damage
under an ultra violet source from a UV Trans-illuminator
(Spectoline Bi-O-Vision Model TVD-1000R/F, USA) in complete darkness. Photographs were taken using digital camera

Material and methods

Fish
For monitoring the safety and efficiency of fluorescein to detect the skin ulcers, experimental studies were performed on
O. niloticus. Nile tilapia (20 ± 0.2 g) (240 fish) were carefully
collected alive from a semi-intensive fish farm to avoid any injury and transported alive in tanks to FDML. Fish were acclimatised to laboratory conditions for two weeks prior to the

experiment. They were maintained in 40 l tanks containing

Potency of the fluorescein dye to fish
Naturally affected fish


Diagnosis of ulcerations using fluorescein dye

363

with automatic adjustment (Fujinon ptical zoom lens camera;
Model No. F 7U2540; China). Fluorescein-positive areas from
Nile tilapia were fixed for histopathological examination [16].
Experimentally ulcerated Nile tilapia
The pre-acclimatised fish were transferred to identical aquaria
containing 40 l of water (10 fish/aquarium, two replications of
the treatment). Water temperature was adjusted to 23 °C. Fish
were collected one at a time from each aquarium and anesthetised with MS222. A few scales were hand-removed from each
fish; then fluorescein dye was used for the detection of the induced ulcers via the regime described above.
Experimentally infected Nile tilapia
A culture suspension of A. hydrophila (ATCC 7966) was prepared by spreading onto Tryptic soya agar for 24 h at 25 °C;
4–5 colonies were suspended in sterile saline 0.85% and matched
to contain 108 bacteria mlÀ1 using McFarland standard tubes.
The test fish were held in replicate aquaria containing 40 l of
thermostatically controlled water at 23 °C (10 fish/aquarium,
two replications of the treatment); fish were infected intraperitoneally with 0.2 ml of the culture suspension. The infected fish
were examined at 8, 12, 18, 24, 48 and 72 h post infection by fluorescein dye for ulceration via the regime described above.
Bacteriological investigations
Smears from skin ulcers, kidney and liver of naturally examined
Nile tilapia and African sharptooth catfish and experimentally

infected Nile tilapia were spread onto Trypticase Soya Agar
(TSA) (Oxoid) and blood agar plates. Culture plates were incubated at 25 °C for 24 h and then inspected for bacterial growth.
Further morphological and biochemical identification of the retrieved isolates was done according to Whitman [17].
Histopathology
For histopathological examination, skin lesions from naturally
ulcerated Nile tilapia were fixed in 10% neutral buffered formalin, and then processed in paraffin embedding method sections
of 5 lm were stained with hematoxylin and eosin (H&E) [16].
Results and discussion
Safety of fluorescein dye to fish
In the first group, fish were subjected to a single application
of a high concentration of fluorescein dye (0.5 mg fluores-

cein/ml water for 15 min) and then monitored for seven
days. The dye proved safe as no adverse clinical or behavioural abnormalities appeared in Nile tilapia post exposure.
The present results coincide with those of Noga and Udomkusonsri [10] who used the same stain with rainbow trout
(Oncorhynchus mykiss), channel catfish (Ictalurus punctatus),
goldfish (Carassius auratus) and hybrid striped bass (Morone
saxatilis male X M. chrysops female), and found it non toxic
with no evidence of health hazards to the examined fish.
Pouliquen et al. [18] found that exposure of turbot (Scophthalmus maximus) to 700 mg fluorescein/l for 96 h was not
lethal. As it will eventually find its way to the gills and gastrointestinal tract, the potential effects of fluorescein dye on
fish were discussed. O’goshi and Serup [19] systematically reviewed literature on fluorescein and concluded that it was
useful in cutaneous research; the intradermal injection of
fluorescein is being used increasingly to investigate skin conditions in vivo with non-invasive devices such as con focal
scanning laser microscopy. Sodium fluorescein was used
intravenously for decades for the examination of the vasculature of the ocular fundus (fluorescein angiography) and
as eye drops for diagnosis of corneal erosions, and proved
its safety. Watson and Rosen [20] stated that the injection
of fluorescein intravenously for fundal angiography in humans is associated with a high incidence of minor adverse effects with a very low incidence of serious (life threatening)
reactions. There are no reports of oral fluorescein causing

a serious reaction, and minor adverse effects are uncommon.
Furthermore, under the diagnostic procedures used for identifying ulcers in fish, it is unlikely that any significant concentration of fluorescein would be taken up by humans. In
addition, in a farming setting, only a sample of the population may be tested at one time.
In vitro antimicrobial assay (agar spot assay)
This test was intended to evaluate the possible risk of fluorescein dye in masking the diagnosis of skin injury due to bacteria. The results showed that there was no difference between
the growth of A. hydrophila in the culture plates and when exposed to fluorescein dye. Such a result indicates that fluorescein had no bactericidal effects in our studied bacterium.
Thus, the dye does not appear to mask the presence of A.
hydrophila during diagnosis of skin ulceration. The agar spot
assay was used by a number of researchers to study the
in vitro activity of bacteria against other bacteria or biological
agents [21,22]. In the present study, it was essential to evaluate
the risk of fluorescein masking the bacteria in ulcerated skin.
Fluorescein is a water soluble and diffusible agent [4,5] and
can readily diffuse into the agar; if it has a bactericidal effect
it would be expected to inhibit the growth of A. hydrophila,
resulting in a clear inhibition zone.

Table 1 Summary of field studies on fish obtained from an aquaculture facility, showing the number of gross ulcers, number of
fluorescein-positive ulcers and the number of microscopic ulcers.
Nile tilapia (Oreochromis niloticus)

African catfish (Clarias gariepinus)

No. of
fish

No. of
gross
lesions

No. of fluoresceinpositive lesions

No. of
microscopic
ulcers

No. of
fish

No. of
gross
lesions

No. of fluoresceinpositive lesions

No. of
microscopic
ulcers

10

0

11

11

10

2

5

5


364

M.D. Ibrahem and S. Mesalhy

Fig. 4 Lateral view of Nile tilapia; the eroded/ulcerated areas
appear as apple green coloured areas at the dorsal aspect of the
abdomen (body region).
Fig. 1 Lateral view of African catfish; the eroded/ulcerated areas
appeared as apple green coloured areas at the lateral aspect of the
abdomen (body region).

A dose of 0.10 mg fluorescein/ml for 5 min was sufficient to
identify even minute undetected skin ulcers. The optimum
fluorescein exposure concentration was different from that of
Noga and Udomkusonsri [10] who used a concentration of
0.2 mg/ml for 3 min sufficient to stain the ulcers and recorded
it. They stated that a lower concentration of fluorescein
resulted in weaker staining of the ulcers. This difference in
the effectiveness of the concentrations may be attributable to
the different fish species and the time of exposure, although
our tested dose lies on the same safety margins that were tested
by Noga and Udomkusonsri [10].
Bacteriological investigations

Fig. 2 Head of African catfish (isthmus and operculum) showing
apple green coloured areas indicating eroded/ulcerated areas.

Fig. 3 Lateral view of Nile tilapia; the eroded/ulcerated areas
appear as apple green coloured areas at the dorsal aspect of the
abdomen (body region).

Potency of fluorescein dye to fish
A bright, apple-green fluorescent colour appeared after
fluorescein treatment, indicating ulcerated areas in naturally
collected scaled (Nile tilapia) and scale-less fish (African sharptooth catfish), see Table 1. Ulcerated spots were mainly observed on the ventral aspect of the head region (isthmus and
operculum((Fig. 1) as well as on the lateral aspect (body region) of African catfish (Fig. 2). Meanwhile, the ulcerated
areas appeared mainly at the dorsal aspect of the abdomen
in Nile tilapia (Figs. 3 and 4).

The importance of detecting skin damage early in bacterial
infections is of major concern [21]. In the present study,
A. hydrophila and Streptococcus feacalis were isolated from
naturally collected Nile tilapia skin ulcers with a prevalence
of 20%. In African sharptooth catfish, no bacterial growth
that was negative was obtained from the ulcerated skin samples, (Table 2). This can be attributed to several causes of ulcers including the aggressive behaviour of African catfish.
However, these ulcers can be a portal of entry for opportunistic bacteria. Being able to detect and then culture from the earliest invisible lesions, will definitely improve the ability to
identify important pathogens, hence bacterial diagnosis. The
importance of detecting early skin damage in bacterial infections is exemplified by the studies of Elliott and Shotts [23]
who found that Aeromonas salmonicida, the primary bacterial
pathogen of atypical furunculosis in goldfish, was only present
in the earliest stages of the disease.
Histopathology
Histopathological examination of lesions detected by fluorescein from naturally affected Nile tilapia revealed ulceration
in the skin as shown by superficial to deep desquamation of

the epidermal epithelium. In some cases, vacuolar degeneration with focal erosion was evident in the cells of the stratum
spinosum (Fig. 5). Remarkable vacuolation with mononuclear
cell infiltration was evident (Fig. 6). In severely affected cases,
a complete loss of the epidermis with ulcer formation was seen
where the underlying dermis was exposed to the exterior
(Fig. 7).
The results of the histopathological examination assessed
the efficiency of the fluorescein dye in detecting various stages
of skin ulcers of the naturally affected Nile tilapia. Such


Diagnosis of ulcerations using fluorescein dye
Table 2

365

Summary of field studies on fish, showing the number and percent of the bacterial isolates from fluorescein-positive ulcers.

Nile tilapia (Oreochromis niloticus)

African catfish (Clarias gariepinus)

Fish Fluorescein-positive lesions Bacterial isolates

Fish Fluorescein-positive ulcers Bacterial isolates

A. hydrophila % Strept. faecalis %
10

5

1

20 1

Fig. 5 Skin of naturally affected Nile tilapia showing vacuolation with superficial ulceration (H&E stain, ·100).

A. hydrophila % Strept. faecalis %

20 10

11

0

0

0

0

Fig. 7 Skin of naturally affected Nile tilapia showing a complete
loss of the epidermis and ulcer formation where the underlying
dermis was exposed to the exterior (H&E stain, ·100).

Experimental infection on Nile tilapia with A. hydrophila
Experimental infection of Nile tilapia using A. hydrophila was
carried out to assess the ability of fluorescein dye to detect
early skin ulcers induced by bacterial infection. Exposure of
the infected fish to the dye (starting from 8 h post infection) resulted in detection of the minute skin ulcers and beyond the

site of injection at 12 h post infection. Re-isolation of A. hydrophila from the lesions was successful at 12 h post infection.
Aeromons hydrophila is a ubiquitous, opportunistic bacterial pathogen that produces ulcerative dermatitis under stress
conditions and inflicts severe losses, manifested by both high
mortality and deterioration of product quality from global
fisheries and fish culture [24–27]. The anticipation of A. hydrophila infection as early as 12 h will definitely lower the economic losses and thus amplify the net gain from the farm.
A sealed package of 250 g of fluorescein stain powder costs
130 Egyptian pounds, and can produce up to 2500 litres of the
diagnostic stain. Therefore, in addition to its reliability as diagnostic procedure, it is considered to be an inexpensive and cost
effective early disease detection method.
Fig. 6 Skin of naturally affected Nile tilapia showing edema and
inflammatory cell infiltration (H&E stain, ·100).

Acknowledgments
findings proved that the stain is safe for use as there were no
obvious tissue reactions.

We thank Dr. Omar El-Tookhy, Department of Surgery, Faculty of Veterinary Medicine, Cairo University, for supplying


366
the fluorescein dye. We also thank Dr. Rawhia Doghaim and
Dr. Osama El-Shazly, Department of Pathology, Faculty of
Veterinary Medicine, Cairo University, for revising and evaluating the gross figures and histopathology results in this study.
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