Research in Microbiology 157 (2006) 355–359

www.elsevier.com/locate/resmic

Optimization and validation of a colorimetric assay

for Tetrahymena sp. survival
Dina Zilberg ∗ , Tamar Sinai
Albert Katz Department of Dryland Biotechnologies, The Jacob Blaustein Institute for Desert Research, Ben-Gurion University of the Negev,
Sede Boqer Campus, 84990 Midreshet Ben Gurion, Israel
Received 24 June 2005; accepted 29 September 2005
Available online 27 October 2005

Abstract
A colorimetric assay based on the reduction of 3-(4,5-dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2h-tetrazolium bromide (MTT), for the quantifi-
cation of Tetrahymena sp. survival is described. An increase in the concentration of Tetrahymena sp. cells from 0 to 1 × 106 cells/ml produced a
linear (R 2 = 0.9965) increase in the optical density (OD, 570–630 nm), and dead cells (pre-exposed to 250 mg/l formalin for 4 h) did not produce
a background reading. Cells exposed to sublethal concentrations to formalin (100 mg/l or less for 4 h) recovered their growth. Using the MTT
assay, we determined that Tetrahymena sp. is sensitive to formalin, chloramine-T, hydrogen peroxide, copper sulfate and NaCl. The sensitivity
increased with increasing chemical concentrations and exposure time. Tetrahymena sp. was resistant to bromex and malachite green. The use
of this assay in drug screening for the development of treatments for tetrahymenosis and as a bioassay to evaluate the toxicity of environmental
toxicants is discussed.
 2005 Elsevier SAS. All rights reserved.

Keywords: Tetrahymena; MTT; Survival assay; Treatment; Chemicals

1. Introduction fection in a commercial farm in southern Israel (I. Paperna, per-

The ciliated protozoan, Tetrahymena sp., is a major disease-
causing agent of guppies, also known as the guppy-killer
parasite. The genus Tetrahymena includes mainly free-living
holotrichous, generally saprozoic ciliates, which feed on or-
ganic matter and bacteria in natural habitats. It is presumed to
infect fish that are stressed by adverse environmental condi-
tions [9]. The infection is characterized by whitish lesions on
the body surface, eroded fins and lethargic swimming [7,8,17].
Aside from guppies, species that have been reported to be
susceptible to this parasite include Atlantic salmon, Salmo
salar [6], zebrafish, Brachidanio rerio [1], pristella, Pristella
maxillaries, neontetra, Paracheirodon innesi and cherry barb,
Puntius titteya [14].
Infection with Tetrahymena sp. presents a serious problem
in guppy production in Thailand [7,14]. A severe systemic in-
* Corresponding author.
E-mail address: dzilberg@bgu.ac.il (D. Zilberg).
0923-2508/$ – see front matter  2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.resmic.2005.09.012
sonal communication) necessitated its drying out. An effective
treatment for tetrahymenosis is not available once it becomes
systemic. Apart from its significance as a fish-disease prob-
lem, Tetrahymena pyriformis is used to assess the cytotoxicity
of chemicals and environmental pollutants [16].
Tetrahymena sp. viability assays are conducted to test the ef-
fect of different therapeutic agents for treatment development,
and as aforementioned, to determine the cytotoxicity of en-
vironmental pollutants. Methods used thus far have involved
direct observation and differential counting of live cells [5,11,
15,16], or an assessment of proliferation rate by repeated enu-
meration using a Coulter counter [16]. In the present study, we
describe the use of a colorimetric assay, based on the reduction
of the tetrazolium salt MTT [3-(4,5-dimethyl-2-thiazol-2-yl)-
2,5-diphenyl-2h-tetrazolium bromide] in live cells to produce
a dark blue formazan product that is colorimetrically quanti-
fied [10]. The assay, originally developed for use with mam-
malian cells [10], was modified and calibrated for use with
Tetrahymena sp.
356 D. Zilberg, T. Sinai / Research in Microbiology 157 (2006) 355–359
2. Materials and methods
2.1. Tetrahymena sp. isolation and culture
Diseased guppies (Poecilia reticulata) displaying whitish
skin lesions and eroded fins were obtained from a commer-
cial aquaculture farm in Israel. The causative agent, a proto-
zoan parasite, was identified as Tetrahymena sp. based on its
morphological characteristics [13]. The Tetrahymena sp. was
isolated from the fish and transferred to ATCC 357 culture
medium (ATCC, Manassas, VA) in a petri dish at 25 ◦ C. Peni-
cillin G (3 mg/l) and streptomycin sulfate (3 mg/l) were added
to prevent bacterial growth. A clean Tetrahymena sp. culture
was obtained 2–4 weeks after the initial isolation. The cells
were transferred to a 24-well cell-culture cluster (Corning In-
corporated, Acton, MA) and subcultured weekly by transferring
10–30 µl to 2 ml of sterile ATCC 357 culture medium, without
antibiotics. Subculturing was performed under sterile condi-
tions in a biological hood (ADS Laminar, Cedex, France).
2.2. Colorimetric MTT (tetrazolium) assay
The assay was based on a previously described colorimetric
assay for cellular growth and survival [10]. Cultured Tetrahy-
mena sp. were inoculated into sterile ATCC medium and grown
at 25±1 ◦ C, with stirring, for 2 days. Ciliates were then concen-
trated by centrifugation at 235 g for 3 min at 10 ◦ C (Labofuge
400R, Heraeus, Hanau, Germany), the supernatant was dis-
carded and the pelleted Tetrahymena sp. was resuspended in
ATCC medium to the desired concentration. The assay was
conducted in flat-bottom, 96-well plates. To each well, 50 µl
of the cell suspension and 25 µl of the material being tested
for toxicity were applied and incubated for 1–4 h at 25 ◦ C.
As a negative control, ATCC medium was used. MTT [3-(4,5-
dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2h-tetrazolium bromide]
(ICN Biomedicals Inc., Irvine, CA) was added and incubated
for 4 h at 25 ◦ C. Finally, 100 µl of a stopping solution (9.5%
(w/v) SDS, 48% (v/v) DMF (dimethyl formamide), 1.9% (v/v)
acetic acid and 0.02 N HCl) were added and the plate was in-
cubated in the dark, overnight at 25 ◦ C. The plates were read
on a microplate reader (model 550, Bio Rad, Hercules, CA),
at 570 nm and a reference wavelength of 630 nm (the reading
of which was subtracted from the 570-nm reading). To calibrate
the MTT assay, different cell concentrations were used, ranging
between 0 and 1 × 106 cells/ml. At each concentration, live and
dead cells, killed by the addition of formalin at a concentration
of 250 ppm for 4 h, were separately assayed. The experiment
was performed in eight replicates.
To verify the reliability of the MTT assay, cells were exposed
to formalin at 0, 2.5, 25, 50, 100 and 250 mg/l and the MTT as-
say was performed after 1, 2 and 4 h in eight replicates. Growth
recovery was tested at 0, 100, 150, 200 and 250 mg/l of forma-
lin for 1, 2 and 4 h of incubation and analyzed by adding 50 µl
of the well content to fresh medium (2 ml) in a 24-well plate
which was incubated at 25 ◦ C, in four replicates. The wells were
observed after 2–4 days for the presence of live Tetrahymena
sp. and the percentage of wells in which growth had recovered
was calculated. Dead/live cells were counted by hemocytometer
(Neubauer improved, Brand Ltd., Germany) after 4 h of incu-
bation using the trypan blue exclusion assay (exposure to 0.2%
(w/v) trypan blue), in eight replicates.
2.3. Testing the effect of different chemicals on
Tetrahymena sp.
The colorimetric MTT assay was used to evaluate the effect
of commonly used chemotherapeutic agents on Tetrahymena
sp. The ciliate was exposed to formalin (Romical, Be’er-Sheva
Israel), malachite green (Katzat, Petah-Tikva, Israel), bromex
(Bromex 50, Machteshim, Beer Sheva, Israel), chloramine-T
(Halamid, Dorvet Ltd. Nes-Tziona, Israel), hydrogen perox-
ide (35% solution, Merck, Darmstadt, Germany), copper sulfate
(Z.M.M. Laboratory Equipment, Be’er-Sheva, Israel) and NaCl
(Riedel de Haen, Seelze, Germany). Exposure was carried out
at different concentrations for 1, 2 and 4 h, except for malachite
green and bromex, where only the 4-h exposure was used. No
chemical was added to the control treatment. Experiments were
carried out with a cell concentration of 1 × 106 cells/ml, in
eight replicates. To calculate percent survival, OD was divided
by the OD of the control and multiplied by 100.
2.4. Statistical analysis
Two-factor analysis of variance (ANOVA) was used to an-
alyze the effects of chemical concentration and time of expo-
sure on Tetrahymena sp. survival. Statistical analyses were per-
formed with Sigma Stat (SPSS Inc., 1992–1997). Differences
were considered significant at P < 0.05.
3. Results
Under light microscopy, untreated cells show pyriform mor-
phology, ‘spiraling football’ movement and high mobility. In
the colorimetric MTT assay, live cells produce a dark blue
formazan product from the pale yellow MTT substrate. The
OD at a wavelength of 570 nm is indicative of the amount of
formazan produced. An increase in the number of cells per
well produced a linear increase (R 2 = 0.9965) in OD reading
(Fig. 1). The OD in control wells (with no added cells) was zero
(Fig. 1). Exposing the cells to 250 ppm formalin for 4 h pro-
duced 100% mortality, as determined by trypan blue exclusion
and growth recovery assays (Table 1). Under direct microscopic
observation, dead cells were rounded and did not move (not
shown). Wells with dead cells gave a low OD reading in the
MTT assay, close to the level in the control wells (maximum
of 0.053 at 1 × 106 cells/ml; Fig. 1). A cell concentration of
1 × 106 cells/ml was used in subsequent experiments.
Survival rates following exposure to formalin are presented
in Fig. 2a. Formalin concentration and length of exposure sig-
nificantly affected survival at concentrations of 50 mg/l and
over. Survival following exposure to 250 mg/l for 2 and 4 h was
very low (4–6%), whereas it was significantly higher (23%) af-
ter 1 h of exposure. Survival was markedly higher following
 D. Zilberg, T. Sinai / Research in Microbiology 157 (2006) 355–359 357

exposure to formalin at 100 mg/l for 4 h (56%) and shorter ex-

posures or exposure to lower concentrations yielded over 70%
survival. Exposure to 2.5 mg/l had little or no effect.
In the trypan blue exclusion assay, dead cells, which were
rounded and did not move, are stained blue, whereas live cells

Table 1
Percentage of Tetrahymena sp. survival following incubation with different con-
centrations of formalin, as determined by the MTT assay, direct cell count using
the trypan blue exclusion assay and growth recovery
Formalin MTT assay Direct cell count Growth recovery (n = 4)
(mg/l) (4 h, n = 8) (4 h, n = 8) 1h 2h 4h
0 1001 100 ± 0 100 100 100
50 65.8 ± 2.1 99.5 ± 0.5 100 100 100
Fig. 1. Linearity of the OD reading in the MTT assay (R 2 = 0.9965) with in- 100 60.9 ± 5.5 98.8 ± 1.2 100 100 100
creasing concentration of Tetrahymena sp. cells, and the OD produced by dead 150 7.5 ± 0.1 ND 100 75 0
cells, killed by exposure to formalin (250 ppm for 4 h). 200 4.6 ± 0.4 ND 100 75 0
250 4.4 ± 0.3 0±0 100 0 0
ND, not determined;
1 used as reference for 100% survival in the MTT assay.

Fig. 2. Effects of formalin (a), chloramine-T (b), hydrogen peroxide (c), copper sulfate (d), NaCl (e), bromex and malachite green (f) on relative survival of
Tetrahymena sp., as determined by the MTT assay. a–e: ( ) 1, ( ) 2, ( ) 4 h.
358 D. Zilberg, T. Sinai / Research in Microbiology 157 (2006) 355–359
(pyriform shape and movement) remain unstained. In results
obtained from this assay, exposure to 0, 50 and 100 mg/l
formalin yielded close to 100% survival, whereas exposure
to 250 mg/l produced 100% mortality (Table 1). Live cells
showed growth recovery (Table 1).
Survival rates following exposure to chloramine-T are pre-
sented in Fig. 2b. An increase in concentration and time of
exposure significantly reduced survival, except at 600 mg/l,
where survival was not significantly different (1 and 2 h of
exposure) or was lower (4 h of exposure) than survival at
1500 mg/l. Although statistically significant, the effect of expo-
sure time was small, especially at 600 and 1500 mg/l. Survival
at 600 and 1500 mg/l chloramine-T ranged between 10 and
23%. At 300 mg/l, survival was over 65% and a concentration
of 150 mg/l or less had little or no effect.
Survival rates following exposure to hydrogen peroxide are
presented in Fig. 2c. An increase in concentration and time of
exposure significantly reduced survival. Survival of 20% or less
was obtained following exposure to 20 mg/l for 4 h, 40 mg/l
for at least 2 h or 100 mg/l at all tested time periods. Exposure
to 1 mg/l had little effect.
Survival rates following exposure to copper sulfate are pre-
sented in Fig. 2d. Increase in copper sulfate concentration sig-
nificantly reduced survival, as did increased exposure time, al-
though the effect of the latter was relatively small. Survival
following exposure to 150 mg/l was below 7%. Survival fol-
lowing exposure to 60 mg/l was markedly higher, over 30%.
A concentration of 1.5 mg/l had little or no effect on survival.
Survival rates following exposure to NaCl are presented in
Fig. 2e. NaCl at a concentration of 16.5 g/l reduced survival to
11% or less, but at 0.17 g/l, it had no effect. There was also
no clear concentration effect at exposure to 1.7, 3.3 and 6.6 g/l
NaCl, and survival at these concentrations ranged between 50
and 72%. Exposure time only significantly affected survival at
6.6 and 16.5 g/l.
Bromex and malachite green exhibited low toxicity to
Tetrahymena sp., with 75 and 74% survival following expo-
sure to 20 mg/l, respectively (Fig. 2f). With malachite green,
survival at 20 mg/l was significantly lower than it was at lower
concentrations of this chemical.
4. Discussion
In this report, we describe the use of the colorimetric
MTT assay to quantify Tetrahymena sp. survival. The assay,
originally developed to quantify the survival of mammalian
cells [10], was calibrated and validated for Tetrahymena sp. An
increase in the concentration of Tetrahymena sp. cells produced
a linear increase in the OD reading. Dead cells (by pre-exposure
to 250 mg/l formalin) produced a low or no background read-
ing as the OD remained close to that of the control with no
added cells. Results from the direct live/dead cell count using
the trypan blue exclusion assay revealed a higher percentage of
live cells compared to the MTT assay. Some of the dead cells
may have disintegrated and therefore have been excluded from
the direct counting. The growth-recovery assay confirmed that
cells that survive the chemical exposure can proliferate. The
low percentage of live cells obtained in the MTT assay at for-
malin concentrations of 150 to 250 mg/l after 4 h of exposure
may have resulted from the low background reading or very low
activity in the damaged cells, because at these concentrations,
cells were not able to recover their growth.
Using the MTT assay, Tetrahymena sp. was found to be
sensitive to formalin, chloramine-T, hydrogen peroxide, cop-
per sulfate, and NaCl. The sensitivity increased with increased
chemical concentration and exposure time. With chloramine-T
and copper sulfate, most of the effect occurred over the first
hour of exposure and further reduction in viability over time
was low, likely due to quick consumption and/or inactivation
of these chemicals [2]. Formalin, hydrogen peroxide and NaCl
could serve as treatment options for external Tetrahymena sp.
infections, as concentrations that markedly reduced the survival
of this parasite were close to those recommended for treatment
in fish [12]. Pompornpisit [15] showed the effectiveness of these
three chemicals against T. pyriformis in vitro, but to prevent
infection in fish it was necessary to use a combination of chem-
ical and immunostimulatory treatments. Tetrahymena sp. was
resistant to bromex and malachite green, even at high concen-
trations. Resistance of the parasite to malachite green has also
been reported by Pompornpisit [15].
Methods previously used to assess Tetrahymena sp. survival
include live/dead cell counts based on observations of viabil-
ity and motility [15,16], and differential fluorescent staining
using calcein AM [5] or feeding on fluorescently labeled par-
ticles [11]. All of these methods require direct observation and
counting, and are therefore quite labor-intensive. Recently, fluo-
rescent indicators dyes, such as alamar blue, were used success-
fully to assess the viability of Tetrahymena sp. [3,4], and like
MTT method did not require microscopic observation. How-
ever, unlike with MTT, a fluorescent plate reader was required.
Results from this study suggest that the MTT assay is a reliable
method of evaluating the survival of Tetrahymena sp. in vitro,
and can be used for drug screening as an initial step in the de-
velopment of treatments for tetrahymenosis. The assay can also
be used to evaluate the toxicity of environmental toxicants [16].
Acknowledgements
We would like to thank Dr. Rivka Ofir and Dr. Tatianna
Rabinski for their useful advice in setting up the assay, and
Dr. Raanan Ariav for providing the Tetrahymena sp. infected
fish.
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