Use of sodium hypochlorite solutions to obtain axenic culturesof Nostoc strains (Cyanobacteria)

Marcelo Gomes Marcal Vieira Vaz • Rafael Wesley Bastos •

Guilherme Paier Milanez • Mariana Neves Moura • Eder Galinari Ferreira •

Celia Perin • Marısia Cyreti Forte Pontes • Antonio Galvao do Nascimento

Received: 31 January 2014 / Accepted: 7 April 2014 / Published online: 9 May 2014

� Botanical Society of Sao Paulo 2014

Abstract A method based on the treatment of akinetes

using diluted solutions of sodium hypochlorite (SH solu-

tion) was developed to obtain axenic cultures of Nostoc

strains (Nostoc Vaucher ex Bornet & Flahault, 1886:181).

Three strains were independently grown on liquid BG-110

medium (BG-11 without a nitrogen source) until a massive

differentiation of akinete cells took place. Samples of these

akinete-rich cultures were homogenized and treated with

diluted SH solutions (1, 2, and 3 %) for 10, 20, and 30 s.

Subsequently, the treated akinetes were spread onto BG-

110-agar plates and incubated under standard conditions for

at least 2 weeks. Both the axenicity and the degree of

contamination were monitored for each treatment by

inoculating spring colonies in nutrient-broth or R2A-agar

plates. Axenic cultures were obtained for the strains Nostoc

sp. CCLFM I (with 1 % SH solution), Nostoc sp. CCLFM

VIII (2 and 3 %) and Nostoc sp. CCLFM XXI (3 %), only

applying 10 s of treatment exposure. This strategy was

proven to be efficient for Nostoc cultures, as all of the

tested strains became axenic. This method can be applied

to virtually any strains that are capable of performing

massive akinete differentiation; furthermore, it is a fast,

inexpensive, and antibiotic-free approach.

Keywords Akinete differentiation � Antibiotic-free

approach � Axenic cyanobacteria � Pure cultures �Soil Nostoc strains

Introduction

The task of obtaining axenic cultures of cyanobacteria is

considered to be time-consuming, laborious, and only

marginally successful (Choi et al. 2008). Generally, these

difficulties are the consequence of differences in the mor-

phological and physiological characteristics among the

cyanobacteria groups. However, some of these features are

related to the habitat of the cyanobacteria (free living or

symbiotic) and its interactions with other bacteria, proto-

zoa, and microalgae (Frontier 1985; Smith et al. 1998;

Rejmankova et al. 2000; Vasquez-Martınez et al. 2004).

These interactions and modifications on morphology and

physiology are relevant for species of the filamentous

genus Nostoc (Vaucher ex Bornet & Flahault, 1886:181),

which are found in a great variety of niches. Furthermore,

most cyanobacteria exhibit a complex multilayer envelope

around the cell (Flores and Herrero 2010) that includes an

external layer of exopolysaccharides of variable composi-

tion and size (Hoiczyk 1998; Vasquez-Martınez et al.

2004). Consequently, some filamentous cyanobacteria can

form rigid colonies, making it difficult to isolate single

filaments that are free of associated bacteria (Hoiczyk and

Baumeister 1995). Additionally, new filaments produced

by filamentous strains in solid media are free of

M. G. M. V. Vaz (&)

Laboratory of Molecular Ecology of Cyanobacteria, Center for

Nuclear Energy in Agriculture, University of Sao Paulo,

Piracicaba, Sao Paulo 13400-970, Brazil

e-mail: marcelovaz@usp.br

M. G. M. V. Vaz � R. W. Bastos � G. P. Milanez �M. N. Moura � E. G. Ferreira � C. Perin �M. C. F. Pontes � A. G. do Nascimento

Laboratory of Microorganism Physiology, Microbiology

Department, Federal University of Vicosa, Vicosa,

Minas Gerais 36570-000, Brazil

R. W. Bastos

Laboratory of Ecology and Physiology of Microorganism,

Microbiology Department, Institute of Biological Sciences,

Federal University of Minas Gerais, Belo Horizonte,

Minas Gerais 31270-901, Brazil

123

Braz. J. Bot (2014) 37(2):115–120

DOI 10.1007/s40415-014-0055-4

heterotrophic contaminants only for a short period because

the accumulation of exopolysaccharides in the medium

stimulates the growth of contaminants (Caire et al. 1997).

Establishing laboratory axenic cultures is critical for

understanding certain fundamental features of cyanobac-

teria and for performing in vitro manipulations (Watanabe

et al. 1998; Vasquez-Martınez et al. 2004). Many studies

have been conducted on axenic cultures of cyanobacteria,

e.g., to screen bioactive compounds (Araoz et al. 2005) or

to analyze genome-based diversity (Shih et al. 2013).

Several methods have been proposed based on various

approaches: mechanical separation of the cyanobacteria

and bacteria contaminants by micromanipulation (Bowyer

and Skerman 1968); gliding motility of the cyanobacteria

strains (Vaara et al. 1979); repeated transfer of cells (Allen

1973; Stanier et al. 1971; Rippka et al. 1981); antibiotic

treatment (Rippka 1988; Ferris and Hirsch 1991; Cho et al.

2002; Choi et al. 2002; Vasquez-Martınez et al. 2004; Choi

et al. 2008; Sena et al. 2011); lysozyme treatment (Kim

et al. 1999); thermal treatment (Wieringa 1968); and

treatment with other agents, such as phenol (McDanile

et al. 1962; Carmichael and Gorham 1974), detergents

(McDanile et al. 1962), sodium sulfite (Parker 1982),

sodium azide, and sodium fluoride (Melo et al. 2011).

Chlorinated water (or sodium hypochlorite) was used to

obtain axenic cultures of Nostocales strains in two studies.

Anabaena sp. was purified by immersing a small sample of

biomass in chlorinated water 0.025 g/L for 120 s (Fogg

1942), and an axenic culture of Mastigocladus laminosus

was obtained after treating akinetes with sodium hypo-

chlorite 20 g/L for 300 s (Tassigny et al. 1969).

Despite the availability of these methods, obtaining

axenic cultures of cyanobacteria remains difficult, mainly

because of the initial degree of contamination and the

variations in sensitivity to the established treatments due to

the morphological and physiological differences among

various species/genera (Sena et al. 2011). Consequently,

there is no common method for obtaining axenic cultures in

cyanobacteria.

Species of the genus Nostoc are capable of differentiating

normal vegetative cells into heterocysts (nitrogen fixing

cells), hormogonium (a motile undifferentiated trichome), or

akinetes (resting cells) (Meeks et al. 2002; Argueta et al.

2006; Flores and Herrero 2010). The latter cells are larger

than the vegetative types and have conspicuous granulation;

they are known as spore-like cells (Meeks et al. 2002).

Cyanobacteria akinetes maintain significant metabolic

activity in a different manner from that of the endospores of

gram-positive bacteria (Thiel and Wolk 1983; Adams and

Duggan 1999). Some studies have also reported that akinete

cells can survive 5–7 years of desiccation (Yamamoto 1975;

Sili et al. 1994), over a month of cold stress (4 �C), and under

conditions of darkness (Sutherland et al. 1979).

The presence of akinetes associated with a treatment

that is not sufficiently harmful to them can offer an alter-

native method to obtain axenic cultures. As a result, the

aim of this study was to design a strategy to obtain axenic

cultures of Nostoc strains by treating their akinetes with

diluted sodium hypochlorite solutions. This is a fast and

inexpensive method that does not require antibiotics and

may be applied to virtually any genera that are able to

undergo akinete differentiation.

Materials and methods

Cyanobacterial strains tested

Three Nostoc strains were tested (Nostoc sp. CCLFM I,

Nostoc sp. CCLFM VIII, and Nostoc sp. CCLFM XXI)

(Figs. 1–6). These strains were isolated from soil samples

collected at the campus of Federal University of Vicosa

(UFV), Vicosa (2084501400S, 4285205400W), Minas Gerais

State, Brazil. These strains have been deposited and

maintained at the ‘‘Colecao de Cianobacterias do Labo-

ratorio de Fisiologia de Micro-organismos’’ under the code

CCLFM.

Culture conditions

The three unicyanobacterial Nostoc strains were grown in

liquid BG-110 medium (Allen 1968). All of the strains

were inoculated in 50 mL Erlenmeyer flasks that contained

20 mL of medium. They were kept in an orbital shaker

(110 rpm) at 27 �C under white light with a photon irra-

diance of 30 lmol photon m-2 s-1, provided by fluorescent

lamps (24 h of exposure), until a massive differentiation of

akinetes was observed (Fig. 7–9) in the culture medium.

Daily microscopic analyses were carried out using an

Olympus� BX-50 microscope to follow the differentiation

of akinetes for each culture (approximately 3 weeks).

Treatment with diluted sodium hypochlorite solutions

To obtain a homogenous suspension of the akinetes, the

total volume (20 mL) containing filaments was homoge-

nized using syringe flows and sonication. From these sus-

pensions, aliquots of 0.5 mL were spread onto a Millipore

filter membrane (0.22 lm) coupled with a vacuum filter

system. The akinetes that adhered to the filter surface were

washed with 2 mL of sodium hypochlorite solution VE-

TEC� (1, 2, and 3 %) for 10, 20, and 30 s and then washed

with 5 mL of distilled sterile water. The biomass present on

the filter membrane was removed using a transfer loop and

streaked onto Petri dishes containing BG-110 solidified

medium (1.5 % w/v). These plates were incubated under

116 M. G. M. V. Vaz et al.

123

the same light and temperature conditions described above

until the cyanobacteria colonies reemerged.

Axenic culture verification

The cyanobacteria colonies grown on the BG-110 solid

medium were selected using a transfer loop and then

streaked into nutrient-broth or onto R2A-agar plates

(1.5 % w/v agar). These plates were incubated at 30 �C for

at least 7 days. The analyses for contaminant heterotrophic

growth were performed as described by Choi et al. (2008)

and Sena et al. (2011).

Results

Axenic Nostoc cultures were obtained for all of the strains

tested applying 10 s of exposure time. When 20 and 30 s

treatments were used, no cyanobacterial growth was

observed (Table 1).

Figs. 1–6 Macroscopic (strains

growing in solid medium) and

microscopic view of Nostoc sp.

CCLFM I (1 and 2); Nostoc sp.

CCLFM VIII (3 and 4) and

Nostoc sp. CCLFM XXI (5 and

6). In Figs. 1, 3 and 5, scale bars

= 1 cm. In Figs. 2, 4 and 6, scale

bars = 10 lm

Fig. 7–9 Cultures of Nostoc strain CCLFM XXI for akinete enrich-

ment. 7 Culture after 7 days of cultivation, showing the prevalence of

vegetative trichomes; 8 Culture after 14 days of cultivation, with

some sparse akinetes; and 9 Culture after 21 days of cultivation,

showing a large amount of akinetes. Scale bars = 10 lm

Axenic cultures of Nostoc strains 117

123

For Nostoc sp. CCLFM I and Nostoc sp. CCLFM XXI,

axenic colonies were obtained using the 1 and 3 % of

sodium hypochlorite solutions, respectively. Pure colonies

of Nostoc sp. CCLFM VIII were produced using the 2 and

3 % sodium hypochlorite concentrations (Table 1).

Using 1 % sodium hypochlorite, axenic cultures were

obtained for only the strain Nostoc sp. CCLFM I. For the other

Nostoc strains, the colonies that emerged after treatment with

1 % SH showed some degree of heterotrophic contamination.

Three phases of akinete differentiation are shown for the

Nostoc sp. CCLFM XXI (Figs. 7–9). In addition, micrographs

of this strain, before and after the treatment with 3 % of

sodium hypochlorite, per 10 s, demonstrate the efficiency of

the proposed method (Figs. 10–12).

Discussion

The production of axenic cultures in Nostoc sp. CCLFM I

using the lowest sodium hypochlorite concentration can be

explained by the morphological characteristics of this

strain (Figs. 1–6). It exhibits trichomes with a low self-

aggregation pattern. The low self-aggregation pattern

shown by this strain can be observed during the earlier

phases of growth (vegetative cells and trichomes) and, also,

in the aged phases. In the aged cultures, this pattern is

intensified, since the akinetes chains are more suitable to

breakage and, consequently, almost all of the trichomes

(with akinetes chains) or the isolated akinetes were reached

using hypochlorite solutions. Once the aggregation has

been eliminated (or reduced), the adhesion surface for

heterotrophic contaminants (Hoiczyk and Baumeister

1995) is equally accessible to sodium hypochlorite action.

Additionally, the akinetes of the strains evaluated in this

study seem to show differential resistance to sodium

hypochlorite. Therefore, the use of highly concentrated

sodium hypochlorite solutions (2 and 3 %) seemed to be

harmful for both contaminants and the Nostoc trichomes, as

no colony growth was observed following these treatments.

Conversely, the Nostoc strains CCLFM VIII and

CCLFM XXI presented colonies composed of trichomes

that overlapped (Figs. 1–6). As a result, even after dis-

ruption using syringe flows and sonication, the highly

aggregated trichomes/colonies decreased the efficiency of

sodium hypochlorite. This observation reinforces the need

for highly concentrated sodium hypochlorite solutions (2

and 3 %) to achieve an axenic state (Figs. 10–12). The

non-growth of the Nostoc strains after the treatments con-

ducted with the higher exposure times indicates that the

akinetes are sensitive when the exposure time exceed cer-

tain thresholds. In the case of our strains, this threshold was

10 s, since all the axenic cultures were obtained only when

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118 M. G. M. V. Vaz et al.

123

The sodium hypochlorite concentration of 1 % SH with

a contact time of 10 s showed to be sufficient to obtain

axenic cultures of Nostoc sp CCLFM I. These concentra-

tion and time were, respectively, 2 and 30 times smaller

than those reported by Tassigny et al. (1969). On the other

hand, despite using a sodium hypochlorite solution more

concentrated than that applied by Fogg (1942), in this

work, the contact time was 12 times shorter. The effec-

tiveness of this method was clearly demonstrated, consid-

ering high rate of success in the different treatments and

also the low time consumption.

The efficiency of using sodium hypochlorite as a dis-

infecting agent to obtain axenic cultures is associated with

the morphology of the colonies and the cell resistance

(presence or absence of akinetes), both of which are highly

variable during the various stages of the life cycle (Flores

and Herrero 2010). The akinetes of Nostoc strains, in

nearly all cases, were resistant to the treatments used,

whereas their vegetative cells did not support this type of

treatment (data not shown).

This strategy presents many advantages compared to

strategies based on antibiotics, which generally depend on

two or more antimicrobial agents (Ferris and Hirsch 1991;

Choi et al. 2008; Vasquez-Martınez et al. 2004), or other

approaches that rely on several steps, including mechanical

separation, centrifugation, and micromanipulation. No

antibiotics were used in our strategy, and only two steps are

needed to obtain axenic cultures. Furthermore, no expen-

sive chemical reagents are required, and the time con-

sumption is minimal. Consequently, using diluted sodium

hypochlorite, solutions can offer a fast and inexpensive

strategy to obtain axenic cultures from akinetes of cyano-

bacteria, such as Nostoc species.

Acknowledgments This study was funded by grants from The

Brazilian National Research Council (CNPq) and M.G.M.V.Vaz was

supported by CNPq scholarship (135154/2008-1) and by State of Sao

Paulo Research Foundation (FAPESP) graduate scholarship (2010/

18732-0). The authors would like to thank Dr. Rosane Aguiar (in

memorian) for her great contribution on cyanobacterial studies at

Federal University of Vicosa. M.G.M.V.Vaz would like to thank Msc.

Diego Bonaldo Genuario for his constructive and helpful comments

on the manuscript.

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