J. Microbiol. Biotechnol. (2013), 23(3), 289–296 http://dx.doi.org/10.4014/jmb.1203.03057 First published online November 24, 2012 pISSN 1017-7825 eISSN 1738-8872 Improved Methodology for Identification of Cryptomonads: Combining Light Microscopy and PCR Amplification Xia, Shuang1,2, Yingyin Cheng3, Huan Zhu1,2, Guoxiang Liu1*, and Zhengyu Hu1 1Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China 2Graduate School of Chinese Academy of Sciences, Beijing 100039, China 3Center for Water Environment and Human Health, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China Received: March 26, 2012 / Revised: August 6, 2012 / Accepted: October 16, 2012 Cryptomonads are unicellular, biflagellate algae. Generally, Key words: Cryptomonad, fixative, glutaraldehyde, Lugol’s cryptomonad cells cannot be preserved well because of solution, morphology, PCR amplification their fragile nature, and an improved methodology should be developed to identify cryptomonads from natural habitats. In this study, we tried using several cytological Cryptomonads are unicellular, biflagellate algae, which are fixatives, including glutaraldehyde, formaldehyde, and cosmopolitan in distribution, living as important primary their combinations to preserve field samples collected producers in both freshwater and marine habitats [4, 15, from various waters, and the currently used fixative, 25, 26, 32, 37]. They often assume dominant phytoplankton Lugol’s solution was tested for comparison. Results status in various waters [27], and sometimes even form showed that among the fixatives tested, glutaraldehyde nuisance blooms [1, 9, 34]. However, the cell density of preserved the samples best, and the optimal concentration cryptomonads was low in preserved samples and their of glutaraldehyde was 2%. The cell morphology was well importance was often neglected. Cryptomonads cells cannot preserved by glutaraldehyde. Cells kept their original color, be preserved well since their cells either rupture or distort volume, and shape, and important taxonomic features drastically when fixatives such as formaldehyde are added such as furrow/gullet complex, ejectosomes, as well as [25]. This is probably because their cell shape is maintained flagella could be observed clearly, whereas these organelles by a delicate surface termed periplast, which is probably frequently disappeared in Lugol’s solution preserved proteinaceous [5]. samples. The osmotic adjustments and buffers tested could Ever since the 1980s, the taxonomy of Cryptophyta has not preserve cell density significantly higher. Statistical been drastically changing, with new genera set up and old calculation showed the cell density in the samples preserved ones eliminated [7, 18, 39]. Specifically, when phylogenetic by 2% glutaraldehyde remained stable after 43 days of analyses based on DNA sequences were incorporated into the fixation procedure. In addition, DNA was extracted taxonomic studies, new hypotheses on the taxonomy of from glutaraldehyde preserved samples by grinding with Cryptophyta were raised [6, 11, 19-21]. However, those liquid nitrogen and the 18S rDNA sequence was amplified works were focused on a relatively small group collected by PCR. The sequence was virtually identical to the and cultured in a few western counties. There are numerous reference sequence, and phylogenetic analyses showed unrecognized genera and species to be described in the very close relationship between it and sequences from the natural habitats [2, 35]. same organism. To sum up, the present study demonstrated At the present time, Lugol’s solution is routinely used to that 2% unbuffered glutaraldehyde, without osmotic preserve cryptomonads samples from natural habitats adjustments, can preserve cryptomonads cells for identification, because it provides the least distortion of cell shape [27]. in terms of both light microscopy and phylogenetic analyses However, Lugol’s solution has two obvious disadvantages based on DNA sequences. in the morphological study. For one thing, it colors cells purple because of the iodine in it, but cell color is a main *Corresponding author Phone: +86-027-68780576; Fax: +86-027-68780123; generic feature of cryptomonads; for another, some important E-mail: [email protected] morphological features of cryptomonads, such as ejectosomes 290 Xia et al. and flagella, frequently disappear in samples fixed with The osmotic adjustments of sucrose and sorbitol as well as Lugol’s solution. More recently, PCR were successfully the buffer of PBS and Hepes were tested. Different performed on protists and microalgae from plankton samples methods of DNA extraction and PCR amplification were preserved in Lugol’s solution, but a complicated washing performed on preserved specimens. Phylogenetic analyses step with thiosulfate solution was needed to overcome were performed. Results of the present study clearly showed PCR inhibition caused by iodine, and there was no that glutaraldehyde without any buffer or osmotic adjustment information about DNA damage [3]. could preserve the morphology of cryptomonads well for In addition, experimenting on live cells collected from light microscopy at the final concentration of 2%, and cell natural habitats is often impractical, because the change of lysis due to fixation was negligible after 43 days of the temperature during transportation can distort or even fixation procedure. For the first time, DNA extraction disrupt the cells [27]. Thus, an improved fixation procedure and PCR amplification were successfully performed on proper for the identification of cryptomonads should be cryptomonads samples preserved with glutaraldehyde. developed. Formaldehyde is a conventional fixative for phytoplankton [44], whereas glutaraldehyde has been used widely as a MATERIALS AND METHODS fixative in electronic microscopy. More recently, glutaraldehyde was also utilized to preserve plankton Samples of seven cryptomonad species were used in the present samples for light microscopy because it was considered study. Samples were collected by phytoplankton nets from various to be able to preserve delicate cell structures [31, 48]. kinds of water bodies in Hubei Province of China from January 2009 to July 2012. Samples of Campylomonas reflexa were collected However, there was no record about fixation of cryptomonads o o from Lake Nanhu (30 29'46''N, 114 20'12''E) in the city of Wuhan. with glutaraldehyde for light microscopy and DNA Cryptomonas tetrapyrenoidifera was collected from a small pond extraction from algae preserved with glutaraldehyde up till (30o29'09''N, 114o23'14''E) in a campus, where it had become a now. Besides, the combination of glutaraldehyde and dominant species. Cryptomonas pyrenoidifera was collected from a formaldehyde has been tried to preserve several algal eutrophic fishpond (30o31'47''N, 114o21'16''E), where it bloomed. samples, including Raphidophytes, and positive results Cryptomonas sp., Komma caudata, and Chroomonas sp. were were recorded [12, 24]. Thus, it may be practical to use collected from Lake Donghu (30o32'55''N, 114o21'16''E). Plagioselmis glutaraldehyde or the combination of glutaraldehyde and sp. was collected from the reservoir of the Three Gorges Dam o o formaldehyde to preserve cryptomonad samples. (31 07'21''N, 110 47'01''E), where it bloomed and the water presented Moreover, buffer and osmotic adjustment are two a red-brown color. important factors that affect the preservation of cell Glutaraldehyde (25~28% solution, BR grade) and formaldehyde (37~40% solution, AR grade) was purchased (Sinopharm Chemical morphology during the fixation procedure [24]. PBS o Reageal Company, China) and stored at 4 C in a refrigerator. (phosphate-buffered saline) is a buffer solution commonly Lugol’s solution was prepared as follows: 5 g iodine (I2) and 10 g used in biological research. Hepes [N-(2-hydroxyethyl)- potassium iodide (KI) were added to 85 ml of distilled water. Then piperazine-N-(2-ethanesulfonic acid)] has been successfully the mixture was stirred with a magnetic bar for dissolution. PBS was used as a buffer for the fixation of algae [28]. Sorbitol and prepared as follows: 8 g sodium chloride (NaCl), 0.2 g potassium sucrose have been used to avoid breakage induced by chloride (KCl), 1.44 g disodium hydrogen phosphate (Na2HPO4), fixation, and sucrose has been applied to the fixation of and 0.24 g potassium dihydrogen phosphate (KH2PO4) were added algae [24]. to 800 ml of distilled water, and the pH was adjusted to 7.2 with In the present study, in order to develop a simple and hydrochloric acid (HCl). convenient procedure for the identification of cryptomonads, At first, samples of Campylomonas reflexa and Cryptomonas pyrenoidifera various fixatives, including glutaraldehyde, formaldehyde, were preserved using the combinations of glutaraldehyde and the combinations of them with different concentration and formaldehyde with different concentration ratios at the final concentration of 1% and 2%, respectively (Experiment A). The final ratios were tested to preserve field samples. The optimal concentrations of the combined fixatives tested are shown in Table 1. concentration of the fixative was found by gradient tests. The fixatives were prepared just before use. After 24 h of fixation, The currently used fixative, Lugol’s solution, was tested cells were observed under light microscopy. To evaluate the effects for comparison both qualitatively and quantitatively. Fixed of the fixation on cell