Parasitol Res (2009) 104:935–937 DOI 10.1007/s00436-008-1304-x
SHORT COMMUNICATION
A simple method for long-term storage of Acanthamoeba species Diana Axelsson-Olsson & Jenny Olofsson & Patrik Ellström & Jonas Waldenström & Björn Olsen
Received: 3 September 2008 / Accepted: 1 December 2008 / Published online: 17 December 2008 # Springer-Verlag 2008
Abstract We present a novel and simple technique for storing live Acanthamoeba for long periods of time. The amoebae are maintained at refrigerator temperatures in a peptone–yeast extract–glucose (PYG) medium normally used for cultivation. Using this method, we obtained survival rates of at least 4 years for Acanthamoeba polyphaga and 3 years for Acanthamoeba castellanii and Acanthamoeba rhysodes. Advantages of this storage method are: (1) it is quick and simple, (2) inexpensive, (3) does not require encystment before storage, (4) resuscitation of cysts can be achieved within a week of culture in PYG medium at 27°C, and does not require co-culture with bacteria or any special equipment.
Introduction Acanthamoeba are abundant protozoa in freshwater and soil, and are also common in laboratories as model species in protistology and in interaction studies with pathogenic bacteria (Axelsson-Olsson et al. 2005; Greub and Raoult 2004; Khan 2006; Rowbotham 1983). Due to their ability to convert from the trophozoite stage into a more resistant cyst, they are refractory to environmental changes such as dehydration and starvation. Their cysts can withstand harsh D. Axelsson-Olsson (*) : J. Olofsson : P. Ellström : J. Waldenström : B. Olsen Section for Zoonotic Ecology and Epidemiology, University of Kalmar, 391 82 Kalmar, Sweden e-mail:
[email protected] J. Olofsson : B. Olsen Section of Infectious Diseases, Department of Medical Sciences, Uppsala University Hospital, Uppsala, Sweden
conditions such as chlorination, antimicrobials, and disinfectants (Borazjani et al. 2000; Kilvington 1990; Kilvington and Price 1990) as well as extremes in temperatures, pH, and osmolarity (Ahearn and Gabriel 1997). In all environments where protozoa occur, there will also be bacteria present. Interactions between bacteria and protozoa are usually in the form of predation, where protozoa utilize bacteria as food, but may take other forms as either symbiosis or endoparasitism. In the latter case, bacteria infect and multiply within the protozoa which may cause the death of the infected cell. However, for certain bacteria and protozoa, the intracellular bacterial cells are maintained, at least transiently, without causing large harm to the protozoan host (reviewed in Greub and Raoult 2004). This has been utilized in Acanthamoeba to develop co-culture methods for cultivation of fastidious bacteria such as Campylobacter spp. (Axelsson-Olsson et al. 2007). Acanthamoeba are fast-growing, easy to handle experimentally, and inexpensive. However, to preserve laboratory strains, it is important with safe and easy methods for longand short-term storage. There are a number of protocols available, with varying demands of labor before and after storage. The methods also differ in the maximum storage time after which it is still possible to recover the cysts. It has been shown that Acanthamoeba can stay viable for 8– 24 months on agar slopes (Biddick et al. 1984) and up to 5 years on clay pellets (Lorenzo-Morales and Maciver 2006). However, both these methods include laborious steps before storage as well as co-culture with bacteria to recover the cysts after storage. Low temperature freezing and cryo-preservation (Menrath et al. 1995; John et al. 1994) can also be effective but demand prolonged cultivation periods post-freezing. In this study, we report a simple and inexpensive long-term storage method that does not require any special treatments of
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cells before or after storage. We show that refrigerating of axenic Acanthamoeba in peptone–yeast extract–glucose (PYG) medium maintain viable cysts for at least 3 years for Acanthamoeba castellanii and Acanthamoeba rhysodes, and for at least 4 years for Acanthamoeba polyphaga. This storage method would be available for any laboratory that works with Acanthamoeba.
Materials and methods A. polyphaga was provided by Bernard La Scola, Université de la Méditerranée, Marseille, France, and A. castellanii and A. rhysodes by Jadwiga Winiecka-Krusnell, Swedish Institute for Infectious Disease Control (SMI), Sweden. All Acanthamoeba strains were initially seeded in PYG medium (Rowbotham 1983, with later modifications of Greub and Raoult 2002) in 25-ml cell culture flasks and let to grow aerobically at 27°C until they formed a confluent layer at the bottom of the flask. From these cultures, samples for storage were obtained by detaching amoebae through a gentle freezing method: first, the medium was replaced with 2 ml of fresh PYG medium. The flasks were then put in a freezer at −20°C for 10 min to allow trophozoites to detach. After this treatment, a 1-ml sample was taken from each culture and put individually in 2-ml screw cap tubes and stored at 4– 8°C in a normal refrigerator. This procedure was repeated between three and four times, approximately 1 year apart, using the same original strains. As a result, we had a collection of samples that had been stored between 1 and 4 years at refrigerator temperatures: in total, seven tubes of A. polyphaga (1 to 4 years old) and six each of A. castellanii and A. rhysodes (1 to 3 years old). The viability of stored cells were assessed by dispensing 10 μl of the cultures in 8 ml of fresh PYG medium in new 25-ml cell culture flasks and growing them aerobically at 27°C. The excystment of the amoebae varied from 24 h to 5 days, and the rational for scoring cultures as positives (i.e., viable) was based on the presence of trophozoites as determined by microscopy. Additionally, the oldest tubes, 4 years for A. polyphaga and 3 years for A. castellanii and A. rhysodes, were cultured and observed once a week during 12 weeks for the presence of viable amoebae cells. We also investigated changes in amoebae morphology of cells stored at 4°C by letting trophozoites of A. polyphaga, A. castellanii, and A. rhysodes grow in 12-well plates at 27°C until the cultures reached confluent layers. The plates were subsequently incubated at 4°C and were observed under the microscope for morphological changes at weekly intervals during 8 weeks. Observations were performed with a Leitz dialux 20 EB phase contrast microscope at a
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magnification of ×100 and an inverted Leica Microsystems light microscope at a magnification of ×40.
Results and discussion The survival of the Acanthamoeba strains was very good using this simple method of long-term storing. All three samples of A. polyphaga that had been refrigerated for 4 years gave viable trophozoites when cultured in fresh PYG medium, and similarly all three 3-year-old samples of both A. castellanii and A. rhysodes were viable (Table 1). Actually, only one out of 19 tubes of Acanthamoeba stored for more than 1 year failed to grow when cultured, this being a 2-year-old sample of A. polyphaga. Although this equals a failure rate of only 5%, it would be recommendable to store at least duplicate samples of precious Acanthamoebae strains. Moreover, when followed for 12 weeks, the oldest tube of each of the three different species gave positive cultures every week for all species, except one sample of A. castellanii at week 5. However, from this tube, we obtained a positive culture at week 6 and throughout the rest of the experiment. One reason for the negative sample might be that with a small sample volume (10 μl) it is easy to overlook the amoebae if the tube is not thoroughly stirred before sampling. In all positive samples, viable trophozoites appeared within 1 to 5 days and readily grew to a confluent layer in the flasks. In the last experiment, we followed the encystment process by checking the morphology of aging cells stored at refrigerator temperatures. For the first 3 weeks, there were no differences between the amoebae species and they all maintained a confluent layer of trophozoites at the bottom of the wells. A. rhysodes cells detached at week 3 and began to form cysts at week 6, while A. castellanii where adherent until week 6 and then started to detach, but did not encyst. Finally, A. polyphaga grew adherent to the bottom of the wells throughout the 8-week study period. From the older cultures, we have observed that A. castellanii and A. rhysodes were completely encysted after 1 year, whereas A. polyphaga were still in a trophozoite-like form after 1 year.
Table 1 Acanthamoeba survival in PYG medium at 4°C Storage time (years)
1 2 3 4
Survival (# viable samples/# tested samples) A. polyphaga
A. castellanii
A. rhysodes
2/2 0/1 1/1 3/3
2/2 1/1 3/3
2/2 1/1 3/3
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This indicates that the encystment process can be very slow in PYG medium for this species. The long-term storage of Acanthamoeba species presented here is an easy, time-saving, and cost-beneficial method available for any laboratory and has been proven efficient for axenic cultures from three species of Acanthamoeba. In protistology Acanthamoeba cultures are used for a number of different purposes, including studies on host bacterial interactions (Axelsson-Olsson et al. 2005), and strains in a given laboratory will vary in pathogenicity as well as in other factors. Some isolates of Acanthamoeba are more difficult to get axenic than others, but usually bacterized cultures can be turned axenic using medium supplemented with antibiotics (Schuster 2002). Depending on the scientific question posed, the issue of xenic or axenic cultures will be of varying importance and it remains to be seen how well xenic strains can be maintained by our storage method. One potential benefit with our system is that bacterial coculture is not necessary to recover the cysts, and hence stored axenic cultures will remain axenic also after storage. The method allows for easy back-up storage as well as fast recovery, making prolonged passaging of viable cultures unnecessary. Acknowledgements This work was supported financially by the Royal Swedish Academy of Agriculture and Forestry (KSLA-H482), the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS 221-2007-438), and Sparbankstiftelsen Kronan. All experiments comply with the national guidelines.
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