CRYOPRESERVATION OF BIFIDOBACTERIA AND BACTERIOPHAGES IN BELARUSIAN COLLECTION OF NON-PATHOGENIC MICROORGANISMS

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1 JOURNAL OF CULTURE COLLECTIONS Volume 6, , pp CRYOPRESERVATION OF BIFIDOBACTERIA AND BACTERIOPHAGES IN BELARUSIAN COLLECTION OF NON-PATHOGENIC MICROORGANISMS Galina Novik*, Anastasia Sidarenka, Denis Rakhuba and Emilya Kolomiets Institute of Microbiology, National Academy of Sciences, Minsk, Kuprevich str., 2, Belarus *Corresponding author, Summary The paper deals with cryopreservation of bifidobacteria and bacteriophages. Effect of high rates of cooling on survival ability and physiological activity of bifidobacteria is evaluated. The possibility to use rapid freezing for cryopreservation of stock Pseudomonas phage cultures is examined. The study indicates that rapid cooling kinetics appeared to lead to minimal losses in viability and acidification activity of bifidobacteria. Media traditionally used for bifidobacteria cultivation proved to have good cryoprotective properties and could be effective freezing media for preservation of these organisms. The study reveals that cryopreservation with high rates of freezing ensures high survival and maintenance of lytic ability of Pseudomonas bacteriophages. 10 % glycerol and 5 % DMSO showed good results for preservation of this group of phages. Key words: collection, cryopreservation, bifidobacteria, bacteriophages. Introduction 76 The main aim of any collection of microorganisms is maintenance of stock cultures in viable state with stable characteristics. Belarusian Collection of Non-pathogenic Microorganisms was entered into the State Register of scientific objects estimated as National Asset. In 2007 Collection was registered in World Data Centre for Microorganisms (WDCM) as BIM WDCM909. Nowadays, Collection guarantees maintenance of viability of more than 1100 microorganisms, including bacteria, bacteriophages, filamentous and yeast-like fungi. Depending on the aims and genus or species affiliation of microorganisms we use different methods of preservation: subcultivation, storage under mineral oil, freezing at -20 o C, freeze-drying, etc. Cryopreservation of microorganisms in liquid nitrogen at -196 o C is considered superior to other preservation methods. Proper cryopreservation allows for the generation and maintenance of bacterial stocks and decreases the need for repeated subculturing, which can lead to contamination, genetic drift or mutation as each next time smaller portions of population are selected. Low-temperature storage significantly reduces phenotypic and genotypic drifts. Microorganisms preserved in liquid nitrogen usually show high survival rates and good strain stability. Moreover, cryopreservation is a simple method, relatively cheap and not time-consuming [8, 9, 15]. Several factors are critical for successful cryopreservation. Freezing kinetics, storage and recovery conditions, composition of growth and cryoprotective media, cell age, population size, type of cryoprotector used all affect viability and stability of bacterial cultures [8, 9].

2 The objectives of this study were to determine the effect of rapid freezing kinetics (direct immersion in liquid nitrogen), type of cryoprotective additive and composition of cryoprotective media on viability of Bifidobacteria and bacteriophages during cryopreservation. Bifidobacteria cryopreservation. The strains of bifidobacteria Bifidobacterium adolescentis 94 BIM, B. adolescentis GO-13 and B. longum V379M deposited in the stock culture collection of the Institute of Microbiology, NASB (Belarusian Collection of Non-pathogenic Microorganisms) were used in this study. Bacteria were maintained by routine subculturing in de Man, Rogosa and Sharpe medium, supplemented with 0.05% L-cysteine (MRS-C). Prior to experiments bifidobacteria were twice subcultured in fresh MRS-C broth. For bacteria concentrates preparation, cultures were grown anaerobically at 37 o C in liquid MRS-C, tryptone-lactose (TLM), Bifidobacterium, supplemented with 0.5 % yeast extract (BMY) and thioglycolic (TGM) media for h. Cells were harvested by centrifugation (6,000xg, 10 min) and pellets were suspended in half-volume of sterile pre-cooled (+4 o C) cryoprotective medium. Fresh growth media with or without protective additives were used as cryoprotective. Sterile solutions of sucrose and lactose (final concentration 10 % w/v) as well as Tween-80 (1 %) were used as cryoprotectants. The cells were allowed to equilibrate for min at +4 o C, divided into aliquots, aseptically placed in sterile cryogenic vials and frozen by direct immersion in liquid nitrogen. Determination of viability, morphological properties and physiological activity was performed on 0, 1, 14 days and 6 months after freezing. At designated time the vials were removed from the liquid nitrogen and placed in 37 o C water bath to thaw. Viability of bifidobacteria was expressed via viable cell counts (CFU/ml) and survival rate (%). Viable bacteria counts (CFU/ml) were performed from serial dilutions, inoculated into semi-liquid (0.1% agar) TGM (ph 7.2). Bacterial counts were calculated after h anaerobic incubation at 37 o C. Survival rate (%) was determined as (ln viable cells after freezing / ln viable cells before freezing) x 100%. Physiological activity was evaluated via growth rate and acidification activity in nutrient media (MRS-C, TGM). Growth in milk was estimated by time of coagulation and ph of milk. Growth rate was measured as optical density of 24-h culture at λ=600 nm (OD 600 ) using spectrophotometer KFK-3 (Russia). ph was determined using ph-tester Piccolo plus Materials and Methods ( HANNA Instruments, Belarus). Cell morphology was examined by light microscopy, using microscope LOMO ( Biolam, Russia), phase-contrast microscopy, using microscope Nicon Eclipse E200 ( Nicon Corporation, Japan), and scanning electron microscopy, using JEM-100CX-II ( ). Each test was carried out in duplicate. Statistical analysis was performed using Statistics 6.0 software package. Statistical significance was set at P<0.05. Cryopreservation of bacteriophages. The following strains of phages were used in the study: BIM BV-1, BIM BV-6, BIM BV-12, BIM BV-16, BIM BV-22, and a preparation Pentaphage, which represents a mixture of indicated bacteriophages. Pseudomonas fluorescens BIM B-86 was used as a host strain. Phages were propagated in sprat broth overnight. 1/5 V of the night culture was inoculated in fresh medium and incubated for 2 h at 30 o С. Then bacteria in exponential phase of growth were infected with phage suspension at multiplicity of 0.1. The resulting lysate was treated with chloroform at a rate 1:10 and freed of cell debris by centrifugation at 8000 rev./min. For each phage to be frozen, three 1-ml samples were prepared: without cryoprotective additive, with 10 % glycerol and 5 % dimethylsulphoxid (DMSO). Samples were dispensed into 1.8-ml cryovials and cooled to 0 o С in ice bath. Then vials with phages were plunged directly into liquid nitrogen. After 24 h samples were thawed in water bath at 37 o С. Each sample was titered before and after freezing by standard agar-layer method. To assay the lytic ability of Pentaphage, the mixture of five bacteriophages was frozen in liquid nitrogen and re-warmed after 24 h of storage as described above. 1-ml of thawed Pentaphage was added to 50 ml of exponential phase host culture and incubated at 27 o С on the shaker. At 5 min intervals small samples of culture broth were taken and the optical density was measured at λ=590 nm. Lytic ability of Pentaphage was determined by the change of optical density of bacterial culture. Unfrozen phage mixture was used as a control. Phage morphology was studied using electron microscopy. For this purpose thawed lysates of bacteriophages were exposed to differential centrifugation (8000 rev./min for 20 min 77

3 followed by 90 min at rev./min). Pellets were suspended in phosphate buffer and centrifuged at 8000 rev./min. Resulting phage suspensions were placed on cooper grids covered with collodion film and stained with 2 % uranyl acetate. Specimens were examined, using JEM-100CX-II electron microscope with magnifications Results and Discussion Bifidobacteria cryopreservation Freezing is a particularly critical step during cryopreservation of microorganisms because it can have a negative impact on cell survival and restoration of activity after thawing [4]. Rapid cooling minimizes the solute concentration effects as ice forms uniformly, but leads to more intracellular ice. Slow cooling, on the other hand, results in a greater loss of water from the cell and less internal ice, but increases the solution effects [11]. Mazur et al have postulated that ice crystal formation and solution effects both play a role in cell inactivation, and that an optimum cooling rate minimizes the effect of each [10]. A recent study demonstrated that rapid freezing rates obtained by direct immersion in liquid nitrogen lead to minimal losses in viability and acidification activity of commercial strains Lactobacillus delbrueckii subsp. bulgaricus [5]. Another research showed that high rates of cooling improve cryotolerance of Streptococcus cremoris and give maximal viable counts of this microorganism [18]. Starting from these data, the effect of rapid freezing kinetics on the viability, morphological and physiological properties of bifidobacteria was evaluated. Three model strains B. adolescentis 94 BIM, B. adolescentis GO-13 and B. longum V379M were used in the study. It was established that high rates of cooling provide good (80-99 %) survival ability, stability of morphological and physiological characteristics of bifidobacteria. There were no significant differences (p>0.05) in growth rate and acidification activity of cultures before and after freezing. No morphological changes (shape, size, arrangement) resulting from the cell injury during cooling were observed. Morphologically, bifidobacteria were Grampositive straight or bifid-shape rods, often assuming V or Y patterns (Fig. 1). a b c Fig. 1. Electron microphotography of B. adolescentis 94 BIM before (a) and 6 month after cryopreservation (b, c) (1x8000). Using cryoprotective additives or chemicals that protect the cells during freezing can minimize the detrimental effects of increased solute concentration and ice crystal formation. Many compounds have been tested as cryoprotective agents, either alone or in combination, including sugars, serum and solvents. The choice of a cryoprotective agent is dependent on the type of cell to be preserved. Sucrose at a concentration of 5 % and lactose at concentrations 1-10 % were reported to protect concentrated starter cultures of Lactococcus lactis ssp. lactis better than 10 % glycerol [6]. 1 % Tween-80 significantly increased survival ability of Escherichia coli during cryopreservation at high rates of cooling [6]. Large bonus of these cryoprotectants is their non-toxicity, which is of great importance for industrial strains preservation. Therefore, effect of these protective additives on the viability of B. adolescentis 94 BIM, B. adolescentis GO-13 and B. longum V379M was studied. Table 1 summarizes the data concerning protective effect of tested cryoprotectants on microbial survival, evaluated by enumeration of CFU and measurement of acidification activity. No significant differences (p>0.05) in viability and acidification activity of bifidobacteria after freezing with or without cryoprotective agents were observed. These results correspond to data [12], which showed that concentrated cultures of B. adolescentis MS-42 have good survival ability during free- 78

4 zing at rapid speeds even without cryoprotective additives. This phenomenon may be explained by high cryotolerance of Bifidobacterium spp. due to some morphological or functional properties of these organisms. On the other hand, in our study we use fresh growth medium with or without cryoprotectants for bifidobacteria freezing. Bifidobacteria are fastidious microorganisms and require complex media for their cultivation. These bacteria are usually grown in rich media containing glucose, lactose, peptone, meat extract, free amino acids (cysteine, glycine and tryptophan), several vitamins, etc. The literature data suggest that most of these compounds have good cryoprotective properties, which may explain the high protective effect of growth media during freezing even without additional cryoprotectants. In our study, the possibility for cryopreservation of bifidobacteria in media, traditionally used for cultivation of these microorganisms, without any cryoprotectants was established. Table 1 Viability* of bifidobacteria after freezing in presence of tested cryoprotectants. Strain/ freezing medium before freezing 1 day after freezing 14 days after freezing 6 months after freezing CFU/ ml % CFU/ ml % CFU/ ml % CFU/ ml % MRS-C 8.0 х х х х B. adolescentis 94 BIM MRS-C sucrose 8.0 х х х х MRS-C lactose 8.0 х х х х MRS-C Tween х х х х TGM 8.0 х х х х TGM sucrose 8.0 х х х х TGM lactose 8.0 х х х х TGM Tween х х х х MRS-C 3.0 х х х х B. adolescentis GO-13 MRS-C sucrose 3.0 х х х х MRS-C lactose 3.0 х х х х MRS-C Tween х х х х TGM 2.2 х х х х TGM sucrose 2.2 х х х х TGM lactose 2.2 х х х х TGM Tween х х х х MRS-C 1.4 х х х х MRS-C sucrose 1.4 х х х х B. longum V379М MRS-C lactose 1.4 х х х х MRS-C Tween х х х х TGM 1.2 х х х х TGM sucrose 1.2 х х х х TGM lactose 1.2 х х х х TGM Tween х х х х *Survival rate (%) was determined as (ln viable cells after freezing/ln viable cells before freezing) x 100%. 79

5 It was noted that survival ability of bifidobacteria grown and frozen in MRS-C was higher than in TGM ( % and %, respectively). Therefore, effect of composition of freezing medium on viability and stability of physiological activity of bifidobacteria was examined. B. adolescentis 94 BIM was used as a model in this study. Four different media for bifidobacteria cultivation, namely MRS-C, TGM, TLM and BMY, were tested. The results obtained are summarized in Tables 2, 3. Correlation between medium used for freezing and viable cell counts after cryopreservation (p<0.05) was established. Thus, the least decline in CFU number was recorded using TLM. Growth activity in milk was also the highest after cryopreservation in this medium (there were no differences between time of coagulation and ph before and after freezing). Small decline in viable counts was observed after freezing in MRS-C and BMY, but activity in milk was slightly reduced as compared to culture before freezing. The greatest decrease in viability and physiological activity was registered when TGM was used for cryopreservation. The data suggest that the effect of media was related to their chemical composition. Thus, TLM, MRS-C and BMY, which contain many compounds with cryoprotective properties, such as lactose, glucose, peptone, trypton, yeast extract, Tween- 80, etc., appeared to have good cryoprotective activity even without addition of any protective additives. At the same time, TGM was less effective, since it comprises less cryoprotective compounds. In our study TLM proved to be the optimal media for bifidobacteria cryopreservation using high rates of cooling. Table 2. Survival rate and physiological activity of B. adolescentis 94 BIM after freezing in different media. Medium before freezing after freezing 1 day 14 days 6 months % рн OD 600 % рн OD 600 % рн OD 600 % рн OD 600 MRS-C TGM TLM BMY Table 3. Growth activity of B. adolescentis 94 BIM in milk after freezing in different media. Medium before freezing 1 day after freezing 14 days after freezing time, h рн time, h рн time, h рн MRS-C TGM TLM BMY The analysis of literature suggests that for successful cryopreservation of different species and strains of bacteriophages selection of optimum modes of freezing is essential. In some cases stabilization of phage particles with cryoprotectors is also required [2, 20]. Research carried out at the Institute of Cryobiology and Cryomedicine (Kharkov, Ukraine) demonstrated that for maintenance of E. coli phages (T3, T4 and ФХ174) cryopreservation Cryopreservation of bacteriophages 80 with narrow intervals of cooling rate was suitable [19]. Experiments performed at American Type Culture Collection revealed that cooling and re-warming rates were not critical for cryopreservation of E. coli T2 bacteriophage [7]. For the majority of phages as well as microorganisms of other taxonomic groups freezing at rates ºС/min proved to be optimal. Cryoprotectants are widely used to protect microorganisms during freezing. Protective action of

6 DMSO at a concentration of 5 % on phage T4 was established [19]. Cryoprotective effect of glycerol at a concentration of 10 % was shown for many phages [3, 6]. Starting from these data, we examined the effect of high cooling rates ( o С/min) and the presence of 5 % DMSO and 10 % glycerol in freezing media on viability and morphology of bacteriophages from our collection. The results on survival ability of bacteriophages during freezing and subsequent 24 h storage in liquid nitrogen are presented in Table 4. The analysis of results revealed that phages of P. fluorescens possess marked cryosensitivity. Despite the fact, that lysate contained various substances (carbohydrates, peptides, etc.), which could provide protective effect at freezing, 100 % survival rate during cryopreservation in lysate without cryoprotective additives was observed only for one strain BIM BV-12. In all other cases single-order decrease of the titer of phages was recorded. On the other hand, bacteriophages survived well during freezing in the presence of cryoprotectants: 10 % glycerol and 5 % DMSO appeared to be suitable protective agents during phage cryopreservation. It was established that DMSO provides prtective effect toward three strains BIM BV-1, BIM BV-6, BIM BV-22. At the same time, glycerol possessed good protective activity during freezing of BIM BV-1, BIM BV-6, BIM BV-22, but demonstrated marked toxic action on BIM BV-12. After cryopreservation in the presence of glycerol the titer of this strain was two orders of magnitude lower, than after freezing without addition of any cryoprotectant. Table 4. Survival of P. fluorescens bacteriophages after freezing in liquid nitrogen. Phage Phage titer (PFU/ml) control* lysate lysate + glycerol (10%) lysate + DMSO (5%) BIM BV x x x x BIM BV x x x x BIM BV x x x x BIM BV x x x x 10 9 BIM BV x x x x Pentaphage 2.20 x x x x 10 8 *Before freezing. Optical density (590 nm) 0,6 0,5 0,4 0,3 0,2 0,1 control lysate lysate + 10%DMSO lysate + 10%glycerol t (min) Fig. 2. P. fluorescens cell lysis by Pentaphage before and after freezing. 81

7 During freezing of Pentaphage preparation, 100 % survival rate was observed in the presence of 10 % glycerol. Protective effect of DMSO was expressed to a lesser degree (Table 4). Study of Pentaphage lytic ability revealed that decrease in titer did not affect the rate of P.fluorescens cell lysis (Fig. 2). Within 20 min after addition of phage preparation to the host culture, the increase of optical density caused by duplication of bacteria during the phage latent period of growth was observed. Further, a sharp clearing of the cultural liquid due to bacterial lysis was detected and minutes later Pseudomonas cells were completely plasmolysed. When studying old lysates with electron microscope damages of phages are usually observed in the form of DNA loss ("ghosting"), head disruption, tail contraction, etc. [1]. Study of phage morphology after freezing by electronic microscopy did not reveal any changes in phage structure in all variants, even in those, where the decrease in phage titer was recorded. Phages had an isometric head nm in diameter and short cone-shaped tail nm in length (Fig. 3). Probably phages inactivation during freezing occurred without visible structural failures of virus particles. Thus, rapid cooling proved to be suitable kinetics for Pseudomonas phages cryopreservation, providing high viability and stability of lytic activity of these microorganisms. Survival ability of phages during freezing can be improved using 10 % glycerol and 5 % DMSO as cryoprotective additives. Fig. 3. Morphology of phage particles BIM BV-1 before and after freezing: a), b) unfrozen phages; c), d), e), f), g), h) phages frozen without additives; i), j), k) phages frozen with 5% DMSO; l), m) phages frozen with 10% glycerin. Magnification: a), c) ; b), d), e), g), h), j), k), m) ; i) ; f) ; l)

8 Cryopreservation in liquid nitrogen was successfully applied for bacteria of Bifidobacterium genus and bacteriophages. High rates of cooling (direct immersion in liquid nitrogen) appeared to lead to minimal losses in viability and acidification activity of bifidobacteria. Media traditionally used for cultivation of bifidobacteria proved to be effective cryoprotective media for preservation of these microorganisms. Trypton-lactose medium seemed to be optimal for bifidobacteria cryopreservation. The possibility to use rapid cooling kinetics for cryopreservation of Pseudomonas phages was shown. Survival rate of bacteriophages after freezing was higher when 5 % 1. Ackermann, H. W., D. Tremblay, S. Moineau, WFCC Newsletter, 38, Ash, R. J., E. R. Barnhart, J. Clin. Microbiol., 2 (3), Clark, W. A., W. Horneland, A. G. Klein, Appl. Environ. Microbiol., 10 (5), Fonseca, F., B. Corrieu, G. Corrieu, J. Dairy Res., 67, Fonseca, F., M. Marin, G. J. Morris, Appl. Environ. Microbiol., 72 (10), Hubalek, Z., Cryobiology, 46, Janet, S. M., J. E. Kempf, Appl. Microbiol., 12 (5), Kerrigan, L., PMF Newsletter, 13 (2), Malik, K. A., World J. Microbiol. Biotechnol., 7 (6), Mazur, P., S. P. Leibo, E. H. Y. Chu, Experimental Cell Research, 71, Nei, T., T. Araki, T. Matsusaka, Freezing injury to aerated and non-aerated cultures of Escherichia coli. In: Freezing and drying of microorganisms, T. Nei (Ed), Tokio: University of Tokio Press. 12. Novik, G. I., N. I. Astapovich, N. G. Kadnikova, N. E. Ryabaya, Microbiology (Moskva). 67 (5), (in Russian). 13. Novik, G., International scientific confe- Conclusion DMSO and 10 % glycerol were used as cryoprotective additives. Decrease in titer of phages from Pentafage during freezing did not affect their lytic ability, and cryopreservation proved to be an effective method for storage of this preparation. Currently, we use cryopreservation for conservation of bifidobacteria and lactic acid bacteria, Lactococcus and Pseudomonads bacteriophages and some groups of fungi [12, 13, 14, 16, 17]. Acknowledgements. This work was supported by Belarusian Republican Foundation for Fundamental Research within the framework of Project N B07K-024. References rence S. P. Kostychev and contemporary agricultural microbiology, 8-12 Oct., Yalta, Ukraine, abstracts, Novik, G., I. Vazhynskaya, A. Kanterova, International scientific conference S. P. Kostychev and contemporary agricultural microbiology, 8-12 Oct., Yalta, Ukraine, abstracts, Reilly, S. S., S. E. Gilliland, J. Food Sci., 64 (2), Sidarenka, A. V., G. I. Novik, th International school-conference Biology science of the XXI century, 29 Oct. 2 Nov., Pushchino, Russia, abstracts, Sidarenka, A. V., G. I. Novik, International scientific conference Microorganisms and biosphere, Nov., Moskow, Russia, abstracts, Tsutsaeva, A. A., V. G. Popov, K. M. Sitnik, Cryobiology and biotechnology, A. A. Tsutsaeva (Ed), Kiev: Nauk. Dumka (in Russian). 19. Visekantsev, I. P., Biological properties of cryopreserved Esherichia coli bacteriophages. Abstract of Ph.D. thesis, Kharkov, Institute for problems of cryobiology and cryomedicine, National Academy of Sciences, Ukraine (in Russian). 20. Wallis, G., J. L. Melnick, J. Virol., 2 (9),

9 КРИОКОНСЕРВИРАНЕ НА БИФИДОБАКТЕРИИ И БАКТЕРИОФАГИ В БЕЛО- РУСКАТА КОЛЕКЦИЯ ЗА НЕПАТОГЕННИ МИКРООРГАНИЗМИ Галина Новик*, Анастасия Сидаренка, Денис Ракхуба, Емилия Коломиец Резюме Публикацията разглежда криоконсервирането на бифидобактерии и бактериофаги. Оценен е ефектът на високите скорости на охлаждане върху преживяемостта и физиологичната активност на бифидобактерии. Изследвана е възможността за бързо замразяване при криоконсервиране на изходни култури фаги по Pseudomonas. Проучването показва, че бързата кинетика на охлаждането води до минимални загуби на жизнеността и подкиселяващата активност на бифидобактериите. Доказва се, че традиционно използваните среди за култивиране на бифидобактерии имат добри криопротектиращи свойства и могат да бъдат ефективни среди за замразяване при консервиране на тези организми. Изследването разкрива, че криоконсервирането с високи скорости на замразяване осигурява висока преживяемост и запазване на литичната способност на бактериофагите по Pseudomonas. Добри резултати за протектиране на тази група фаги показват 10 % глицерол и 5 % DMSO. 84