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The effect of storage in liquid nitrogen on the isolation of oral yeasts in human saliva
AI&S oral Biol. Vol. 37, No. 3, pp. 237-239, 1992 Printed in Great Britain. AI1rightsrcsemd
OQO3-996992 55.00 + 0.00
copyrightQ 1992Pergamcn Press plc
THE EFFECT OF STORAGE IN LIQUID NITROGEN ON THE ISOLATION OF ORAL YEASTS IN HUMAN SALIVA E. BRAMBILLA,L. STRO~NGER
and G. VOGEL
Department of Dentistry and Stomatology, School of Medicine, University of Milan, Milan, Italy (Accepted 22 August 1991) Summary-Stimulated whole saliva samples were collected from a group of 127 6-yr-old schoolchildren. Each sample was divided into three parts. The first two, one of which contained added glycerol, were immediately frozen in liquid nitrogen and stored at - 196°C for 2 months. The third part was transferred to the laboratory and plated on selective medium for yeasts. Colony counts of the frozen and non-frozen samples were then compared. Statistical analysis showed a highly significant correlation between the counts for frozen and unfrozen samples. A high (40.1%) prevalence of yeast carriers was found, and Candidu albicans was the most frequently recovered yeast. Key words: dental caries, saliva, oral yeasts, liquid nitrogen.
MATERIALS AND METHODS
INTRODUCTION In spite of its lethal effects on many biological systems, freezing can preserve viable cells and their constituents for a long time. Several micro-organisms are able to survive this dramatic environmental change, while others cannot. The usefulness of liquid nitrogen for storage of both aerobic and anaerobic micro-organisms is well documented (Jarvis, Wynne and Telfer, 1967; Gilmour et al., 1978; AshwoodSmith, 1985). In microbiology, cryopreservation of various types of samples assures availability of the specimens without temporal or geographical restrictions. Because of large variability between subjects, clinical/microbiological studies require relatively large samples. Furthermore, laboratory facilities and staff are seldom easily available for immediate microbiological processing of large numbers of samples. Yeasts are a common and variable part of the commensal flora of man. They are more frequent in the mouth than at any other site (Barlow and Chattaway, 1969; Arendorf and Walker, 1979, 1980). Estimated prevalence of yeasts in the mouths of healthy schoolchildren have varied from 6 to about 60% (Clayton and Noble, 1966; Russell and Lay, 1973; Pienihakkinen et al., 1985). Salivary yeast counts, especially of the genus Cundidu, are now frequently made. The prevalance of Candida spp. in saliva correlates well with risk of caries and its prevalence is not affected by oral hygiene (Pienihakkinen, Scheinin and Banoczy, 1987; Pienihakkinen, 1988). The aim of our study now was to evaluate the effects of storage in liquid nitrogen on the prevalence and number of yeasts in salivary samples. The availability of a simple method for sample preservation that would not affect the isolation of microorganisms would help to overcome the problems encountered in large epidemiological surveys. We also investigated the rate of Cundidu carriage in the population.
A group of 127 normal, healthy, 6-yr-old schoolchildren involved in a programme of fluoroprophylaxis was studied. The children chewed paraffin wax for 5 min, and then saliva was collected from each of the subjects in a sterile plastic container. After vortexing for 15 s, each salivary sample was divided into three portions of 1 ml. Two portions were transferred to cryovials (Nunc, Gibco Europe Ltd. Paisley, U.K.), one of them containing sterile glycerol (autoclaved at 121°C for 15 mitt) to give a final 10% VjV concentration of glycerol in saliva. After equilibration for 30 min at 24’C to allow permeation of the suspension with glycerol, the two vials to be frozen were vortexed for 15 s. Afterwards, these subsamples were frozen in liquid nitrogen on a freezing tray (Taylor Warton Int., Theodore, AL, U.S.A.) at a cooling rate of lO’C/min. The samples were stored in liquid nitrogen for 2 months. The third subsample was transferred to the laboratory within 20 min and vortexed for 15 s; 50 ~1 of undiluted saliva were plated on Sabouraud’s agar and Biggy agar plates (Difco, Detroit, MI, U.S.A.), which allow the differentiation of Cundidu spp. by colonial morphology. After aerobic incubation at 30°C for 7 days the colonies were counted and identified. A germtube test was made on at least 10 colonies belonging to each recognized morphotype. This test gives rapid presumptive identification of Cundidu ulbicuns (Mackenzie, 1962), although other species of Cundidu have been reported to produce a positive test. Yeast colonies with negative or doubtful germ-tube tests were finally identified by biochemical tests, using the API32C system (API system, Montalieu Vercieu, France). To confirm these results, a second biochemical identification was done with an automatic device (Vitek, BioMerieux, Marcy-L’Etoile, France). After the 2 months, the frozen subsamples were processed in the same way.
237
E. BRAMBILLA et al.
238
sccc
c
1odoc
ISdCO iccoo
25iCO
?5) .:,:
3&
Number of yeasts in frozen salivary samples (c.f.u./ml) Fig. 1. Frequency distribution of yeasts in frozen salivary samples. Statistical
analysis
which the concentration of yeasts in unfrozen fresh saliva can be calculated from the data obtained from frozen samples.
The data were analysed statistically with a software package (Stat View, Brainpower, Ventura, CA, U.S.A.) Logarithmic transformations were made and Pearson’s linear correlation coefficients were calculated. A linear regression model for the data was used to evaluate the degree of correlation between the data for the frozen and thawed and unfrozen samples. Significance was set at p < 0.01.
DISCUSSION
We have shown that freezing samples in liquid nitrogen is a satisfactory technique for storing oral yeasts, with little or no effect on their presence in saliva. With this technique a large number of frozen saliva specimens can be stored. Counts comparable to those obtained from unfrozen, fresh samples can be obtained at a later date. As salivary yeasts are almost unaffected by this method of preservation, the technique may be useful for clinical/microbiological screening of subjects at high risk of caries. Salivary yeast concentrations have been considered to be a better indicator of caries risk than salivary counts of Streptococcus mutans or Lactobacillus spp. (Pienihakkinen et al., 1987; Pienihakkinen, 1988). This technique would also appear to be useful for epidemiological studies, in which sampling of a large population requires collection of a large number of salivary samples. The immediate processing of these samples would need many skilled technicians and equipment that is often not readily available. Cryopreservation allows delayed examination of the sample. It also allows examination of the samples without modifying their relative compositions. The use of glycerol as a cryopreservative gave results almost comparable to those obtained without glyc-
RESULTS Fifty-one (40. I %) of the 127 children were found to have yeasts in their saliva. C. albicans was the yeast in 97% of these; C. guillermondii was the only other species recovered. Frequency distribution analysis (Fig. I) described the trends for yeast counts
in unfrozen samples. Pearson’s correlation coefficients showed a significant correlation between the yeast counts of frozen and unfrozen samples (r = 0.957, p < 0.0001) and between those frozen with glycerol and unfrozen (r = 0.816, p < 0.0001). The difference between the two correlation coefficents may be explained by the difference in the size of the experimental groups, owing to the loss of some frozen samples containing glycerol (53 missing data). Linear regression analysis confirmed the results previously indicated by the correlation coefficients and showed a first-degree function (Fig. 2), from
y= 1.096x + 0.283, R - squared: 0.915
!A
_i
0
2
d
6
8
/
IC
I2
Number of yeasts in unfrozen salivary samples (log c.f.u./ml) Fig. 2. Linear regression of number of yeasts in frozen samples and number of yeasts in unfrozen samples
Storage of salivary yeasts in liquid nitrogen we cannot say whether or not it should be used when salivary samples are frozen for longer periods cf time. Further studies may reveal the reliability of this technique for preservation of samples for medium intervals of time and may extend the spectrum of microbial and yeast species for which it can be used (Gilmour er al., 1978; Wilson, Woods and Ashley, 1984). An additional purpose of our study was to analyse the salivary yeasts in a sample of northern Italian schoolchildren. The prevalence of yeast carriers (40%) was about that anticipated for normal individuals in the collected data of Odds (1988). The yeast flora of most of our subjects was found to be simple, with three or fewer morphotypes. C. albicans was the species most frequently found (96.8% of isolates). These data are in agreement with the results of several previous studies (Clayton and Noble. 1966; Mackenzie, 1962; Arendorf and Walker, 1979; Martin and Wilkinson, 1983). Even more recently, Russell et al. (1990) examined a group of 372 adolescents and found that 50% of the subjects were oral yeast carriers and that the micro-organisms were C. al&cans in 99% of cases. They obtained erol. On the basis of these results,
results comparable to those of Pienihakkinen et al. (1987). The lack of other species may be due to either
the type and geographical location of the population sampled, or that these species are not present in the oral flora of younger children. rickno,~ledgemenrs-We wish IO thank Drs Enrico Carbone, Giuseppe Belluomo and Livio Sacchi for skilful technical assistance. This work was supported by National Research Council (C.N.R.&Targeted Project “Prevention and Control of Disease Factors” (F.A.T.M.A). REFERENCES Arendorf T. and Walker D. (1979) Oral candidal populations in health and disease. Br. dent. J. 147, 267-272. Arendorf T. Xi. and Walker D. M. (1980) The prevalence and intra-oral distribution of Cundidu olbicans in man. .-lrchs oral Biol. 25, I-10.
239
Ashwood-Smith M. J. (1985) Genetic damage is not produced by normal cryopreservation procedures involving either glycerol or dimethyl sulfoxide: a cautionary note, however, on possible effects of dimethyl sulfoxide. Cryobiology 22, 427-433.
Barlow A. J. and Chattaway F. W. (1969) Observations the carriage of Candida albicans in man. Br. J. Dermar. 103-106. Clayton Y. M. and Noble W. C. (1966) Observations the epidemiology of Cundidu albicans. J. clin. Path.
on 81, on 55,
580-590.
Gilmour M. N.. Turner G., Berman R. G. and Krenzer A. K. (1978) Compact liquid nitrogen storage system yielding high recoveries of Gram-negative anaerobes. Appl. Environ. Microbial. 35, 84-88.
Jarvis J. D., Wynne C. D. and Telfer E. R. (1967) Storage of bacteria in liquid nitrogen. J. med. Lab. Tech. 24, 84-88.
Mackenzie D. W. R. (1962) Yeasts from human sources. Sabouraudia 1, 8-l 5. Martin M. V. and Wilkinson G. R. (1983) The oral yeast flora of IO-year-old schoolchildren. Sabouraudia 21, 129-135.
Odds F. C. (1988) Candida and Candidosis-a Review of Bibliogrphy, 2nd edn. Bailliere Tindell-W. B. Saunders, London. Pienihakkinen K. (1988) Salivary lactobacilli and yeast in relation to caries increment. Annually repeated measurements versus a single determination. Acta odonf. Scund. 46, 57-62.
Pienihakkinen K., Gabris K., Nyarasdy I., Rigo O., Scheinin A. and Ban&y J. (1985) Longitudinal counts of lactobacilli and yeast in saliva. Acra odonr. scund. 43, 359-365.
Pienihakkinen K., Scheinin A. and Banoczy J. (1987) Screening of caries in children through salivary lactol bacilli and yeast. Stand. J. dent. Rex 95. 397-404. Russell C. and Lay K. M. (1973) Natural history of Cundida species and yeast in the oral cavities of infants. Archs oral Biol. 18, 957-962.
Russell J. I., MacFarlane T. W., Aitchison T. C., Stephen K. W. and Burchell C. K. (1990) Salivary levels of mutans streptococci, Lactobacillus. Candida. and Veillonellu species in a group of Scottish adolescents. Communiry dent. oral Epidetniol. 18, 17-21. Wilson R. F., Woods A. and Ashley F. P. (1984) The effect of storage in liquid nitrogen on the recovery of human dental plaque bacteria. Archs ornl Bio/. 11, 941-944.
OQO3-996992 55.00 + 0.00
copyrightQ 1992Pergamcn Press plc
THE EFFECT OF STORAGE IN LIQUID NITROGEN ON THE ISOLATION OF ORAL YEASTS IN HUMAN SALIVA E. BRAMBILLA,L. STRO~NGER
and G. VOGEL
Department of Dentistry and Stomatology, School of Medicine, University of Milan, Milan, Italy (Accepted 22 August 1991) Summary-Stimulated whole saliva samples were collected from a group of 127 6-yr-old schoolchildren. Each sample was divided into three parts. The first two, one of which contained added glycerol, were immediately frozen in liquid nitrogen and stored at - 196°C for 2 months. The third part was transferred to the laboratory and plated on selective medium for yeasts. Colony counts of the frozen and non-frozen samples were then compared. Statistical analysis showed a highly significant correlation between the counts for frozen and unfrozen samples. A high (40.1%) prevalence of yeast carriers was found, and Candidu albicans was the most frequently recovered yeast. Key words: dental caries, saliva, oral yeasts, liquid nitrogen.
MATERIALS AND METHODS
INTRODUCTION In spite of its lethal effects on many biological systems, freezing can preserve viable cells and their constituents for a long time. Several micro-organisms are able to survive this dramatic environmental change, while others cannot. The usefulness of liquid nitrogen for storage of both aerobic and anaerobic micro-organisms is well documented (Jarvis, Wynne and Telfer, 1967; Gilmour et al., 1978; AshwoodSmith, 1985). In microbiology, cryopreservation of various types of samples assures availability of the specimens without temporal or geographical restrictions. Because of large variability between subjects, clinical/microbiological studies require relatively large samples. Furthermore, laboratory facilities and staff are seldom easily available for immediate microbiological processing of large numbers of samples. Yeasts are a common and variable part of the commensal flora of man. They are more frequent in the mouth than at any other site (Barlow and Chattaway, 1969; Arendorf and Walker, 1979, 1980). Estimated prevalence of yeasts in the mouths of healthy schoolchildren have varied from 6 to about 60% (Clayton and Noble, 1966; Russell and Lay, 1973; Pienihakkinen et al., 1985). Salivary yeast counts, especially of the genus Cundidu, are now frequently made. The prevalance of Candida spp. in saliva correlates well with risk of caries and its prevalence is not affected by oral hygiene (Pienihakkinen, Scheinin and Banoczy, 1987; Pienihakkinen, 1988). The aim of our study now was to evaluate the effects of storage in liquid nitrogen on the prevalence and number of yeasts in salivary samples. The availability of a simple method for sample preservation that would not affect the isolation of microorganisms would help to overcome the problems encountered in large epidemiological surveys. We also investigated the rate of Cundidu carriage in the population.
A group of 127 normal, healthy, 6-yr-old schoolchildren involved in a programme of fluoroprophylaxis was studied. The children chewed paraffin wax for 5 min, and then saliva was collected from each of the subjects in a sterile plastic container. After vortexing for 15 s, each salivary sample was divided into three portions of 1 ml. Two portions were transferred to cryovials (Nunc, Gibco Europe Ltd. Paisley, U.K.), one of them containing sterile glycerol (autoclaved at 121°C for 15 mitt) to give a final 10% VjV concentration of glycerol in saliva. After equilibration for 30 min at 24’C to allow permeation of the suspension with glycerol, the two vials to be frozen were vortexed for 15 s. Afterwards, these subsamples were frozen in liquid nitrogen on a freezing tray (Taylor Warton Int., Theodore, AL, U.S.A.) at a cooling rate of lO’C/min. The samples were stored in liquid nitrogen for 2 months. The third subsample was transferred to the laboratory within 20 min and vortexed for 15 s; 50 ~1 of undiluted saliva were plated on Sabouraud’s agar and Biggy agar plates (Difco, Detroit, MI, U.S.A.), which allow the differentiation of Cundidu spp. by colonial morphology. After aerobic incubation at 30°C for 7 days the colonies were counted and identified. A germtube test was made on at least 10 colonies belonging to each recognized morphotype. This test gives rapid presumptive identification of Cundidu ulbicuns (Mackenzie, 1962), although other species of Cundidu have been reported to produce a positive test. Yeast colonies with negative or doubtful germ-tube tests were finally identified by biochemical tests, using the API32C system (API system, Montalieu Vercieu, France). To confirm these results, a second biochemical identification was done with an automatic device (Vitek, BioMerieux, Marcy-L’Etoile, France). After the 2 months, the frozen subsamples were processed in the same way.
237
E. BRAMBILLA et al.
238
sccc
c
1odoc
ISdCO iccoo
25iCO
?5) .:,:
3&
Number of yeasts in frozen salivary samples (c.f.u./ml) Fig. 1. Frequency distribution of yeasts in frozen salivary samples. Statistical
analysis
which the concentration of yeasts in unfrozen fresh saliva can be calculated from the data obtained from frozen samples.
The data were analysed statistically with a software package (Stat View, Brainpower, Ventura, CA, U.S.A.) Logarithmic transformations were made and Pearson’s linear correlation coefficients were calculated. A linear regression model for the data was used to evaluate the degree of correlation between the data for the frozen and thawed and unfrozen samples. Significance was set at p < 0.01.
DISCUSSION
We have shown that freezing samples in liquid nitrogen is a satisfactory technique for storing oral yeasts, with little or no effect on their presence in saliva. With this technique a large number of frozen saliva specimens can be stored. Counts comparable to those obtained from unfrozen, fresh samples can be obtained at a later date. As salivary yeasts are almost unaffected by this method of preservation, the technique may be useful for clinical/microbiological screening of subjects at high risk of caries. Salivary yeast concentrations have been considered to be a better indicator of caries risk than salivary counts of Streptococcus mutans or Lactobacillus spp. (Pienihakkinen et al., 1987; Pienihakkinen, 1988). This technique would also appear to be useful for epidemiological studies, in which sampling of a large population requires collection of a large number of salivary samples. The immediate processing of these samples would need many skilled technicians and equipment that is often not readily available. Cryopreservation allows delayed examination of the sample. It also allows examination of the samples without modifying their relative compositions. The use of glycerol as a cryopreservative gave results almost comparable to those obtained without glyc-
RESULTS Fifty-one (40. I %) of the 127 children were found to have yeasts in their saliva. C. albicans was the yeast in 97% of these; C. guillermondii was the only other species recovered. Frequency distribution analysis (Fig. I) described the trends for yeast counts
in unfrozen samples. Pearson’s correlation coefficients showed a significant correlation between the yeast counts of frozen and unfrozen samples (r = 0.957, p < 0.0001) and between those frozen with glycerol and unfrozen (r = 0.816, p < 0.0001). The difference between the two correlation coefficents may be explained by the difference in the size of the experimental groups, owing to the loss of some frozen samples containing glycerol (53 missing data). Linear regression analysis confirmed the results previously indicated by the correlation coefficients and showed a first-degree function (Fig. 2), from
y= 1.096x + 0.283, R - squared: 0.915
!A
_i
0
2
d
6
8
/
IC
I2
Number of yeasts in unfrozen salivary samples (log c.f.u./ml) Fig. 2. Linear regression of number of yeasts in frozen samples and number of yeasts in unfrozen samples
Storage of salivary yeasts in liquid nitrogen we cannot say whether or not it should be used when salivary samples are frozen for longer periods cf time. Further studies may reveal the reliability of this technique for preservation of samples for medium intervals of time and may extend the spectrum of microbial and yeast species for which it can be used (Gilmour er al., 1978; Wilson, Woods and Ashley, 1984). An additional purpose of our study was to analyse the salivary yeasts in a sample of northern Italian schoolchildren. The prevalence of yeast carriers (40%) was about that anticipated for normal individuals in the collected data of Odds (1988). The yeast flora of most of our subjects was found to be simple, with three or fewer morphotypes. C. albicans was the species most frequently found (96.8% of isolates). These data are in agreement with the results of several previous studies (Clayton and Noble. 1966; Mackenzie, 1962; Arendorf and Walker, 1979; Martin and Wilkinson, 1983). Even more recently, Russell et al. (1990) examined a group of 372 adolescents and found that 50% of the subjects were oral yeast carriers and that the micro-organisms were C. al&cans in 99% of cases. They obtained erol. On the basis of these results,
results comparable to those of Pienihakkinen et al. (1987). The lack of other species may be due to either
the type and geographical location of the population sampled, or that these species are not present in the oral flora of younger children. rickno,~ledgemenrs-We wish IO thank Drs Enrico Carbone, Giuseppe Belluomo and Livio Sacchi for skilful technical assistance. This work was supported by National Research Council (C.N.R.&Targeted Project “Prevention and Control of Disease Factors” (F.A.T.M.A). REFERENCES Arendorf T. and Walker D. (1979) Oral candidal populations in health and disease. Br. dent. J. 147, 267-272. Arendorf T. Xi. and Walker D. M. (1980) The prevalence and intra-oral distribution of Cundidu olbicans in man. .-lrchs oral Biol. 25, I-10.
239
Ashwood-Smith M. J. (1985) Genetic damage is not produced by normal cryopreservation procedures involving either glycerol or dimethyl sulfoxide: a cautionary note, however, on possible effects of dimethyl sulfoxide. Cryobiology 22, 427-433.
Barlow A. J. and Chattaway F. W. (1969) Observations the carriage of Candida albicans in man. Br. J. Dermar. 103-106. Clayton Y. M. and Noble W. C. (1966) Observations the epidemiology of Cundidu albicans. J. clin. Path.
on 81, on 55,
580-590.
Gilmour M. N.. Turner G., Berman R. G. and Krenzer A. K. (1978) Compact liquid nitrogen storage system yielding high recoveries of Gram-negative anaerobes. Appl. Environ. Microbial. 35, 84-88.
Jarvis J. D., Wynne C. D. and Telfer E. R. (1967) Storage of bacteria in liquid nitrogen. J. med. Lab. Tech. 24, 84-88.
Mackenzie D. W. R. (1962) Yeasts from human sources. Sabouraudia 1, 8-l 5. Martin M. V. and Wilkinson G. R. (1983) The oral yeast flora of IO-year-old schoolchildren. Sabouraudia 21, 129-135.
Odds F. C. (1988) Candida and Candidosis-a Review of Bibliogrphy, 2nd edn. Bailliere Tindell-W. B. Saunders, London. Pienihakkinen K. (1988) Salivary lactobacilli and yeast in relation to caries increment. Annually repeated measurements versus a single determination. Acta odonf. Scund. 46, 57-62.
Pienihakkinen K., Gabris K., Nyarasdy I., Rigo O., Scheinin A. and Ban&y J. (1985) Longitudinal counts of lactobacilli and yeast in saliva. Acra odonr. scund. 43, 359-365.
Pienihakkinen K., Scheinin A. and Banoczy J. (1987) Screening of caries in children through salivary lactol bacilli and yeast. Stand. J. dent. Rex 95. 397-404. Russell C. and Lay K. M. (1973) Natural history of Cundida species and yeast in the oral cavities of infants. Archs oral Biol. 18, 957-962.
Russell J. I., MacFarlane T. W., Aitchison T. C., Stephen K. W. and Burchell C. K. (1990) Salivary levels of mutans streptococci, Lactobacillus. Candida. and Veillonellu species in a group of Scottish adolescents. Communiry dent. oral Epidetniol. 18, 17-21. Wilson R. F., Woods A. and Ashley F. P. (1984) The effect of storage in liquid nitrogen on the recovery of human dental plaque bacteria. Archs ornl Bio/. 11, 941-944.