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Appl Environ Microbiol. Feb 2006; 72(2): 1702–1704.
PMCID: PMC1392932

Freezing Induces Biased Results in the Molecular Detection of Flavobacterium columnare

Abstract

Specific PCR detection and electron microscopy of Flavobacterium columnare revealed the risk of false-negative results in molecular detection of this fish pathogen. Freezing and thawing destroyed the cells so that DNA was for the most part undetectable by PCR. The detection of bacteria was also weakened after prolonged enrichment cultivation of samples from infected fish.

DNA-based techniques revolutionized microbial ecology because they are not biased by selective cultivation. Another important reason for the fast spread of molecular methodologies is that molecular recognition samples can be stored for later research. Freezing is commonly used for storing samples for molecular analysis, and freeze-thaw cycles have been used for extraction of DNA. Very little attention has been paid to the freezing effect, although there have been indications that it can increase the number of false-negative results in PCR detection of pathogens (4, 8). In this paper we show that freezing and thawing of samples can negatively affect the molecular detection of a specific bacterial pathogen, as well as diversity analyses in general.

Flavobacterium columnare is a fish pathogen that causes economic losses in fish farming worldwide. There are several PCR protocols that can be used to analyze pathogens in either carrier or infected fish and in the incoming water of fish farms. The detection limit of these methods varies from 29.7 CFU mg−1 tissue (12) to 100 CFU mg−1 tissue (1). The number of bacteria may be increased above the detection limit by using enrichment, if it is essential to detect the pathogen (e.g., in carrier fish or in water samples). However, we observed that most enriched and frozen (at −20°C) fish mucus samples collected from F. columnare-infected fish were false-negative in the diagnostic PCR assay (unpublished results). In this study our aim was to investigate whether freezing of samples can affect the detection of enriched flavobacterial cells.

Cotton swab samples from skin mucus of fingerling rainbow trout (Oncorhynchus mykiss Walbaum) suffering from F. columnare infection and of the tank water (1 ml) were enriched for 1, 2, and 3 days with constant agitation (150 rpm, 23°C) in 5 ml of Shieh medium (9), which was specifically designed to support the growth of F. columnare. The enriched samples were then either analyzed directly or frozen at −20°C and thawed at 23°C. One milliliter of a sample was immediately pelleted by brief centrifugation (13,000 × g for 10 min) and used for DNA extraction with a Biosprint 15 DNA blood kit (QIAGEN) and a KingFisher magnetic particle separator (Labsystems). F. columnare-specific primers FvpF1 and FvpR1 (1) were used to detect the pathogen with a PCR. The reaction mixture (20 μl) contained 0.3 μM of each primer, 0.2 mM of a deoxynucleoside triphosphate mixture (MBI Fermentas), 1× PCR buffer, 1 μl of template DNA, and 0.5 U of DNA polymerase (Biotools), and the reaction conditions described previously were used (1). The positive PCR control mixture included 50 ng of F. columnare DNA, and the negative control mixture contained no template. Ten microliters of the PCR product was subjected to agarose gel electrophoresis.

The effect of freezing on the morphology of F. columnare strain HTAN5/03 cells was studied by electron microscopy. Escherichia coli ATCC 1147 was used as a reference strain. Bacterial cultures were grown in 5 ml Shieh broth for 1, 3, and 5 days. At each time a sample was frozen at −20°C. The specimens were thawed and washed quickly with 0.1 M phosphate buffer (pH 7.4, 4°C). After this they were fixed in 2.5% glutaraldehyde for 1 h and then postfixed in 1% osmium tetroxide for 1 h in the same buffer. Samples were dehydrated in an ethanol series, stained with uranyl acetate, and embedded in LX-112 (Ladd). Thin sections were cut with a diamond knife and mounted on Formvar-coated copper grids. Double staining on the grids was performed with uranyl acetate and lead citrate. The specimens were examined using a Jeol JEM-1200 electron microscope.

F. columnare-specific PCR amplified weaker products from samples enriched for 2 or 3 days, which may indicate overgrowth of other bacteria and/or destruction of F. columnare DNA (Fig. (Fig.1).1). Freezing had an even more drastic effect, eliminating the PCR signal almost completely (Fig. (Fig.11 and and2).2). The growth of F. columnare is slow, and this organism can be outgrown by other bacteria occurring on fish skin or in water (e.g., pseudomonads) (10, 11), which might explain the slight weakening of the PCR results during prolonged enrichment. Addition of selective antibiotics, such as tobramycin, to the culture has been shown to increase the probability of growth of pure F. columnare (5). However, there is no evidence that all F. columnare strains can grow with tobramycin.

FIG. 1.
Effect of incubation time on PCR detection of F. columnare enriched from skin samples of infected fish (lanes A and B) and from tank water (lanes W). The numbers indicate the time of enrichment in Shieh medium (in days). f indicates that a sample was ...
FIG. 2.
Effect of sample freezing on PCR detection of F. columnare from infected fish skin. Samples (lanes A to I) were enriched for 3 days in Shieh medium prior to analysis. f indicates that a sample was frozen between enrichment and DNA extraction. Lane λ/HE ...

Electron microscopy (Fig. (Fig.3)3) revealed the reason for the total fading of the PCR signal from the freeze-stored samples. Freezing caused severe damage to the cells of F. columnare, and in frozen samples the cell walls were completely disintegrated (Fig. (Fig.3B).3B). However, freezing had no effect on E. coli cell morphology (data not shown). It is very well known that F. columnare cells produce vast amounts of DNases (2), lyases (7), and proteases (3), which are most likely connected to its pathogenity and erosion of the external surfaces of fish. On the other hand, the same products may also cause damage to the cell itself. The destruction of F. columnare cell walls, together with the apparent release of DNase from the disrupted cells of a dense F. columnare culture, could explain the damage to the PCR-amplifiable DNA. Electron microscopy also revealed that vegetative F. columnare cells started to turn very quickly (in 5 days) into “spheroplasts,” the fate or meaning of which is not completely understood (2, 6). The electron micrographs of older F. columnare cells showed that these cell forms were formed by outer membrane protrusion (Fig. (Fig.3A3A).

FIG. 3.
F. columnare HTAN5/03 cells observed by electron microscopy. (A) Cells observed after 7 days of cultivation. (B) Frozen cells observed after 3 days of cultivation and sample freezing. (C) Cells observed after 3 days of cultivation. The arrows indicate ...

Taken together, the data from PCR experiments and electron microscopy show that some of the difficulties in detecting F. columnare in environmental samples (10) may be attributed to sample freezing. This kind of bias may also hamper analysis of other bacterial strains which are difficult to cultivate from frozen stocks and which produce DNases, as F. columnare does. How samples should be stored and how widespread this phenomenon is among the Cytophaga-Flexibacter-Bacteroides group should be studied, especially because the Cytophaga-Flexibacter-Bacteroides group is widely dispersed in aquatic environments.

Acknowledgments

This work was funded by grants from the University of Jyväskylä and the Graduate School of Biological Interactions, University of Turku, to L.-R.S. M.T. was supported by postdoctoral research funding from the Academy of Finland.

REFERENCES

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