Scandinavian Journal of Infectious Diseases

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Selective Culturing of Yersinia Enterocolitica at a Low Temperature James Eiss To cite this article: James Eiss (1975) Selective Culturing of Yersinia Enterocolitica at a Low Temperature, Scandinavian Journal of Infectious Diseases, 7:4, 249-251, DOI: 10.3109/ inf.1975.7.issue-4.05 To link to this article:

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Date: 16 March 2016, At: 03:24

Scand J Infect Dis 7:249-251,1975

Selective Culturing of Yersinia enterocolitica at a Low Temperature JAMES EISS

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From the Department of Clinical Bacteriology. University of Lund. Allmiinna Sjukhuset , Malmo. Sweden

ABSTRACT. Stool specimens from patients with suspected Yersinia enterocolitica (Y. ent.) infection were cultured at +4°C in a nutrient broth for 5 days and at the same time the same stool specimens were cultured by the routine method of the laboratory. The routine method gave 34 Y. ent, isolations. Selective culturing at +4°C succeeded in isolation of the same 34 Y. ent, strains but also 15 further strains or a 44% increase in yield. Serial dilutions in broth of 34 strains of Y. ent., 7 strains of Y. pseudotuberculosis and 14 strains of other gram-negative enterobacteria were cultured at +4°C. Colony counts were performed from each dilution after 2 and 5 days. Human pathogenic strains of Y. ent. (serotype 3, 8, 9) increased markedly at +4°C in contrast to the other gram-negative enterobacteria.

INTRODUCTION Different bacteria have different optimal growth temperatures. The selective culturing of bacteria at a specific temperature which promotes the growth of those bacteria while hindering the growth of other bacteria in a mixed bacterial flora is an old and proven method for recovering the desired organism. Yersinia enterocolitica (Y. ent.) has been recognised as a type of bacteria that behaves differently at + 25° and + 370C as regards motility, biochemical reactions and sensitivity to phages (2, 3). In an effort to improve the cultivation of Y. ent. from human faeces we have examined the ability of this organism to grow in a laboratory refrigerator at +4°C. It has previously been noted that Y. ent. is able to survive long periods of time at low temperatures (6). It was observed that Y. ent. was also able to increase in numbers in a nutrient broth at a low temperature. Therefore we started selection of Y. ent. at +4°C in routine culturing of faeces. Also the degree of growth of these strains at +4°C in contrast to several other enterobacteria was studied. MA TERIAL AND METHODS Media

All examinations were done on LSU (I) and SS (Difco) agar media plates. Rappaport broth (4, 5) and our standard nutrient broth with the following formula were also used:

Beef extract, Difco 0.5%, NaCI 0.3%, Na2HP04+2H~O 0.2 %, Peptone Orthane 1%, glucose 0.1 %. Routine cultivation method Stool specimens, sent to the bacteriological laboratory for cultivation of pathogenic enterobacteria, were examined for the presence of Y. ent. by being plated directly on LSD and SS agar plates and incubated for 48 hours at +25°C and also on another LSU agar plate incubated I'm 48 hours at +37°C. At the same time the faeces specimens were added to Rappaport broth and incubated for 48 hours at +25OC. Samples from the broth were thereafter cultivated on LSD and on SS agar plates and incubated for 48 hours at +25OC. Suspect colonies were confirmed as Y. ent. by biochemistry according to Nilehn (2) and serotyping according to Winblad (7, 8, 9). Low temperature cultivation method Faecal specimens were transferred directly to 4.5 ml of our standard nutrient broth by a wooden applicator. The broth was then incubated in a refrigerator at +4OC. After 2 and 5 days of incubation, the broth was plated out with a wire loop on LSD as well as on SS agar plates and incubated for 2 more days at +25OC. Suspect colonies were confirmed as mentioned above. Clinical specimens All stool specimens sent to the bacteriological laboratory with a specific request for culturing of Y. ent. were cultured both by the routine method described above and in an enrichment broth at +4°C. Some of these specimens were from patients with high antibody titres against Y. ent. or with clinical symptoms suggestive of Y. ent. infection. Scand J Inject Dis 7


J. Eiss

Table I. Examples of various patterns of growth at +4°C Number of colonies counted are given. Day O=growth from broth which had not been incubated Dilutions of inoculated broth

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10- 1


10- 5

10- 6

10- 7

10- 6

Y. enterocolitica serotype Day 0 CGa Day 2 CG Day 5 CG


3000 10000 CG

349 1000 DG

27 157 10000

3 17 1300

0 3 175

0 0 36

Y. pseudotuberculosis Ib Day 0 CG Day 2 CG Day 5 CG


2500 3000 DG

252 290 2000

20 21 183


3 16

I I 0

0 0 0

E. coli Day 0 Day 2 Day 5

700 476 252

78 44 25

13 8 5

2 0 0

0 0 0

0 0 0

0 0 0

a b

DG 5000 3000

CG=Confluent growth. DG=Dense growth but still individual colonies.

Evaluation of bacterial growth at loll' temperature Strains examined. The following gram-negative bacteria were examined: 5 different strains of Y. ent. serotype 3, 4 different strains of Y. ent. serotype 9, one strain each of Y. ent. serotype 1-2,4, 5a, 5b, 6-8,10-20,22-25, 26a, and 26b, one strain each of Y. pseudotuberculosis serotype la, Ib, lIa, lib. III. IV and V, 4 strains of Escherichia coli, 2 strains of Enterobacter cloacae, 2 strains of Proteus vulgaris, one strain of P. mirabilis and P. morganii, 2 strains of Klebsiella pneumoniae and 2 strains of Salmonella typhimurium. Procedure. The strains were cultured on a peptone human blood agar plate overnight at +37°C. Three colonies from this plate were suspended into 5 ml 0.1 % NaCI, and 0.5 ml of this suspension was added to 4.5 ml of nutrient broth (tube I). From tube I again 0.5 ml was added to tube 2. which also contained 4.5 ml of nutrient Table II. Number of strains showing different patterns of growth with colony counts on LS U agar plates from 0.1 ml of nutrient broth with a dilution of 10- 4 after 5 days of cultivation at +4°C Coefficients of growth increase at +4°C Strains examined

Y. enterocolitica serotype 3, 8, 9 (human pathogens) Y. enterocol itica of other serotypes, mostly nonhuman pathogens Y. pseudotuberculosis Other gram-negative bacteria Scand J Infect Dis 7


broth. The same procedure was carried out 8 times, the dilution of the last tube being 10- 8 • The different broth tubes were incubated at +4°C. From each tube, 0.1 ml of broth was plated out after 2 and 5 days on LSD agar plates, and the colonies were counted after 48 hours of incubation at +25°C. RESULTS

Comparison between routine cultivation and cultivation at +4°C During a period of 8 months (1973-1974) the routine culturing of faeces frum patients with enteritis, abdominal pains, appendicitis-like symptoms or high yersinia antibody titres resulted in 34 isolations of Y. ent., all serotype 3. During the same period, cultivation of the same specimens at +4°C yielded 49 isolations of Y. ent. The low temperature method confirmed the same

34 Y. ent. strains found by the routine cultivation method. There was no strain of Y. ent. isolated by the routine method, that was not isolated by the low temperature method. The additional 15 strains included 13 of serotype 3, I of serotype 5 and I of serotype 6. Thus, the low temperature method gave an increase in successful isolations of 44 %.








Growth capacity of Y. ent. and other enterobacteria at +4°C

9 2

7 3

5 2





The results of the experiments with 55 strains showed three different patterns of growth (Table I). Y. ent. serotype 3 illustrates the first type of growth pattern, i.e. a large increase in growth while

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Selective culturing of Yersinia enterocolitica

being incubated at +4°C for 5 days. Y. pseudotuberculosis illustrates the second type of growth pattern, i.e. a moderate growth at +4°C. The third type is illustrated by a strain of E. coli, which showed a reduction in the number of colonies while being incubated at +4°C. Table II shows the different degrees of growth at +4°C of the 55 examined bacteria strains. In order to illustrate the degree of growth after 5 days at +4°C the dilution of 10-4 in broth was chosen and the number of colonies were counted on a LSU agar plate. Y. enterocolitica of serogroups 3, 8 and 9 (human pathogenic strains) all showed good growth at +4°C. The other serotypes of yersinia, the nonpathogenic and low-pathogenic serotypes, varied in growth. More than half of the examined strains increased at the low temperature. Five of the 7 examined Y. pseudotuberculosis strains showed a large increase at +4°C. A quite opposite result was observed among most of the other examined gramnegative enterobacteria. Only one of these strains showed a large increase at +4°C; a strain of Enterobacter cloacae. There was no appreciable difference in growth of Y. ent. on LSU and SS agar plates. DISCUSSION Y. ent. is characterized by a somewhat slower growth in the usual diagnostic media than the common enteropathogenic bacteria such as salmonella and shigella. There is thus a risk that the dominant gram-negative enterobacteria, for example E. coli or proteus, will outgrow and obscure the recognition of Y. ent. which has small colonies and requires 48 hours at room temperature to achieve the same colony size as the other enterobacteria. Therefore a selective procedure for the diagnosis of Y. ent. has a practical value. Low temperature selections, as described above, has resulted inabout a 40% increase in identification of Y. ent. The most notable increase was observed among the human pathogens of serotype 3, 8 and 9. These results would indicate that selective growth at +4OC can be successfully used "to isolate human pathogenic strains of Y. ent. from faeces. LSU agar (I) is routinely used only in a few laboratories, while SS agar is commonly used for faecal specimens. Isolation of Y. ent. on SS agar after incubation at low temperature has been shown to be practical and could easily be adapted in many laboratories.


Another advantage of this method is that the broth used for low temperature culturing is a standard nutrient broth without any special components. The selective diagnostic advantage of this system more than outweighs the delay that the diagnosis requires. Within the species Y. ent. are many different serotypes and even biotypes (6, 7, 8, 9). From a clinical point of view it is of practical value that the human pathogenic serotypes (serotype 3, 8 and 9) grow well at +4°C compared with the other serotypes which are mostly of low- or nonpathogenic character. ACKNOWLEDGEMENTS The author wishes to thank Professor Sten Winblad for encouragement and most valuable help with this article. The skilful technical assistance of Mrs Eva Karlsson is gratefully acknowledged.

REFERENCES I. Juhlin, I. & Ericson, C.: A new medium for the bacteriologic examination of stools (LSU-agar). Acta Pathol Microbiol Scand 52: 185, 1961. 2. Nilehn, B.: Studies on Yersinia enterocolitica with special reference to bacterial diagnosis and occurrence in human acute enteric disease. Acta Pathol Microbiol Scand, Suppl. 206, 1969. 3. Nilehn, B. & Ericson, C.: Studies on Yersinia enterocolitica. Bacteriophages liberated from chloroform treated cultures. Acta Pathol Microbiol Scand 75: 177, 1969. 4. Rappaport, F., Konforti, N. & Navon, B.: A new enrichment medium for certain Salmonellae. J Clin Pathol 9:261,1956. 5. Rappaport, F. & Konforti, N.: Selective medium for paratyphoid bacteria. Appl Microbiol 7: 63, 1959. 6. Wauters, G.: Contribution a l'etude de Yersinia enterocolitica. Thesis. Vander, Louvain 1970. 7. Winblad, S.: Studies on serological typing of Yersinia enterocolitica. Acta Pathol Microbiol Scand, Suppl. 187: 115, 1967. 8. - Studies on O-antigen factors of "Yersinia enterocolitica". International Symposium on Pseudotuberculosis, Paris 1967. 9. - Studies on the O-serotypes of Yersinia enterocolitica. In: Contribution to microbiology and immunology, vol. 2. Yersinia, Pasteurella and Francisella, p. 27. Karger, Basel 1973.

Address for reprints: J. Eiss, M. D., Department of Clinical Bacteriology, Allmiinna sjukhuset, 5-2/40/ Malmo, Sweden Scand J Infect Dis 7

Selective culturing of Yersinia enterocolitica at a low temperature.

Stool specimens from patients with suspected Yersinia enterocolitica (Y. ent.) infection were cultured at +4 degrees C in a nutrient broth for 5 days ...
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