1054 CAN. J. MlCROBlOL. VOL. 19. 1973

neuartiger Antibiotika aus Fusarien. Helv. Chim. Acta, 31: 594-602.

2. WIPF, H.-K., L. A. R. PIODA, Z. STEFANAC, and W. SIMON. 1968. Komplexe von Enniatinen und anderen Antibiotics mit Alkalimetall-Ionen. Helv. Chim. Acta, 51 : 377-381.

3. Qurrr, P., R. 0. STUDER, and K. VOGLER. 1963. Syn- these in der Depsipeptid-Reihe. 2. Synthese von Enniatin A. Helv. Chim. Acta, 46: 1715-1720.

4. SHEMYAKIN, M. M., Y. A. OVCHINNIKOV, V. T. IVANOV, V. K. ANTONOV, E. I. VINOGRADOVA, A. M.

SHKROB. G. G. MALENKOV, A. V. EVSTRATOV, I. A. LAINE, E. I. MELNIK, and I. D. RYABOVA. 1969. Cyclo- depsipeptides as chemical tools for studying ionic transport through membranes. J. Membrane Biol. 1: 402-430.

5. MACDONALD, J. C. 1969. Production of valinomycin. Can. J. Microbiol. 15: 236-238.

6. CAPPELLINI, R. A., and J. L. PETERSON. 1969. Sporula- tion of Gibberella zeae. 11. The effects of pH on macro- conidium production. Mycologia, 61 : 481-485.

An efficient ultrafiltration method for enterovirus recovery from ground beef1

JACK KONOWALCHUK AND JOAN I. SPEIRS Microbiology Diuisiotz, Departmetrt of National Health and Welfare, Otta~va KIA OL2, Cattarla

Accepted April 30, 1973

KONOWALCHUK, J., and J. I. SPEIRS. 1973. An efficient ultrafiltration method for enterovirus recovery from ground beef. Can. J. Microbiol. 19: 1054-1056.

An efficient and simple method is described for recovering coxsackievirus B5 from a 100-g sample of ground beef. After extraction of virus from the meat, the sample is clarified by a simple acid-base treatment enabling concentration of virus by ultrafiltration. Virus recoveries with this method, enu- merated as plaques, averaged about 67%.

KONOWALCHUK, J., et J. I. SPEIRS. 1973. An efficient ultrafiltration method for enterovirus recovery from ground beef. Can. J. Microbiol. 19: 1054-1056.

On dtcrit une methode simple et efficace pour rCcupCrer le virus coxsackie B5 a partir d'un Cchantillon de 100 g de boeuf hacht. Apres avoir isole le virus de la viande, 1'Cchantillon est clarifie par un simple traitement acide-base, ce qui permet la concentration du virus par ultrafiltration. Par cette methode nous avons rtcupert en moyenne 67% des virus, tel que dCterminC par le nombre de plages.

[Traduit par le journal]

Fresh commercial ground beef may contain ratory (5) indicated a 40% recovery of poliovir~ls enteroviruses and therefore present a health seeded into 100-g samples using ultrafrltration as hazard. The detection of such contamination has a method of virus concentration. We applied recently received some attention. In one report (4), where 5-g samples were tested, 25% contained one or more enterovirus types; in a different study (2), where 100-g samples were tested, 22% were positive for virus. The similar proportion of positive recoveries does not indicate that both methods were of equal efficiency. Enteroviruses are more likely to be detected in larger samples of ground beef than in small samples.

The relatively high proportion of positive samples referred to above, especially in view of the fact that ground beef is at times undercooked when eaten, stresses the importance of establish- ing reliable methods for isolation of virus.

An important feature of an isolation method is its efficiency. Sullivan et al. (4) recovered 75% of coxsackievirus B2 seeded into I-g samples of ground beef. A later report from the same labo-

'Received December 28,1972.

ultrafiltration for the practical and efficient recovery of coxsackievirus B5 added to 100-g samples of ground beef.

Portions of 100 g of fresh ground beef samples from local supermarkets were weighed in 1000- ml Erlenmeyer flasks. Coxsackievirus type B5 in 0.1 ml of phosphate-buffered saline was thor- oughly mixed into the meat samples by using a glass rod. (To determine the virus input a similar inoculum was added to 10 ml of medium 199 containing 10% fetal bovine serum.) Virus was recovered by shaking a 100-g sample with 200 ml of phosphate-buffered saline, pH 7.2, containing 0.1% peptone (Difco) on a ~nechanical shaker for 30 min at room temperature (about 22°C). The meat slurry was centrifuged at 1000 x g for 15 min to remove meat solids. The supernatant fluid was filtered through glass wool to relnove floating fat particles.

The filtrate (unclarified beef extract) contained

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fine particulate matter that restricted ultrafil- tration. This material could not be sedimented by centrifugation at 15 000 x g for 15 min but could be removed by the following acid-base treatment. Beef extract, at room temperature (lower temperatures hindered clarification), was adjusted to pH 3.0 with 2 N HCl and stirred for 2 min. The pH was readjusted to the original (6.1 to 6.4) by the slow dropwise addition, with constant stirring, of 2 N NaOH. Centrifugation at 1000 x g for 10 min resulted in sedimentation of the precipitate formed during alkali addition.

The clear amber supernatant fluid (clarified beef extract) was concentrated to a volume of 10 rnl with a type PM30 ultrafiltration membrane (Amicon Corp., Lexington, Mass.), 76 mm dia- meter, fitted in a magnetically stirred ultrafiltra- tion cell (Diaflo Model 402, Amicon Corp.) using a filtration pressure of 45 lb/in.'. The con- centrate was centrifuged at 15 000 x g for 15 min to remove most of the bacteria (6).

Virus was assayed by pipetting equal portions (2.5 ml) of sample or input control into each of four, 60 x 15 mm plastic dishes. Medium 199 containing 10% fetal bovine serum was added to a volume of 5 ml. HEp-2 cells at a concentration of 3.0 x 106/0.5 ml were added to each dish and virus was adsorbed onto shaken suspended cells as described before (I). Plaques were read in 3 days, averaged, and compared.

Selection of the PM30 membrane, from among several types with a mean pore size of 18 nm or less, was based on preliminary experiments which indicated rapid filtration flux and good coxsackievirus B5 retention (Table 1). Less than 1% of the virus was found in the filtrate. Larsson reported similar retention of poliovirus with the PM30 membrane (3).

Efficiency of extraction, clarification, and

NOTES 1055

TABLE 1 Comparison of filtration flux and coxsackievirus

B5 retention with membranes of different porosities

Membrane Time, min, of Virus as % of input in type* ultrafiltrationt concentrate filtrate

- -. -. . . -..

XMlOOA 35 8 1 14 XM50 1 20 94 0.3 PM30 30 94 0.5

*Mean pore diameter in nm: XM300.18; XMlOOA, 6; XM50, 3 r PM30. 2. Membrane diameter 76 mm. -, -. .... .. ~ ~ ~ . . - ~

?Phosphate-buffered saline containing 10% fetal bovine serum and 8 X 104PFU of virus reduced from 100 to 10 ml with pressures of 12 1b/in.2 for XM300 and XMlOOA and 45 1b/in.2

TABLE 2

Ultrafiltration time and coxsackievirus B5 recovery after concentration of clarified and unclarified

beef extract

Treatment of Time, h, of Virus recovery, beef extract ultrafiltration* % of input

Clarified 2.3(2.&2.5)7 89(8&97) Unclarified 7.0(6.5-7.5):: 26(20-32)

*Beef extract containing 3 x lo4 P F U of virus concentrated from 150 to 10 ml wit11 a PM30 membrane and a pressure of 45 Iblin.2.

?An average of six samples, extremes in parenthesis. $An average of two samples.

ultrafiltration was determined by comparing virus counts before and after each step. (An inoc- ulum of 3 x lo4 plaque-forming units (PFU) of virus was used and portions of .appropriate di- lutions of samples were assayed for total virus determination.) In four experiments, 15% (1 1 to 22%) of the input virus was lost during extraction. The volume of the unclarified beef extract varied from 160 to 180 ml, indicating that 10 to 20% of the diluent remained associated with the meat. Clarification of beef extract reduced virus counts by 7% (5.0 to 8.5%). The volume of extract lost

PROCEDURE TIME (H) TREATMENT

r ~ o o g gro_und beef

EXTRACTION

I 400 p$ virus 2 0 0 rnl diluent

~ o n c e i trate I

Centrifugation 15000Xg p ~ a c t e r i a

Supernate + 2 22" Medium

/'/ \ \ 0 0 0 0

1 I

CLARIFICATION 22O

I Z

!

Plaque count

I Centrifugation IOOOX g

C \ L ~ o l i d s Extract +

HCI (pH 3.0) NaOH (pf i 6.1-6.4)

I Centrifugation IOOOxg

Fprec ip i ta te - Clarified extract

I

FIG. 1. Procedure for the recovery of coxsackievirus B5 from 100-g, laboratory-inoculated sample of ground beef. for XM50 and PM30.

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1056 CAN. J. MlCROBlOL. VOL. 19. 1973

during clarification was also about 7%. In six of ground beef. The method is applicable to experiments, 11% (3 to 16%) of the input virus enteric viruses, which, because of their physical was lost during ultrafiltration. The use of clarified nature, can withstand exposure to pH 3.0. instead of unclarified beef extract reduced fil- tration time by two-thirds and increased virus recovery three times (Table 2).

In four separate experiments, 100-g ground beef samples were inoculated with 400 PFU of coxsackievirus B5 to test the efficiency of the method. A summary of the procedure including performance time is presented diagrammatically in Fig. 1. Virus recoveries as percentage of input virus were 66,73,62, and 66%, an average of 67%. Processing time took about 6 h.

Although virus recovery was reduced by clarification and concentration, the reduction in voluine from 160 to 10 ml enabled the economical assay of the entire sample. In this way ultrafil- tration concentration facilitated the recovery of small numbers of coxsackievirus B5 froin 100 g

1. KONOWALCHUK, J., and J. I. SPEIRS. 1972. Enterovirus recovery from laboratory-contaminated samples of shellfish. Can. J. Microbiol. 18: 1023-1029.

2. LARKIN, E. P., J. T. TIERNEY, A. L. REYES, R. M. E. NOVELLI, R. SULLIVAN, and R. B. READ, Jr. 1972. Microbiological content of ground beef: a survey of a large metropolitan area. Abstr. 72nd Annu. Meet. Am. Soc. Microbiol. E112.

3. LARSSON, B. 1970. Concentration of polio virus. Dialog (Amicon Corp.), 2(3).

4. SULLIVAN, R., A. C. FASSOLITIS, and R. B. READ, JR. 1970. Method for isolating viruses from ground beef. J. Food Sci. 35: 624-626.

5. TIERNEY, J. T., R. SULLIVAN, and E. P. LARKIN. 1972. A comparison of methods for the recovery of virus inoc- ulated into ground beef. Abstr. 72nd Annu. Meet. Am. Soc. Microbiol. E96.

6. WENNER, H. A. 1964. Outline of laboratory procedures for the diagnosis of enterovir~~s infections. 111 Diagnos- tic procedures for viral and rickettsia1 diseases. 3rd. Ed. EditedDy E. H. Lennette and N. J. Schmidt. American Public Health Assoication, New York. p. 247.

The effects of phospholipid depletion on the cleavage pattern of the cell walls of frozen Gram-negative bacteria1

A. FORGE' AND J. W. COSTERTON Depart~netzt of Biology, Utziversity of Calgary, Calgary, AIDertn

Accepted April 25, 1973

FORGE, A., and J. W. COSTERTON. 1973. The effects of phospholipid depletion on the cleavage pattern of the cell walls of frozen Gram-negative bacteria. Can. J. Microbiol. 19: 1056-1057.

Extraction of whole cells of the marine pseudomonad (B-16) with chloroform-methanol causes the disappearance of the cleavage planes, and the cross-sectioned profile of both the cytoplasmic membrane and the double-track layer of the cell wall.

FORGE, A., et J. W COSTERTON. 1973. The el'iects of phospholipid depletion on the cleavage pattern of the cell walls of frozen Gram-negative bacteria. Can. J. Microbiol. 19: 1056-1057.

L'extraction au chloroforme-methanol des cellules entieses d'une pseudomonade marine, (B-16), amene la disparition ~ L I plan de clivage ct ~ L I profil de la coupe transversale de la membrane cytoplas- mique et de la c o ~ ~ c h e B double feuillet de la paroi cellulaire. [Traduit par le journal]

We have reported that there is a single cleavage plane within the cell wall of the Gram-negative marine pseudomonad (B-16) (1). And further studies have established that this cleavage plane lies within the double-track layer of the cell wall (2), and that extraction of the phospholipids of the isolated double-track layer caused the dis- appearance of this plane (3).

We have extracted whole cells of this Gram- negative species with chloroform-methanol to

'Received June 8, 1972. 2Present address: Astbury Department of Biophysics,

University of Leeds, Leeds, Yorkshire, United Kingdom.

remove phospholipids from the cell envelope. Cells were grown and harvested as described before (2), and the cell pellet was suspeilded in 4 volumes of methanol and agitated for 20 min, then 8 volumes of chloroform were added and agitation continued for another 20 min. The extracted cells were pelleted by centrifugation at 35 000 x g for 10 min, the extraction was re- peated twice, and specimens were prepared for embedding and for freeze-etching.

Thin-sectioned preparations show that the cytoplasmic membrane and the double-track layer of the cell wall, which exhibit strong double-

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