IN VITRO 16{9~:751-753;1980 All rightsreserved9

P A S S A G E O F COR Y N E B A C T E R I U M A Q U A T I C U M T H R O U G H MEMBRANE FILTERS WARNER PHELPS,' ROBERT BAUGHN, AND HOMER S. BLACK

Department of Dermatology, Baylor Collegeof Medicine, and PhotobiologyLaboratory, Veterans Administration Medical Center, Houston, Texas 77030 {Received July 18, 1979; accepted January 11, 1980)

SUMMARY Cellulose ester and polycarbonate membrane filters were tested for sterilizing capability with a contaminant, Corynebacterium aquaticum, isolated from cell culture medium. This contaminant, often found in water supplies, routinely passes filters of the polycarbonate type, but not of the cellulose ester type. C. aquaticum grows slowly requiring 7 to 10 days incubation in BME at 37 ~ C before turbidity develops. Cryptic contamination is potentially a serious problem with this organism.

Key words: cell culture; contaminant; medium; sterilization; membrane filters. INTRODUCTION The use of membrane filters to sterilize a culture medium has become a standard procedure (1,2}. Two different filter compositions are commonly used, namely cellulose ester and polycarbonate. The routine examination of cell culture medium demonstrated a slow-growing contaminant after filtration with a polycarbonatetype filter. This source of contamination was eliminated by use of cellulose ester filters. MATERIALS AND METHODS

Filter comparison. One liter of Eagle's Basal Medium (BME) was prepared with 10% fetal bovine serum (Grand Island Biological Company, Grand Island, NY). The components were sterilized before contamination by filtration through disposable cellulose ester filters {Nalgene, Rochester, NY}. This procedure was previously shown to result in sterilization by incubation of the medium for 10 days at 37 ~ C. The contaminant was maintained in serial culture in BME and a four-day-old culture was used to contaminate sterile BME (2 ml contaminant/1000 ml mediumL Two identical 90-mm filter holders {Millipore, Bedford, MA} were pre-

pared with the test filters, either a 0.2-/~m polycarbonate filter or a 0.22-~m cellulose ester filter. More than one lot was evaluated from one supplier of each filter type. The holders were autoclaved simultaneously. Each filter processed 500 ml of the contaminated medium. Medium was collected in 100-ml aliquots and incubated for 10 days at 37 ~ C before being recorded as positive or negative for contaminant growth as evidenced by the appearance of turbidity. Samples of BME were removed for dilution plating before and after filtration for each filter tested. All filters used in this study passed a bubble test at the completion of filtration. Polycarbonate testing. Further experiments were performed to examine the retention capability of the polycarbonate filters. One liter of BME was prepared as previously stated and contaminated with one of two bacterial dilutions (1:500 and 1:50,000, bacterial culture:medium, respectivelyL Two identical 90-mm filter holders with 0.2-#m polycarbonate filters were autoclaved simultaneously. Each filter was used for 500 ml of medium contaminated with the bacterium and the filtrate was collected in 100-ml aliquots that were incubated for 10 days as before. Samples of medium were removed before and after filtration for quantification by dilution plating. Dilution plating. Bacterial growth before and after filtration was followed by plating either 20 #1 or 1 ml of 10-fold serial dilutions, made in

~To whom requests for reprints shotdd be addressed at Department of Dermatology, Baylor College of Medicine, Houston, Texas 77030. 751

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PHELPS, BAUGHN, AND BLACK TABLE 1 T H E RETENTION OF CORYNEBACTERIUM AQUATICUM BY POLYCARBONATE AND CELLULOSE ESTER MEMBRANE FILTERS Cellulose Ester

Polycarbonate

Before filtration a 1.30 x 102 After filtration a <40 b Growth upon incubation (: -a Bacteria per milliliter. b Forty bacteria per milliliter is the limit of detection by this method. c Growth is defined as turbidity in medium after 10 days incubation at 37 ~ C.

1.30 x 10~ 4.2 x 10~ -4-

p h o s p h a t e buffered saline, on blood agar. P l a t e s were i n c u b a t e d for 10 days a n d the n u m b e r of colony-forming units ( C F U ) per milliliter was determined. Bacterial identification. T h e isolate was tested for catalase a n d urease activity, motility, gelatin liquefaction, a n d n i t r a t e reduction, a n d for the ability to f e r m e n t glucose, maltose, a n d sucrose in oxidation f e r m e n t a t i o n (OF) m e d i u m . I t was also cultured on blood a g a r (3).

teria were detected in t h e filtrate by p l a t i n g as well as by i n c u b a t i o n . Bacterial identification. T h e isolate was identified as Corynebacterium aquaticum. N o h e m o lysis was seen on blood agar. T h e o r g a n i s m is catalase positive a n d motile, negative for urease production, gelatin liquefaction, a n d n i t r a t e reduction. I n O F m e d i u m it is positive for sucrose a n d maltose utilization a n d positive for glucose u n d e r aerobic conditions.

EXPERIMENTAL RESULTS

DISCUSSION

T h e c o m p a r i s o n of p o l y c a r b o n a t e a n d cellulose ester filters is s u m m a r i z e d in T a b l e 1. N o b a c t e r i a were detected after filtration with the cellulose ester filter, either b y dilution p l a t i n g or by incubation of the filtrate. Conversely, b a c t e r i a were detected by b o t h m e t h o d s after filtration t h r o u g h p o l y c a r b o n a t e m e m b r a n e s . T a b l e 2 shows the resuits of f u r t h e r tests with the p o l y c a r b o n a t e m e m branes. U s i n g the 20-~1 dilution plating procedure, no b a c t e r i a were detected in the filtrate. However, u p o n i n c u b a t i o n , g r o w t h of the cont a m i n a n t was a p p a r e n t . By plating a larger volume a n d using a slightly h i g h e r challenge, bac-

Since this slowly growing c o n t a m i n a n t requires 7 to 10 days i n c u b a t i o n at 37 ~ C before t u r b i d i t y develops, the p o t e n t i a l for cryptic c o n t a m i n a t i o n of s h o r t - t e r m studies or stock cultures is considerable. A culture of 108 o r g a n i s m s / m l is only slightly t u r b i d . T h u s a c o n t a m i n a t e d culture could easily c o n t a i n more b a c t e r i a t h a n m a m m a l i a n cells. W h i l e the r e m o v a l rate varied somew h a t , in no case did the p o l y c a r b o n a t e filters remove less t h a n 9 9 . 5 % of the b a c t e r i a a n d in no case was the c u l t u r e d m e d i u m negative for g r o w t h at 10 days. F u r t h e r m o r e , in no case did the cellulose ester filters fail to sterilize m e d i u m passed through them. It is not i m m e d i a t e l y a p p a r e n t why two filters with the s a m e pore size would b e h a v e so differently. T h e r e are several possible explanations. One possibility is the m a n u f a c t u r i n g m e t h o d used for each. T h e cellulose ester filters are a m a t of fibers with a n a v e r a g e pore size of 0.22 ~m. T h e p o l y c a r b o n a t e filters contain pores etched from ion tracks i n t r o d u c e d by a radioactive source. As such, the pores pass directly t h r o u g h the filters. T h e more t o r t u o u s p a t h of the cellulose ester filters m a y b e m o r e efficient at t r a p p i n g particles with d i m e n s i o n s close to the r a t e d pore size. F u r ther, since t h e pores in the p o l y c a r b o n a t e filter are b a s e d on a r a n d o m event, t h e r e will be pores t h a t overlap, t h u s p r o d u c i n g a pore with twice or t h r e e

TABLE 2 T I t E PASSAGE OF CORYNEBAC'I'ERIUMAQUATICUM BY POLYCARBONATE MEMBRANES

Before Filtration a

After Filtration a

Growth U pon Incubation b

2.68 • 10~ 2.1 x l 0 '

< 40 c <40

+ -t-

3.4 • 10~ 4.4• 102 + 2.12• 10~ 2.8x 10' + a Bacteria per milliliter. b Growth as turbidity in medium after 10 days incubation at 37 ~ C. c Forty bacterial per milliliter is the limit of detection by this method.

PASSAGE OF COB YNEBA CTERIUM THROUGH FILTERS times the rated size. These large pores could conceivably pass particles at, or slightly larger than, the rated pore size. Additionally, a representative of the manufacturer has suggested that under certain circumstances traces of the etching agent may remain in the membrane pores and enlarge them slightly upon autoclaving. Since Corynebacterium aquaticum approaches the membrane pore size, any alterations of the trapping efficiency will be more critical with this organism than with larger organisms. At any rate, the necessity of thorough routine testing of culture medium is apparent.

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REFERENCES 1. Paul, J. Cell and tissue culture. New York: Churchill Livingstone; 1975: 143-144. 2. Morton, H. J.; Morgan, J. F. Sample preparations of media. Kruse, P. F., Jr.; Patterson, M. K., Jr. eds. Tissue culture methods and applications. New York: Academic Press; 1973: 686-692. 3. Leifson, E. The bacterial flora of distilled and stored water. III. New species of the genera Corynebacterium, Flavobacterium, Spirillum and Pseudomonas. Bull. Bacteriol. Nomencl. Taxon. 12: 161-170; 1962.

The authors gratefully acknowledge the assistance of Mrs. Wanda Lenger and Mrs. Carolyn Wells. This work was supported in part by USPHS Grant 5 F32 CA05635-02 from the National Cancer Institute.