Simple Bacterial Preservation Medium and Its Application to

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1978, p. 487491 0099-2240/78/0035-0487$02.00/0 Copyright i 1978 Amerian Soiety for Mirobiology Vol. 35, No. 3 Printed in U.S.A. Simple Baterial Preservation Medium and Its Appliation to Profiieny Testing in Water Bateriology M.

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1978, p /78/ $02.00/0 Copyright i 1978 Amerian Soiety for Mirobiology Vol. 35, No. 3 Printed in U.S.A. Simple Baterial Preservation Medium and Its Appliation to Profiieny Testing in Water Bateriology M. H. BRODSKY,* B. W. CIEBIN, AND D. A. SCHIEMANN Ontario Ministry ofhealth, Laboratory Servies Branh, Environmental Bateriology, Toronto, Ontario M5W IR5, Canada Reeived for publiation 12 September 1977 A medium omposed of nutrient broth, 1.8% bori aid, and 1% sodium hloride at ph 7.0 was shown to maintain the stability of Esherihia oli ultures for up to 10 days at room temperature. By using this preservation medium for preparing a simulated sample, a suessful profiieny test survey in water bateriology was onduted. A profiieny testing program in water bateriology must be based on enumeration and thus requires the provision of samples with a fixed number of baterial ells. Ideally, natural samples should be used; however, the baterial numbers in suh samples are not stable during storage (1), making natural water samples unsuitable for profiieny tests. A simulated water sample is the most pratial alternative (4, 11). Unfortunately, the baterial preservation tehniques useful in the qualitative profiieny testing programs of linial bateriology, suh as Stuart transport medium (3), Bat-Chek disks (7), and lyophilization (9), are not suitable for quantitative investigations in water bateriology. Although many baterial preservation methods will ensure that the organisms will remain viable, none are able to maintain a onstant number of baterial ells (2, 6, 15, 17). Only two approahes for preparing profiieny testing samples for bateriologial analysis of water ould be loated. Cada (4) employed a system utilizing lyophilized ultures of Esherihia oli at 104 and 106 ells per 100 ml. Analysis of his data indiates that although the ultures maintained their viability during the 7-day holding period, the numbers reovered from eah ampoule were not uniform, varying by as muh as 10-fold in some instanes. Gray and Lowe (10, 11) desribed a formateglutamate medium ontaining 1.8% bori aid (I.F.L.G. La-) for preserving seleted strains of E. oli and Klebsiella aerogenes for 7 to 10 days at room temperature. They used this tehnique for preparing simulated water samples for profiieny testing in Great Britain. During the ourse of their investigation, they examined a number of preservation media using a mostprobable-number tehnique for enumerating the organisms. In addition to the I.F.L.G. La- medium, they noted that nutrient broth with 1.8% bori aid maintained a stable ell number for an equivalent length of time; however, they did not pursue the investigation of this medium. Gray and Lowe also onsidered preserving E. oli ultures by freeze-drying on soluble paper disks. They were, however, unable to produe disks inoulated with relatively small numbers of oliforms that yielded uniform ell numbers when reonstituted in sterile water. They also reported that some of the reonstituted ultures of E. oli had lost their ability to ferment latose at 440C. This paper is a report of the development and pratial appliation of a simple baterial preservation medium for profiieny testing in water bateriology. MATERIALS AND METHODS Test organisms. Feal oliforms isolated from seondary sewage effluent by membrane filtration and M-FC medium (1) and biotyped (IMViC) as E. oli type 1 were used throughout the investigation. Preservation media The following media were investigated for their ability to maintain a onstant ell number during prolonged storage: (i) brain heart infusion broth (BHIB); (ii) distilled water, (iii) sodium hloride, 1% solution; (iv) ulture preservation medium A (CPM-A; in grams per liter): sodium hloride, 3.0; potassium hloride, 0.2; dipotassium phosphate, 1.15; monopotassium phosphate, 0.2; sodium thioglyolate, 1.0; alium hloride, 0.1; ethylenediaminetetraaeti aid, 2.76; glyerol, 270 ml; distilled water, 720 ml, ph 7.2; (v) CPM-A with 1.8% bori aid; (vi) CPM- A with 1.8% bori aid and 0.2% peptone; (vii) nutrient broth with 1.8% bori aid; (viii) nutrient broth with 1.8% bori aid and 1% sodium hloride, ph 7.0 (NSB). Storage suspensions. E. oli isolates were subultured from nutrient agar stok slants into BHIB and grown for 16 to 18 h on an Eberbah osillating shaker (200 osillations per min) at 35 C. All preliminary dilutions were made in 0.1% peptone water. The final dilution of the ultures for storage was made in the preservation medium itself. Culture suspensions 487

488 BRODSKY, CIEBIN, AND SCHIEMANN were stored in 1-oune (a. 29.6-ml) sterile, glass, srew-apped bottles in 10-ml volumes. Storage onditions. Samples held under refrigeration were kept at 4 C.

2 488 BRODSKY, CIEBIN, AND SCHIEMANN were stored in 1-oune (a ml) sterile, glass, srew-apped bottles in 10-ml volumes. Storage onditions. Samples held under refrigeration were kept at 4 C. Room temperature (23 ± 1 C) ultures were plaed in darkened upboards. Enumeration tehniques. Viable ounts were made either by the pour plate tehnique, using standard methods agar (BBL) and inubating at 35 C for 48 h, or by membrane filtration (Millipore HC WG57S3, lot ) on M-Endo LES agar (Difo) or M-FC medium (M-FC broth, BBL) with 1.2% agar (Oxoid). Eah ount was determined with five repliates. All dilutions were made with 0.1% peptone buffer. Statistial analysis. The uniformity of the inoulum for eah series of samples and the equivaleny of baterial reovery during storage were determined by analysis of variane. Profiieny test evaluation. A 16 to 18-h BHIB shake ulture of E. oli was diluted 1:10 in NSB medium. The NSB ulture was subdivided into 22 samples of 3 ml eah in srew-apped vials. The samples were arefully paked in boxes and delivered to the laboratories by either postal or ourier servie. No speial preautions were taken to guard against temperature flutuations during transit. Ten laboratories within the Ontario Ministry of Health partiipated in the survey. Eah laboratory reeived a suspension of the test ulture in NSB medium and instrutions on how to prepare the sample for analysis. The head tehnologist in eah laboratory was direted to add a predetermined volume of the NSB-preserved ulture to 100 ml of a buffer ommonly used, e.g., Butterfield phosphate buffer or 0.1% peptone buffer, to simulate a water sample. These samples were to be given to the benh tehniians with instrutions to prepare an appropriate dilution for doing five repliate filtrations onto M-Endo LES agar and M-FC medium. Nine samples were blindly seleted for referene and used to establish the uniformity of the inoulum and the aeptable limits for baterial reovery. RESULTS Without any other preservative, ells suspended in BHIB, distilled water, or 1% sodium hloride were ompletely unstable regardless of storage onditions. Survival of CPM-A-preserved ultures was poor at 4 C and nonexistent at room temperature, even with the addition of 1.8% bori aid. The shelf life of CPM-A-bori aid-preserved ultures ould not be extended by adjusting the ph to 7.0 or by adding 0.5% peptone to the suspending medium. Only nutrient broth medium with bori aid added was able to maintain the stability of E. oli fluidpreserved ultures during storage at room temperature. The optimum onentration of bori aid to be used as a preservative was determined by assay. The E. oli ulture was suspended in nutrient broth ontaining varying onentrations of bori aid and enumerated after being stored at room temperature (Table 1). Bori aid onentrations of 1.25, 1.5, and 1.8% were equivalent in maintaining ulture stability for 3 days, but only 1.8% bori aid showed promise for extending the shelf life beyond this time. The addition of 1.0% sodium hloride and adjusting the ph to 7.0 were found to improve the performane of the nutrient broth-bori aid preservation medium (NSB). We observed that the E. oli ulture suspended in NSB at 103 ells per ml survived 100% after 3 days of storage at room temperature, whereas the same ulture held at 101 ells per ml showed a 40% loss in viable ount. Table 2 shows the signifiane of initial ell density to the preservation of ulture stability during storage in NSB medium. Stabilization of the E. oli ulture during 10 days of storage was ahieved only when the initial ell density was at least 108 per ml. The reliability of the NSB medium for preserving an E. oli isolate during storage at room temperature was onfirmed by the results of four trials shown in Table 3. Analysis of variane (a = 0.01) of the data for eah trial onfirmed uniform reovery of ells during the 10-day storage period. The survival of different strains of E. oli isolates in NSB medium is shown in Table 4. Isolates 2 and 5 maintained the same TABLE 1. Effet of bori aid onentration in nutrient broth at ph 7.0 for preserving E. oli during room temperature storage Cell onn/fmla at storage time (days) of: Bori aid onn (%) NDb ND ' ND, Not done. TABLE 2. Effet of ell density in maintaining ulture stability of E. oli during storage in NSB medium at room temperature Log initial ell APPL. ENVIRON. MICROBIOL. % Survivala at storage time (days) of: no./mla NDb ND ND b ND, Not done.

VOL. 35, 1978 viable ount over 10 days of storage; isolates 1 and 3 began to show a signifiant redution in viability after day 7; isolate 4 did not survive beyond day 2 of storage.

3 VOL. 35, 1978 viable ount over 10 days of storage; isolates 1 and 3 began to show a signifiant redution in viability after day 7; isolate 4 did not survive beyond day 2 of storage. Profiieny test study. The results of nine samples analyzed in the referene laboratory are shown in Table 5. After 6 days of storage at room temperature, the bateriologial ounts of the NSB ultures were statistially uniform (a = 0.05), regardless of the diluent or reovery medium used, with the exeption of the ombination of phosphate buffer and M-FC medium. However, the mean total and feal oliform ounts were higher (46.6 and 40.2, respetively) when 0.1% peptone was the diluent than when Butterfield phosphate buffer was used (39.2 and 26.7, respetively). Also, the preision of the enumeration tehnique was distintly better when 0.1% peptone was used to reonstitute the ells (oeffiients of variation of 15% for both total and feal oliform ounts) than when Butterfield phosphate buffer was used as the diluent TABLE 3. Reproduibility of NSB ulture preservation method with a strain of E. oli Storage time at room Mean ounts/mla in trial: temp (days) NDb ND ND ND ND Statistial onlu- NSC NS NS NS sion by analysis of variane (a = 0.01) b ND, Not done. NS, Not signifiant. SIMPLE BACTERIAL PRESERVATION MEDIUM (oeffiients of variation of 24 and 34%, respetively). Table 6 shows the results of the inter-laboratory analysis of the simulated water samples. Laboratories A, B, and C, whih used 0.1% peptone water as the diluent, and D, E, F, G, and J, whih used phosphate buffer as the diluent, had total and feal oliform ounts aeptable within the 95% onfidene limits; laboratories H and I, whih also used phosphate buffer, had results unaeptable at the 99% onfidene limits. DISCUSSION We have demonstrated that preservation of stable baterial ultures for up to 10 days at room temperature is pratial using fluid suspensions. Furthermore, ulture stability during prolonged storage was shown to be as dependent on initial ell densities as on the preservation medium itself. CPM-A, whih was modeled after some onventional enteri transport media (8), was moderately suessful in maintaining stable E. oli ultures for short periods of storage; however, this medium ould not be modified to provide stability to the E. oli ulture during prolonged storage at room temperature. Without modifiation, nutrient broth medium with bori aid, as desribed by Gray and Lowe, did not preserve our E. oli ultures beyond 3 days of storage at room temperature. The inorporation of 1% sodium hloride, a preservative reommended by other investigators (5, 14), and the adjustment of the ph to 7.0 resulted in the development of a simple baterial preservation medium (NSB) apable of maint.aiing ulture stability during prolonged storage at room temperature. Our bori aid assay results support the findings of Porter and Brodie (16). A final TABLE 4. Survival of E. oli during storage in NSB medium Storage timne at Mean room temp ounts/mla for isolate: (days) NDb ND ND ND ND ND ND Statistial onlusion NSC up to 7 days NS up to 10 days NS up to 7 days Sd NS up to 10 days by analysis of variane (a = 0.01) b ND, Not done. NS, Not signifiant. d S, Signifiant. 489

490 BRODSKY, CIEBIN, AND SCHIEMANN TABLE 5. Summary of statistial analyses for sample homogeneity and establishment of aeptable bateriologial limits for profiieny testing evaluationa No.

4 490 BRODSKY, CIEBIN, AND SCHIEMANN TABLE 5. Summary of statistial analyses for sample homogeneity and establishment of aeptable bateriologial limits for profiieny testing evaluationa No. of Mean Coeffiient Statistial Aeptable mean ounts SD of variation r ANOVA (a 95%9 Diluent ompari- ountv/ml *0.05) Upper Lower Upper Lower 0.1% Peptone water M-Endo LES NS M-FC NS Butterfield phosphate buffer M-Endo LES NS M-FC S aanalyses were performed by nine tehniians using nine samples from the same lot as those distributed. These samples were held for 6 days in a dark upboard at room temperature. Eah analyst did five repliates, using both 0.1% peptone water and phosphate buffer as diluents. All media, membrane filters, and inubation onditions were kept uniform. b SD, Standard deviation. ANOVA, Analysis of variane. NS, Not signifiant; S, signifiant. di ± 1.96 x SD. ei ± 2.57 x SD. TABLE 6. Summary of results of simulated water sample with the NSB ulture preservation method Medium Laboratory Transit time Diluent useda M-Endo LES M-FC (h) xb SD CVd SD CV A 48 Peptone B 48 Peptone C 48 Peptone D 72 Phosphate E 48 Phosphate F 72 Phosphate G 48 Phosphate H 48 Phosphate I 72 Phosphate J 72 Phosphate Overall a Diluents used were 0.1% peptone water or Butterfield phosphate buffer. b X, Mean ount determined from five repliates. SD, Standard deviation. d CV, Coeffiient of variation as a perentage. onentration of 1.8% bori aid had the best preservative effet. We onsidered using paper disks as a means of holding ultures in preservation media. Although we were able to prepare ulture-saturated disks (Difo ) with equivalent numbers of E. oli per disk, using a miropipette (Pipettman P-200, Gilson), we ould not reover more than 56% of the ells that were added to the disk, despite vigorous shaking in dilution buffer. In addition, we were unable to prolong the shelf life of disk-preserved ultures, regardless of the preservation medium. The use of soluble paper disks might make this approah more feasible. Sourek (17) suggested that every baterial strain of a given serologial type had different viability harateristis. This onlusion was supported by the results of Gray and Lowe and by our own investigation. The shelf life of the E. oli isolates in NSB medium was a variable harateristi. Our searh for an NSB-stable ulture, although not extensive, was, nevertheless, reasonably suessful. Four of five E. oli ultures, isolated from seondary sewage effluent, remained stable in NSB medium for at least 7 days at room temperature, and two of these were stable for 10 days. The evaluation of the ability of a bateriologial preservation medium to maintain stable ul- APPL. ENVIRON. MICROBIOI-

VOL. 35, 1978 SIMPLE BACTERLAL PRESERVATION MEDIUM 491 tures depends, to a large extent, on the methods used to reover and enumerate the ells after storage (5, 15, 17).

5 VOL. 35, 1978 SIMPLE BACTERLAL PRESERVATION MEDIUM 491 tures depends, to a large extent, on the methods used to reover and enumerate the ells after storage (5, 15, 17). Our inability to dupliate the results of Gray and Lowe, using nutrient with bori aid, may be related to our use of a pour plate, rather than a most-probable-number, tehnique for enumeration. Although the mostprobable-number tehnique is less preise than diret enumeration, broth enrihment has been shown to reover injured ells (whih are likely to our in preserved ultures) better than the pour plate tehnique (13). The analysis of the referene ultures demonstrated the effet of the two diluents on the reovery of stored ells. NSB-suspended E. oli, reonstituted in Butterfield phosphate buffer after 6 days of storage at room temperature and enumerated on M-FC medium at 44.5 C, resulted in feal oliform ounts signifiantly lower than those of the same ulture reonstituted in 0.1% peptone water. In addition, the mean total oliform ounts of the ells reonstituted in 0.1% peptone water were numerially higher, and the repliate enumeration was more preise, than those of the same ultures reonstituted in Butterfield phosphate buffer. Straka and Stokes (18) had previously demonstrated improved reovery of baterial ells when 0.1% peptone water rather than phosphate buffer was used as the diluent. The effet of the quality of distilled water on the performane of phosphate buffers was reently reported by Jensen and Hausler (12). Our interlaboratory profiieny test results demonstrate the possibility of this effet. Only three laboratories, A, B, and C, used 0.1% peptone water as the diluent. The overall mean ounts per milliliter pbtained by these laboratories were 43.3 on M-Endo LES agar and 34.2 on M-FC medium. All other partiipating laboratories used Butterfield phosphate buffer as the diluent. The potential toxi effet of this buffer was shown by the variable and partiularly low reovery of E. oli ells on M-FC medium reported by laboratories D (21.8), H (5.8), I (8.2), and J (19.8). The M-FC results from laboratories E (33.8), F (28.8), and G (33.8) were aeptable. Until a tehnique is developed that will ensure the stability of baterial numbers in natural water samples for prolonged periods, NSB medium provides a simple ompromise for profiieny testing purposes. An investigation on the stability of other speies of bateria in NSB medium and other appliations of this preservation tehnique are in progress. LITERATURE CITED 1. Amerian Publi Health Assoiation Standard methods for the examination of water and wastewater, 14th ed. Amerian Publi Health Assoiation, Washington, D.C. 2. Anthieunisse, J Viability of Iyophilized miroorganisms after storage. Antonie van Leeuwenhoek J. Mirobiol. Serol. 39: Barry, A. L., and K. L. Bernsohn The role of quality ontrol in the linial bateriology laboratory. Am. J. Med. Tehnol. 34: Cada, R. L Profiieny test speimens for water bateriology. Appl. Mirobiol. 29: Chane, H. L Salt-a preservative for baterial ultures. J. Bateriol. 85: Devay, J. E., and W. C. Shnathorst Single ell isolation and preservation of baterial ultures. Nature (London) 199: Douglas, G. W., A. Balows, D. Rhoden, K. Tomfohrde, and P. B. Smith In-use evaluation of a ommerially available set of quality ontrol ultures. Appl. Mirobiol. 25: Edwards, P. R., and W. H. Ewing Identifiation of Enterobateriaeae, 3rd ed. Burgess Publishing Co., Minneapolis. 9. Gavan, T. L A summary of the bateriology portion of the 1972 basi, omprehensive, and speial, College of Amerian Pathologists (CAP) Quality Control Program. Am. J. Clin. Pathol 61: Gray, R. D An improved formate latose glutamate medium for the detetion of Esherihia oli and other oliform organisms in water. J. Hyg. 62: Gray, R. D., and G. H. Lowe The preparation of simulated water samples for the purpose of bateriologial quality ontrol. J. Hyg. 76: Jensen, J. P., and W. J. Hausler, Jr Contribution of KH2PO4 to toxiity in phosphate buffered dilution water system. J. Milk Food Tehnol. 39: Koburger, J. A., and J. L Oblinger Organisms from positive MPN tubes inoulated with samples that yielded no growth on pour plates. J. Food Protetion 40: Kropinski, A. M Stability of baterial mutants in saline. Appl. Mirobiol. 29: LaPage, S. P., J. E. Shelton, T. G. Mithell, and A. R. Makenzie Culture olletions and the preservation of bateria, p In J. R. Norris and D. W. Ribbons (ed.), Methods in mirobiology, vol. 3a. Aademi Press In., New York. 16. Porter, I. A., and J. Brodie Bori aid preservation of urine samples. Br. Med. J. 2: Sourek, J Long-term preservation by freeze-drying of pathogeni bateria of the Czehoslovak National Colletion of Type Cultures. Int. J. Syst. Bateriol. 24: Strada, R. P., and J. L Stokes Rapid destrution of bateria in ommonly used diluents and its elimination. Appl. Mirobiol. 5:21-25.

Proliferation of Legionella pneumophila as an Intracellular Parasite

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1984, p. 467-471 0099-2240/84/030467-05$02.00/0 Copyright C) 1984, Amerian Soiety for Mirobiology Vol. 47, No. 3 Proliferation of Legionella pneumophila as