351 DISTILLED WATER SUITABILITY FOR MICROBIOLOGICAL APPLICATIONS E. E. GEIJ>REICH AND H. F. CLARK1 Basic and Applied Sciences Branch Division of Water Supply and Pollution Control U. S. Department of Health, Education, and Welfare Robert A. Taft Sooitary Engineering Center Cincinnati, Ohio (Received for publication June 26, 1965) SUMMARY The pH for distilled water supplies used in labora­ Downloaded from http://meridian.allenpress.com/jfp/article-pdf/28/11/351/2396684/0022-2747-28_11_351.pdf by guest on 25 September 2021 Criteria for distilled water suitable for microbiological use tories in various geographical locations ranges from are discussed with some observations on biological and chemi­ 5.8 to 6.8. The pH of our laboratory distilled water, cal contaminants. An expanded description of a suitability fresh from the supply line, is 6.2; after storage in a test for distilled water based on growth response of Aerobacter glass carboy for 1 week, 6.4; and after conversion aerogenes in a minimal medium is reported and selected into freshly prepared rriple distilled water, 6.8. These examples of its application are given. are the pH values that would be expected if C02 The quality of distilled water for laboratory use were a major source of contamination. has been the subject of some confusion for many Microbiological analysis of a distilled water supply years. Theoretically, distilled water should only may at times show large numbers of Pseudomonas contain H 20. Such a product is not usually attain­ and Achromobacter species. These organisms are able, however, because of limitations on production capable of survival and growth in the presence of and storage methods that influence the amount of minimal concenb'ations of nutrients. Our examination contamination. The cost per unit volume also limits of one distilled water system showed Pseudomonas the degree of purity and costs scale sharply upward counts ranging from 21 to 51 per ml from the build­ when chemical and biological purity are required. ing supply lines. Higher counts were observed when Thus the specifications for a distilled water should samples were examined from individual laboratory be tailored to the laboratory needs. carboys. In one instance, several coliforms per liter Distilled water for microbiological use should be were found in the distilled water supply as a result free of inorganic and organic substances, either toxic of inadequate storage tank protection from dust or nub'itive, that could influence survival or growth contamination. At other times the source of various of bacteria and viruses. For most laboratories, the bacterial contaminants· has been demonsb'ated to be distilled water SQpply should be free of microbiologi­ the deionizing column or the carbon filter used in cal organisms that might conb'ibute inhibitory sub­ conjunction with a given distillation system. stances; and in laboratories producing various bio­ Once species of Pseudomonas have gained enrry and logicals, the distilled water must be pyrogen free. begun to multiply in a distilled water supply, their Many factors can influence the quality of a lab­ antagonistic action toward other organisms may be a oratory supply: (a) design of the distillation appar­ source of laboratory concern. Waksman (10), in are­ atus; (b) source of raw water; (c) condition of the view on bacterial antagonisms, reported that species deionizing column, if used; (d) use of a carbon filter; of Pseudo1T!01UIS can adversely affect the growth of (e) storage chamber for reserve supply; and (f) tern­ Escherichia coli, Salmonella typhosa, Serratia mar­ perature of stored supply and duration of storage be­ cescens, Corynebacterium diphtheria, Mycobacterium fore use. These factors may conrribute varying de­ tuberculosis, and Vibrio comma, among others, and grees of contamination such as metal ions from the . that the active substance is thermostable. Beitler distilla:tion system; ammonium hydroxide, hydro­ and Seligmann (7) reported that Pseudomonas aeru­ chloric acid, and other fumes from the laboratory; ginosa can inhibit E. coli in peptone water within chlorine from the tap water supply; and C02 from a critical range of peptone concenb'ation. the air. Algal spores can also enter the distilled water sys­ The pH of distilled water is rarely 7.0. In studies tem as airborne contaminants and have often been of the bactericidal effect of silver, Chambers, Proctor, observed in carboy water supplies in individual and Kabler (2) reported the pH of distilled water laboratories. This is particularly rrue when glass prepared from Cincinnati tap water averaged 6.2. containers -with loose-fitting cotton stoppers (or no stopper) are located in areas where there is intense 'Deceased. light. Under these conditions and with infrequent 352 DISTILLED wATER SUITABILITY use of the supply, a sizable growth of algae often plied to the committee for Standard Methods for the develops within a few weeks. The necessary nutri­ Examination of Dairy Products, 11th edition (1). ents may come from the distilled water or dust fall­ 1bis approach to a biological test for distilled water ing into the poorly covered opening. In time, the was based on a study of the growth of pure cultures algae may contribute organic products of their me­ of bacteria in a minimal growth medium prepared tabolism; some may be toxic to certain bacterial with distilled water from the source in question. species, whereas others may add sufficient nutrients The present report deals with an expanded descrip­ to stimulate multiplication of bacterial contaminants. tion of the distilled water suitability test and with Spoehr et al. (8) demonstrated that material showing some examples of its application. antibacterical activity could be obtained from killed cells of Chlorella. The authors also found the anti­ MATERIALS AND METHODS bacterial material was produced in larger quantity A medium containing minimal amounts of nutrients was Downloaded from http://meridian.allenpress.com/jfp/article-pdf/28/11/351/2396684/0022-2747-28_11_351.pdf by guest on 25 September 2021 when killed cells were aerated than when they were devised both to support moderate growth of Aerobacter aero­ placed under anaerobic conditions. genes and to be very .sensitive to any "foreign" additives that One criterion of the quality of distilled water is might be introduced by way of the distilled water used to prepare the medium. In fact, with the use of. carefully conductivity. Conductivity measurements can be selected reagents and glassware rinsed with triple distilled used to indicate the presence of inorganic ions of water, the sensitivity of the test could be extended to detect metals, salts, and bases. The specific resistance of irregularities in glassware cleaning procedures that can cause good quality water should exceed 578,000 ohms at difficulties in virological tissue culture studies. 18 C (equivalent to conductivity of 0.5 ppm as NaCl). A. aerogenes was chosen because it can grow in minimal The conductivity measurement, however, does not nutrients and does not require the complex amino acids or other additives that are necessary for E. coli or Streptococcus distinguish between the presence of toxic or nontoxic fecalts. Ps. aerugirwsa in particular, and possibly other psen­ metallic ions and does not reveal any organic con­ domads, will also grow in minimal nutrients, but they would taminants that also may be present. be insensitive to any inhibiting factors from other strains Price and Gare (6) reported that traces of volatile of the same organism present in the unknown distilled water. · The minimal requirements for a medium to support a short-chain fatty acids can occur in distilled water moderate growth~ of A. aerogenes are carbon source ( ci~rate), and can be a serious source of inhibitory interference nitrogen source ( anunonium sulfate), salt mixture {magnes­ in microbiological assays. These organic contamin­ ium, calcium, iron, and sodium), and a buffer (phosphate) ants may be derived from volatilized distillates of solution to keep the pH from shifting into a lethal range. dead organisms accumulating in the distillation re­ All chemicals used in preparation of these stock solutions tort; various endogenous macromolecular constituents were analytical reagent (AR) grade. This was particularly important in preparing potassium dihydrogen phosphate degradated from dead cells in distilled water supplies { KH2PO4 ) since some brands have significant amounts of (9 ), volatile organic fumes in the laboratory, accumu­ chemical impurities. Garvie (3) found the chemical impuri­ lated slugs of organic debris released by a carbon ties in phosphate buffer and single distilled water would sup· filter, solder flux and grease residuals encountered port the growth of Pseudomonas fluorescens without any in new supply installations, or detergent residuals other additives. Preparation of the various nutrients was as following flushing of the system. Thus, if only a follows: specific conductivity measurement is used to check Carton source: Dissolve 0.29 g sodium citrate ( Na3C6H 50 7• on water quality, these contaminants probably will 2H20) in 500 ml double-distilled water. not be detected. Nitrogen source; Dissolve 0.26 g ammonium sulfate (NH ) S0 in 500 ml double-dbtilled water. As a result of these conditions, when any organic 4 2 4 Salt mixture: Dissolve the following mixture of electrolytes ~complexes are present in sufficient amounts to be in 500 ml double-distilled water: either toxic or nutritive, the first evidence of distilled Magnesium sulfate ( MgSO • 7H 0 ) 0.26 g water impurities may be observed in erratiCJeplicate 4 2 Calcium chloride {CaCl ·2H 0) 0.17 g pour plate or membrane filter counts and irregular 2 2 Ferrous sulfate {FeS04·7H20) 0.23 g growth in certain minimal nutrient culture media. Sodium chloride { NaCl) 2.50 g Such difficulties may also be a result of improper Buffer: Use a 1 to 25 dilution of stock phosphate buffer washing procedures, which are related to detergents solution that is prepared by dissolving 34 g of potassium that leave toxic residues on glassware items.