IN VITROVol. 19, No. 9, September1983 9 1983TissueCultureAssociation,Inc.

V A R I A T I O N IN C O M P O S I T I O N O F M E D I A AND R E A G E N T S U S E D IN T H E P R E P A R A T I O N OF CELL CULTURES FROM HUMAN AND OTHER ANIMAL TISSUES: DULBECCO'S, E A R L E ' S , AND H A N K S ' B A L A N C E D S A L T S O L U T I O N S C. N. BURKE ANDG. CROXALL

Department of Pathobiology, Univershyof Connecticut, Storrs, Connecticut06268 (Received February 7, 1983; accepted April 4, 1983)

SUMMARY When comparison was made of directions given for three salt solutions commonly used in cell culture preparations and identified as Dulbecco's, Earle's, and Hanks', variations in composition were found. Some significantly alter the suitability of the materials for the intended use. Other differences may have less effect. This brief review reveals a tendency among researchers to follow procedures obtained from colleagues for the preparation of laboratory reagents and media, to cite the original publication rather than their colleagues' work as the source of the information, and to fail to compare the two for differences. Some failures in cell culture propagation may be attributed to similar instances with other published but incorrectly cited work. Tables are provided that facilitate comparison of the correct original formulations with variants from selected published sources.

Key words: Hanks; Dulbecco; Earle; cell culture media. INTRODUCTION To incorporate the most recent scientific advancements into ongoing or newly initiated studies, heavy reliance is placed upon current publications for procedures, composition of reagents, and sources of materials. Not infrequently insufficient details are given in an important paper because authors, in the interest of brevity, cite previous publications upon which they, in turn, relied for certain aspects of their work. In adopting new procedures, an involved and time-consuming literature search is often required. In addition to the advantage of a thorough (though reluctantly undertaken) review, other benefits are sometimes realized, among which is the discovery of long perpetrated citation errors and unexplained variations in procedures or composition of reagents and materials. Some variations may be attributed to undiscovered author or typographical errors. Most common, however, are variations due to changes that occur in the laboratory and, if not obviously damaging to the research, go undetected. These become part of the literature when the altered procedure is published while still carrying the citation

of the original paper. This is of concern because the origin of the differences between the two is not explained, and other researchers follow the variant procedures while citing the original publication, perpetuating the error. In several previous reports, areas of concern involving various aspects of media preparation were identified. Hayflick and associates (1) called attention to variations that were attributable to the state of the reagents used (purity and methods of combination). Morton 12) identified several instances where the modifications were instituted by the original authors, but published in later, less well-known papers. This report, however, focuses on errors in various publications due to inaccuracies of citation. In the tables that follow, the range of differences between an original formulation and subsequent publications that cite the original are given. These include but are not limited to the addition or omission of components, incorrect gram weights, failures to correct for hydration states, and the listing of incorrect components. These differences will be seen when the composition of the original paper (first column of each table) is compared with that of the publications selected as

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examples. It is important to note that all, however, cite the original paper as the source of the information.

Phosphate Buffered Saline This medium was first described by Dulbecco and Vogt in 1954 (3}. Samples of differences that have evolved since that time are given in Table 1. The formulation of the original paper will be found in Column A. Variations that should be noted are" incorrectly cited gram weights for NaCI and KCI; improperly adjusted weights for hydrated components; the addition of phenol red, which was not in the original formulation; and the omission of MgCL in one publication t4}, persisting in the literature to reappear, uncorrected, 11 yr later under different authorship t5}.

Earle's Balanced Salt Solution Of all the references searched, Earle's balanced salt solution (11~ proved to be one of the most interesting because of the citation date and journal. With one exception, all authors referenced Earle's article of 1943, which appeared in the Journal of the National Cancer Institute ~12}. The original formulation on which so many papers are based was published, however, 9 yr earlier by Earle in the Archiv fiir Experimentelle Zellforschung {11}. Of the many publications examined, only one made reference to the 1934 paper. Among the references included in Table 2, four of the six do not give the corrected gram weights for the hydrated forms of one or more components. Another weight variant occurs with NaHCO3 resulting in an alteration of its buffering molarity in the final preparation. Compositional alterations also occur with glucose, which is omitted from the list of materials in one reference and then occurs in the text with no more guidelines as to the correct amount to be used than "as desired." In addition, phenol red was commonly included in many papers and manuals reviewed. This was not, however, listed as one of the materials included in either of the publications by Earle, although the authors made reference to one or the other in each instance.

Hanks' Basic Salt Solution Of the media surveyed for this study, the fewest variations in composition were found among references citing the early publications by Hanks (14,151. Some confusion arises, however, when

the date of the "original" paper is variously given as 1946 (8}, 1948 (14L and 1949 (15}. Some differences exist between the 1948 and 1949 papers, and these are best seen by examination of the first two columns of Table 3. In one publication, which compares the two papers in the same table (5}, the weight of glucose cited is attributed incorrectly to the 1949 paper rather than the 1948. This error has been found in other references also (16). Another author lists the components and gram weights of both publications, but does not provide a reference for either of the alternative preparations (17}. In addition, there are no indications of the hydration status of any of the components listed inasmuch as descriptive rather than chemical formulations are given. Several of the references (7,8,18} split the source of magnesium between the MgSO4 and MgC12. Although reference is made to Hanks' early publications (14,151, this variation does not, in fact, occur in either. Through 1955, Hanks still indicated his source of magnesium to be by the use of MgSO4 alone (19}. Another variation in composition occurs because of a heading in a table i16} where the monobasic form of sodium phosphate (NaH2PO~'2H~O~ was erroneously substituted for the dibasic form (Na2HPOo'2H20}. These variations are referenced in Table 3.

D ISCUSSION The references included in the tables of this publication represent only a few of the many surveyed. The problem of unexplained variations in the preparation of the three salt solutions was far more frequent than anticipated. Some variations have, in fact, become so common that although the original paper is cited, these variations are more frequently seen than the original formulations. Of the many variations found during this survey, those that affected the concentration of components were the most frequently encountered and were due to the failure to correct for the hydration states of the components used. Of the variations that alter the composition of a preparation, omissions or substitutions were the most frequent source of differences. These errors were most often traced to tal reliance on the accuracy of other authors who quoted or cited an original paper, but who published a laboratory variant of the cited procedure, or (bl a citation that was incorrect for date or content. Commercial sources that publish the composition of their market-ready products were found to be

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a c c u r a t e in citation as well as in f o r m u l a t i o n , a n d v a r i a t i o n s were correctly identified. T a b l e s in this p a p e r not only p r o v i d e t h e correct f o r m u l a t i o n s a n d citations of the original p a p e r s b y H a n k s , D u l b e c c o a n d Vogt, a n d Earle, b u t also will facilitate c o m p a r i s o n with t h e v a r i a n t f o r m u l a t i o n s t h a t h a v e b e e n p u b l i s h e d in promin e n t works.

REFERENCES 1. Hayflick, L.; Jacobs, P.; Perkins, F. A procedure for the standardization of tissue culture media. Nature 204: 146-147; 1964. 2. Morton, H. J. A survey of commercially available tissue culture media. In Vitro 6: 89-108; 1976. 3. Dulbecco, R.; Vogt, M. Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 99: 167-182; 1954. 4. Kuchler, R. J. Milieu for maintaining and growing animal cells in vitro. Biochemical methods in cell culture and virology. Chapt. 2. Stroudsburg, PA: Dowden, Hutchinson and Ross, Inc.; 1977: 45-89. 5. Waymouth, C. Construction and use of synthetic media. Willmer, E. N. ed. Cells and tissues in culture. Methods, biology, and physiology. Vol. 2. New York: Academic Press; 1966: 99-142. 6. Adams, R. L. P. Media formulations. Work, T. S.; Burdon, R. H. eds. Laboratory techniques in biochemistry and mnlecular biology. Cell culture for biochemists. Vol. 8. New York: ElsevierNorth Holland Biomedical Press; 1980: 246-270. 7. Bashor, M. M. Basic methods of dispersion and disruption of tissues. Jacoby, W. B.; Pastan, I. H. eds. Methods in enzymology. General cell culture techniques. Vol. 58. New York: Academic Press; 1979: 119-131. 8. Paul, J. R. Media for culturing cells and tissues. Cell and tissue culture. 5th ed. New York: Churchill and Livingstone; 1975: 95-99.

9. Schmidt, N. J. Tissue culture methods and procedures for diagnostic virology. Lennette, E. H.; Schmidt, N. J. eds. Diagnostic procedures for viral and rickettsial diseases. Chapt. 3. New York: Am. Public Health Assoc.; 1964: 78-176. 10. Whitaker, A. M. Mammalian cell culture media. Wasley, G . D . ed. Animal tissue culture. Advances in technique. Chapt. 2. Baltimore: Williams and Wilkins; 1973: 1-19. 11. Earle, W. R. A technique for adjustment of oxygen and carbon dioxide tensions, and hydrogen ion concentration, in tissue cultures planted in Carrel flasks. Archly fur Experimentelle Zellforschung, Besonders Gewebzuechtung. Jena. 16: 116-138; 1934. 12. Earle, W. R. Production of malignancy in vitro. IV. The mouse fibroblast culture and changes seen in the living cell. J. Natl. Cancer Institute 4: 165-212; 1943. 13. Parker, R . C . Balanced salt solutions and pH control. Methods of tissue culture. Chapt. 5. New York: Hoeber Medical Division; Harper and Row; 1961: 53-61. 14. Hanks, J. H. The longevity of chick tissue cultures without renewal of medium. J. Cell. Comparative Physiol. 31:235 260; 1948. 15. Hanks, J. H.; Wallace, R. E. Relation of oxygen and temperature in the preservation of tissues by refrigeration. Proc. Soc. Exp. Biol. Med. 71: 196-200; 1949. 16. Penso, G.; Balducci, D. Tissue culture media. Tissue cultures in bio|ogical research. Chapt. 4. New York: Elsevier Publishing Co.; 1963: 68 100. 17. Humason, G. L. Solution preparation and laboratory aids. Animal tissue techniques. San Francisco: W. H. Freeman ~ Co.; 1972: 534. 18. Weller, H.; Enders, J . F . ; Robbins, F . C . ; Stoddard, M. B. Studies on the cultivation of poliomyelitis viruses in tissue culture. J. Immunol. 69: 645-671; 1952. 19. Hanks, J. H. Balanced salt solutions, inorganic requirements and pH control. An introduction to cell and tissue culture. New York: Burgess Publishing Co.; 1955.

T h i s research was s u p p o r t e d by Storrs A g r i c u l t u r a l E x p e r i m e n t Station, University of C o n n e c t i c u t , Scientific C o n t r i b u t i o n N u m b e r 1032.