Media and Methods for Isolation and Enumeration of the Enterococci1

Media and Methods for Isolation and Enumeration of the Enterococci1

Media and Methods for Isolation and Enumeration of the Enterococci’ PAULA . HARTMAN. GEORGE W. REINBOLD.AND DEVIS. SARA SWAT^ Departments of Bacteriol...

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Media and Methods for Isolation and Enumeration of the Enterococci’ PAULA . HARTMAN. GEORGE W. REINBOLD.AND DEVIS. SARA SWAT^ Departments of Bacteriology and Dairy and Food Industry. Iowa State Uniuersity. Ames. Iowa

I . Introduction ..................................... I1. Media Available ................................. A . General Information .......................... B. Crystal Violet (Gentian Violet) . . . . . . . . . . . . C . Sodium Azide ................................ D . Azide Plus Dyes .................... E . Azide Plus Elevated Incubation Tempera F. Azide Plus Esculin . . . . . . . . . . G . Precautions when Using AzideH . Thallium Salts .............................. I . Tetrazolium Salts and Thallous Acetate . . . . . . . . . . J . Thallium Salts Plus Other Ingredients . . . . . . . . . . . K . Citrate ............................. .... L . Sodium Chloride ..................... .... M . Tellurite .................................... N . Penicillin and Other Antibiotics . . . . . . . . . ... 0. Sodium Taurocholate and Bile Salts . . . . . . . . . . . . . P . Phenethyl Alcohol ............................ Q. pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R . Selenite ..................................... S . Tetrathionate ................................ I11. Comparative Studies on Media and Methods . . . . . . . . . . A. General Discussion ........................... B. Enterococci in Water ......................... C . Enterococci in Nondairy Foods . . . . . . . . . . . . . . . . D . Enterococci in Dairy Products . . . . . . . . . . . . . . . . . E . Enterococci in Intestinal Contents . . . . . . . . . . . F . Future Needs . . . . . . . . . . . . . . ............. References ..................... .............

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I. Introduction Thiercelin (1899) first used the term “enterococcus” to describe a gram-positive diplococcus of intestinal origin. Thiercelin’s enterococcus 1 Journal Paper No . J-5257 of the Iowa Agricultural and Home Economics Experiment Station. Ames. Iowa . Projects No . 1050 and 1379. This work was supported in part by U . S. Public Health Service grant EF-112 from the Division of Environmental Engineering and Food Protection . 2 Rockefeller Foundation scholar . Present address: Rajasthan College of Agriculture. Udaipur. India .

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has since gained world-wide reknown because of its ubiquity and versatility. Enterococci have assumed a prominent role in studies on the following subjects: infectious diseases of man and other animals ( McCarty, 1958; Krantz and Dunne, 1965); dental caries and oral microbiology (Keyes, 1962; Burnett and Scherp, 1962); food poisoning (Hartman et d., 1965); food fermentations (Niven, 1963; Hartman et al., 1965); silage fermentations (Langston and Bouma, 1960); plant epiphytes (Mundt, 1964); intestinal flora (Rosebury, 1965; Schaedler et d., 1965); pollution of food and water (Niven, 1963; Deibel, 1964); spoilage of foods (Frazier, 1958; Niven, 1963); air and environmental microbiology (R. E. 0. Williams and Hirch, 1950; Rosebury, 1965); enzyme production (Rainbow and Rose, 1963); microbial reduction of vitamin B2 (A. H. Rose, 1961); amino acid and vitamin assay (Gunsalus and Stanier, 1962b; Kavanagh, 1963); nutrition, metabolism, and physiology ( Gunsalus and Stanier, 1961, 1962a; Deibel, 1964); structure and function (Gunsalus and Stanier, 1960; Hartman et al., 1966); serology (Shattock, 1962; Hartman et d., 1966); genetics (Gunsalus and Stanier, 1964; Braun, 1965); taxonomy (Deibel, 1964; Hartman et al., 1966). A large volume of literature has accumulated on media and methods for isolation and enumeration of enterococci. Although the present essay is directed toward determination of enterococci in foods and allied products, the principles discussed are almost equally applicable to other areas of microbial ecology as well as the more distantly related fields of study.

II. Media Available A. GENERALINFORMATION All the agar and broth media known to us to have past or potential use for seZective isolation or enumeration of the fecal streptococci are shown in Tables I and 11. Each medium has been assigned a letter ( A = agar, Table I; B = broth, Table 11) and a number to facilitate subsequent discussion. The agent( s ) and conditions of incubation that impart primary selectivity to the media also are listed, as are the concentrations of each agent. Some common names are given, and a reference designates the origin of each medium. The media are arranged more or less into groups according to the selective agents used. This departure from a purely historical presentation will, we hope, enable better discussion of the comparative properties of each type of medium. Background information relative to each group of media has been included in the discussion so that trends in development can be ascertained. Usually the first use of a selective agent is followed by a flurry of activity in which new media and various formula modifications are made.

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TABLE I SELECTIVE AGAR MEDIAFOR ISOLATION OF FECAL STREPTOCOCCI Medium A-1 A-2 A-3 A-4 A-5 A-6 A-7 A-8 A-9 A-10 A-11 A-12 A-13 A-14 A-15 A-16 A-17

A-18

Selective and differential agent( s ) , a conditions, comments, and synonyms 0.006% NaN,, 0.0001% crystal violet 0.01% NaN, 0.01% NaN,, 2% sodium citrate, 0.001% tetrazolium blue (Citrate Azide agar) 0.01% NaN,, 0.0002% basic fuchsin, anaerobic 0.02% NaN, ( Azide Blood Agar Base) 0.02% NaN,, 0.0002% crystal violet, 0.1% sodium citrate (Streptocel agar) 0.02% NaN,, 5% sucrose 0.029% NaN,, 0.002% acridine orange, 0.93% sodium glutamate, 0.0015% TTCb 0.03% NaN,, 3.25 IU/ml. penicillin 0.03% NaN,, 6.5 IU/ml. penicillin, 0.001% methylene blue 0.04% NaN, 0.04% NaN,, 0.001% methylene blue (after enrichment in medium B-20) 0.04% NaN,, 0.01% TTC (M-enterococcus agar) 0.04% NaN,, 0.0005% ethyl violet, 0.01% TTC (Ethyl Violet Azide or EVA agar) 0.0470 NaN,, 0.05% Tween 80, 0.2% sodium carbonate, 0.01% TTC (Tween-carbonate agar) 0.04% NaN,, 0.05% Tween 80, 0.2% sodium bicarbonate, 0.01% TTC (TC agar) 0.04% NaN,, 0.0015% bromcresol purple, 1.0% sodium glycerophosphate, 0.0636% sodium cmbonate, 0.01% TTC (KF agar) 0.04% NaN,, 2% sodium citrate, 0.001% tetrazolium blue (Citrate Azide agar)

Reference Chapman (1944) Snyder and Lichstein (1940) Reinbold et al. (1953) M. Rogosa (Fitzgerald and Keyes, 1960) Anonymous (1953, 1956) BBL (1963) Sherman et al. (1943) Raibaud et al. (1961) J. C. White and Sherman (1944) Winter and Sandholzer (1946a) A. P. Harrison and Hansen (1950) Fujiwara et a,?. (1956) Slanetz and Bartley (1957) Mallmann and Kereluk (1957); Kereluk ( 1960) Burkwall and Hartman (1964) Lachica and Hartman (1965)

Kenner et al. (1961) Saraswat et al. (1963)

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TABLE I (Continued) Medium ~

A-19

A-20

A-21 A-22 A-23 A-24 A-25 A-26 A-27 A-28 A-29 A-30 A-31 A-32 A-33 A-34 A-35

Selective and differential agent( s ) , a conditions, comments, and synonyms 0.04% NaN,, 0.001% methylene blue (Enterococcus Confirmatory slant); 0.04% NaN,, 0.001% methylene blue, 6.5% NaCI, 6.5 IU/ml. penicillin, pH 8.0 (Enterococcus Confirmatory broth) 0.04% NaN,, 4.5% NaCl, 0.003% water-soluble aniline blue, pH 8.3, 45°C. (for confirmation only; see also medium B-13) 0.05% NaN,, 0.0002'37 crystal violet 0.05% NaN,, 0.0002% crystal violet, 39.5"C. 0.05% NaN,, 0.000125% crystal violet, 0.008% brom thymol blue, 0.5% sorbitol 0.225% NaN,, 0.5% glycine, 0.1% esculin, pH 9.0 0.225% NaN,, 0.5% glycine, 0.1% esculin, 1.0% lactose, pH 9.0 (ELA agar 1 Hydrazoic acid vapor 0.0033% thallium sulfate, 0.000133% crystal violet, 0.1% esculin (TKT agar) 0.08% thallium acetate or thallium nitrate 0.1% thallium acetate, 0.01% TTC (TLTG agar) 0.1% thallium acetate, 0.01% TTC, 45°C. 0.1% thallium acetate, 0.01% TTC, 2% sodium citrate 0.1% thallium acetate, 0.00005% crystal violet 0.1% thallium acetate, 0.01% TTC, 0.5% tyrosine, 0.27% sorbitol, 37" and 45°C. 0.024% sodium selenite, 0.071% aniline blue (see A-20) 1%sodium taurocholate, 0.05% aniline blue, 0.1% esculin, pH 8.0

Reference

Winter and Sandholzer (1946a,b)

Guthof and Dammann (1958) Packer (1943) hlossel et al. (1957) Kjellander (1960) Colobert and Morelis ( 1958); Morelis and Colobert (1958) Horie and Saheki (1960) Gerencser and Weaver (1959) Mieth (1960) Rantasalo ( 1947) Barnes (1956b,c) Franklin and Sharpe (1963) Lachica and Hartman (1965) McKenzie ( 1941) Mead (1963) Guthof (1952) Koch ( 1935)

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TABLE I (Continued) Medium A-36 A-37 A-38 A-39 A-40 A-41 A42 A-43

Selective and differential agent( s ) , a conditions, comments, and synonyms 1% sodium taurocholate, 0.035% potassium tellurite, 0.1% esculin, 2.5% NaCl, pH 8.0-9.0 0.001% potassium tellurite, 0.00025% crystal violet (S-1 ) 0.002% potassium tellurite, 0.00008% crystal violet, 0.0075% trypan blue Mitis-Salivarius agar ) 0.0067% potassium tellurite 0.00005% neomycin sulfate, 0.0025% phenol red, 1% mannitol (MN Trypticase Soy agar) 0.0014% neomycin sulfate, 0.003% polymyxin B sulfate (MSDH agar) 0.25% phenylethyl alcohol 4% NaCl

Reference Schafer ( 1953) R. E. 0. Williams and Hirch (1950) Chapman (1944) C. H. H. Harold (1936), cited in Kjellander ( 1960) Greer and Britt (1959); BBL (1963) Vera (1963) Anonymous ( 1956) Snyder (1940)

Blood (usually 5%) was included in some of these media to serve as an indicator of the type of hemolysis, and, in one medium, to serve as a source of F e + + + . TTC = 2,3,5-triphenyltetrazolium chloride. @

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TABLE I1

SELECTIVE BHOTH MEDIA FOH Medium

-~

...

ISOLATION OF h C A L STHEPTOCOCCI

Selective and differential agents( s ) ,a conditions, comments, and synonyms ~~

~

-

~~

~

0.006% NaN,, 0.0001% crystal violet (confirmation on medium of Bierkowski, 1956) 0.006-0.0077, NaN,, 0.0002% crystal B-2 violet B-3 0.01 NaN,, 0.0001% crystal violet B -4 0.01% NaN,, 0.0025% brom cresol purple B-5 0.02% NaN, B-6 0.02% NaN, (Azide Dextrose or AD broth) B-7 0.02% NaN, (AD broth, 45"C., presumptive test, see also B-33) B-8 0.02% NaN, (presumptive test, see also B-17) B-9 0.02% NaN,, 0.0002% crystal violet, 0.1% sodium citrate (Streptocel broth for enrichment) B-10 0.02% NaN,, 0.0005% crystal violet B-11 0.02% NaN,, 0.003% brom thymol blue B-12 0.02% NaN,, 1% sodium taurocholate, 3% NaCl (N-N broth) B-13 0.025% NaN,, 0.0032% brom cresol purple, 45°C. B-14 0.025% NaN,, 0.0032% brom cresol purple, 37°C. followed by 45°C. B-15 0.025% NaN,, 0.00025% water-soluble aniline blue, 3% NaCI, pH 8.3 (presumptive medium, see medium A-20) B-15a 0.04% NaN,, 0.0032% brom thymol blue, pH 8.0, 45°C. (Enterococcus Presumptive broth; see A-19) B-16 0.04% NaN,, 0.0032% brom thymol blue, 45°C. B-17 0.04% NaN,, 0.0005% ethyl violet (Ethyl Violet Azide or EVA broth) B-18 0.04% NaN,, 1% sucrose, 0.01% TTC,a 0.000012% ethyl violet optional (for membrane filter) B-1

Reference Mastromatteo and Pisu (1959 Pike (1944, 1945a) Edwards (1938) McKenzie ( 1941) Mallmann (1940) Rothe (1948) Splittstoesser et al. (1961) Litsky et al. (1953) BBL (1963) Ritter et d. (1956) Raj et ul. ( 1961)

Micth (1960) Hannay and Norton (1947) Childs and Allen (1953)

Guthof and Dammann (1958 Winter and Sandholzer (1964b Wang and Dunlop (1951) Litsky et al. (1953) Slanetz et ul. (1955)

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TABLE 11 (Continued) Medium B-19

B-20 B-21 B-22 B-23 B-24 B-25 B-26 B-27 B-28 B-29 B-30 B-31 B-32 B-33 a

Selective and differential agents( s ) , a conditions, comments, and aynonyms 0.04% NaN,, 1% sodium glycerophosphate, 0.0015% brom cresol purple, 0.0636% sodium carbonate, 0.01% TTC (KF broth) 0.05% NaN,, 0.0002% crystal violet 0.05% NaN,, 0.0015% brom cresol purple, 0.5% glycerol (BAGG broth) 0.05% NaN,, 0.0032% brom cresol purple, 45.5"C. ( SF medium) 0.05% NaN,, 6.5% NaCI, 45°C. 0.01% potassium tellurite, 0.00005% crystal violet 0.02% potassium tellurite 0.05% thallium acetate 0.1% thallium acetate 0.1% thallium acetate, 0.00005% crystal violet tetrathionate 10% ox bile 0.5% sodium taurocholate, 1.0% mannitol, 44°C. (confirmatory only) 0.05% Tween 80, 0.53% sodium carbonate, pH 10.0

6.5% NaCI, 45°C. (confirmatory test; see B-7)

TTC = 2,3,5-triphenyltetrazolium chloride.

Reference

Kenner et ol. (1961) Packer ( 1943) Hajna ( 1951 ) Hajna and Perry (1943) Ostrolenk and Hunter (1946) Pike (194570) Cooper and Ramadan (1955) Cooper and Ramadan ( 1955) Barnes ( 1 9 5 6 ~ ) McKenzie (1941) Cooper and Ramadan (1955); Cooper et al. ( 1942) Weissenbach (1918) Rycroft (1956) Chesbro and Evans (1959) Splittstoesser et al. ( 1961 )

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After this initial burst of activity, there is little further refinement of the media. Applications of media parallel to a great extent the development of taxonomy of the streptococci. Many early media were probably fairly selective for the enterococci, but the nomenclatural status of the group was not defined sufficiently to recognize the importance of the discovery. Most early works were aimed primarily at isolation of streptococci associated with mastitis and human infections. Subsequent refinements in formula were made to adapt these media to studies on entercocci. Several media that have been used to isolate streptococci, including the enterococci, do not appear in the tables. For example, many nonselective media, such as blood agar or Standard Methods agar, have been omitted. Likewise, a few media, such as the modified Dorsett egg medium of Crowe ( 1913), have been omitted because the extensive manipulations needed for their preparation indicate that they would not attain widespread use. If other omissions occur, they are unintentional. Not all the media listed in Tables I and I1 will be discussed; the original publications give further details concerning these media.

B. CRYSTAL VIOLET(GENTIAN VIOLET) Churchman (1912) was the first to study extensively the selective bactericidal action of gentian violet, although earlier observations had been made by Drigalski and Conradi (1902). Churchman’s work was followed closely by the investigations of Krumwiede and Pratt ( 1914), who found that some streptococci were more resistant to gentian violet than were other bacteria. Haxthausen (1927a,b) added 0.001% crystal violet to glucose broth to produce a selective medium. Edwards ( 1931) modified this liquid medium to contain 0.0005% crystal violet, and devised an agar medium containing crystal violet plus 0.1% esculin (Edwards, 1933) for better colony differentiation. This latter medium was the predecessor of other esculin-containing media (A-24, A-25, A-27). Bryan (1932), apparently unaware of the publications of Haxthausen and the earlier work of Edwards, proposed the addition of 0.000677.1, gentian violet to improve the selectivity of blood agar. Brilliant green was equally satisfactory (Bryan, 1932; see also Litsky et al., 1952). Dick and Hucker (1940) utilized 0.00025% crystal violet to impart selectivity for “S. salivarius” in a broth medium used to detect oral contamination of drinking glasses, while Cooper and Linton (1947) utilized a similar medium (0.0033% crystal violet) for the detection of gonococci. These media served as the basis for development of many different media now used for isolation and enumeration of fecal streptococci. Although there is only one primary inhibitory agent ( a dye) in these media, the experiences of several workers utilizing such media with

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streptococci will be mentioned to illustrate a point common to all selective agents. The workers cited used crystal violet at concentrations of 0.00025% or greater. McKenzie (1941) observed that crystal violet in a concentration of 0.00017, markedly inhibited growth of some streptococci; a level of 0.00005% was finally chosen for his medium. The selective action of crystal violet on streptococci also was noted by Packer (1943), who reported that a concentration of 0.0005% was inhibitory to many streptococci. A level of 0.0002% allowed the growth of all of the streptococci examined except a few strains of Streptococcus luctis. R. E. 0. Williams and Hirch (1950), using a tellurite-crystal violet medium (A-37), noted that a reduction of either ingredient led to some increase in the streptococcal count; an increase in either inhibitor decreased the count. Decreased levels of either inhibitor, however, resulted in a lowering of the selectivity of the medium. Thus, one might assume that the types of streptococci that would grow on a particular medium would vary somewhat with different concentrations of inhibitor (see also Splittstoesser et al., 1961). In addition, using the same medium, some cells of a specific species of streptococcus might be inhibited while others might grow, or the period of incubation might not be sufficiently long for maximum counts to be obtained. R. E. 0. Williams and Hirch (1950) found that the period of incubation considerably affected the results obtained; some strains of enterococci failed to survive for incubation periods that were sufficiently long to permit colony formation by other strains. Media containing dyes alone are not listed in Tables I and I1 because all these media lack the selectivity demanded by present-day workers. C. SODIUMAZIDE

After the studies of Loew (1891) of the inhibitory properties of azide to a variety of microorganisms, Schattenfroh (1896) examined the effects of various levels (0.05-1.0%) of sodium and ammonium azide on bacteria, yeasts, and molds. He noted that the test organisms differed considerably in their susceptibility to these compounds. Over 40 years elapsed, however, until azide was used to advantage in devising a selective medium for use in studies on fecal streptococci. Impetus was given these studies by Hartmann (1937), who examined the relative inhibition of Escherichia coli and streptococci by a variety of compounds. A large number of chemical agents had previously been studied by Kramer and Koch ( 1931 1, Garrod (1933), and Diernhofer (1936); see these publications for other citations. Most of the compounds tested did not lead to development of a practical medium, but some compounds of potential future use were discovered. Azide was the only compound that inhibited E . coli more than the streptococci at a relatively wide range of concentration (Hart-

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mann, 1937). Concentrations of 0.02-0.04% were recommended. Literature regarding the effect of varying concentrations of sodium azide on microorganisms has recently been summarized by Forget and Fredette ( 1962). Mallmann (1940) modified a medium devised by Darby and Mallmann (1939) by adding 0.02% sodium azide (B-5). He was able to estimate selectively the numbers of streptococci in samples of sewage. This was probably the first instance of selective enumeration of enterococci from a grossly contaminated environment without concomitant excessive inhibition of the enterococci themselves. A widely used modification of Mallmann’s broth medium was made by Rothe (1948). This medium (B-6) contained 0.02% sodium azide; changes were made in two of the other constituents. Mallmann and Seligmann (1950) reported on the use of this medium but, through a typographical error, the quantities of sodium chloride and glucose were in error. This latter formulation was used with success, however, by Morris and Weaver (1954). A similar medium (Ritter and Treece, 1948) also contained 0.027, azide, but less glucose was used. The medium supported luxuriant growth of all of the strains of streptococci and staphylococci tested. Commercial azide blood agar bases (A-5) apparently follow generally the work (A-2) of Snyder and Lichstein (1940; Lichstein and Snyder, 1941), with the azide concentration set at 0.02% according to the studies of the other workers cited previously. McKenzie ( 1941) preferred a thallous acetate-crystal violet medium (A-32) to a 0.01% sodium azide medium for isolation of mastitis streptococci. One might suspect from this work that use of the single selective ingredient, azide, leaves much to be desired as far as selectivity for fecal streptococci is concerned when grossly contaminated samples are to be examined. This has been found to be the case by a number of workers, including Burkwall and Hartman ( 1964), who included Azide Blood agar in their study (see also Richards et al., 1945a,b; Ritter and Treece, 1948; Zaborowski et al., 1958). Thus, the use of azide alone in media for food microbiology is usually limited to preenrichment prior to confirmation in a more selective broth or agar medium. In addition, altered hemolytic patterns may be obtained on media containing azide (Snyder and Lichstein, 1940; Packer, 1943), and the large quantities of glucose used in many of the media discussed in this section would tend to reduce the hemolytic properties of many of the streptococci growing there. One notable exception to the use of azide alone as a selective ingredient is the medium devised by Slanetz and Bartley (1957). This medium (A-13), intended for use with the millipore filter in water bacteriology, originated by substantial alteration of a medium (A-1) of Chapman (1944) by Slanetz et al. (1955). The relatively high level of azide, 0.04%,

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might account for the selectivity of the medium, although Saraswat et al. (1963) found that a stock culture each of Streptococcus bovis and Lactobacillus acidophilus grew on this medium. Raibaud et al. (1961) obtained low recoveries and overgrowth by lactobacilli when pig cecal samples were plated in medium A-13. Burkwall and Hartman (1964) also reported that recovery was low when frozen foods were plated on medium A-13; however, greatly increased recovery was obtained when Tween SO ( W . L. Williams et al., 1947) and sodium carbonate (Chesbro and Evans, 1959) were added to medium A-13. Since pH changes were minimal, the increased recovery was not thought to be due to lowered selectivity resulting from higher pH, as has been reported by Edwards (1938) and Packer (1943). Further studies by Lachica and Hartman (1965) showed that medium A-13 yielded highest counts when supplemented with 0.075% Tween 80, 0.2% KH2P04, and 0.2% NaHC03 (medium A-16). If the higher counts are obtained because of increased recovery of fecal streptococci, medium A-16 holds promise of being better for many purposes than any medium tested by Burkwall and Hartman (1964). Whether the use of Tween and carbonate in some manner reverses in part the inhibitory power of sodium azide remains to be determined. Carbonate may even be contributing to the selectivity of the medium, as described in the section that deals with pH. Kenner et aE. (1961) described new solid (A-17) and liquid (B-19) media for enumeration of enterococci. These media contained, among other ingredients, 0.04% sodium azide, 0.0015% bromcresol purple, and 0.01% 2,3,5-triphenyltetrazolium chloride (TTC). The use of TTC in culture media is discussed in a later section. Media A-17 and B-19 seem to permit growth of S. bovis and Streptococcus equinus (Kenner et al., 1961), so they would not be appropriate for use when only enterococci are desired, e.g., when dairy products are to be examined (Saraswat et al., 1963). The broth (B-19) is probably slightly more inhibitory than the agar (A-17) to S. bovis, S. equinus, and Streptococcus mitis (Hall et al., 1963). Hall et al. (1963) and Burkwall and Hartman ( 1964) were favorably impressed with results obtained when frozen foods were plated on medium A-17. The latter workers obtained results using medium A-17 that were equivalent to those obtained using medium A-29. These two media were superior to many other direct-plating media examined for enumeration of fecal streptococci from frozen foods. In a recent study of freeze-dried foods (Saleh et uZ., 1966), higher counts were usually obtained on medium A-15 than on medium A-17; however, medium A-15 was generally the less selective of the two. Mossel et nl. (1957; Mossel, 1964) also were of the opinion that medium A-17 lacked selectivity because almost quantitative recoveries of Staphylococcus aureus were ob-

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tained on the medium. Raibaud et al. (1961) reported that they obtained low recoveries of enterococci and encountered overgrowth by lactobacilli when cecal samples were plated on medium A-17. Regardless of these shortcomings, KF agar (A-17) and broth (B-19) media are among the better yet developed for enterococci (Hall, 1964). Saleh et al. (1966) noticed that colony size on medium A-17 was larger when scrambled eggs were plated than when other freeze-dried foods were examined. This observation might be utilized in future improvement of medium A-17.

D. AZIDE PLUSDYES Edwards (1938) examined the efficacy of 0.0001% crystal violet in combination with 0.01% sodium azide (B-3) for the diagnosis of mastitis. In contrast, Packer (1943) selected higher concentrations of crystal or gentian violet and azide (A-21, B-20). Various concentrations of crystal violet and azide have been used by other investigators (Tables I and 11)

in media developed for different purposes. Other dyes have been used in combination with sodium azide (see Schaedler et al., 1965). Some workers have added pH indicators so that acid production can be detected (see B-4, B-11, B-16, B-22; Zaborowski et al., 1958). For some purposes a great deal of selectivity is not necessary. All these azide-dye media, although more selective than media containing azide or dye alone, still require confirmation under most circumstances. Litsky et al. (1953) developed an ethyl violet-azide medium (B-17) ( Litsky et al., 1952) that reportedly possessed increased selectivity, although Ritter et al. (1956) thought that the new medium was equivalent to their own azide-crystal violet medium (B-10). Litsky et al. (1953) recommended use of the broth (B-5) devised by Mallmann ( 1940) for presumptive identification, followed by transfer into medium B-15 for confirmation, This combination of media has been widely used and is apparently very satisfactory for isolation and enumeration of enterococci from water. Only Streptococcus faecalis and Streptococcus faecium and their varieties were reported to survive such an isolation procedure (Litsky et al., 1953), but Kenner et al. (1961; Hall et al., 1963) noted that Lactobacillus plantarum and Pediococcus cerevisiae gave positive results with ethyl violet-azide medium. Materials, containing these and related bacteria might therefore give erroneously high counts (see also Splittstoesser et al., 1961). Medium B-15 would not be satisfactory if counts of S. bouis, S . salivarius, or S. equinus were among those desired. A similar medium containing agar (A-14) was devised for use by the drop-plate method, and Ferraro et al. (1958) used an ethyl violet-azide plating medium for confirmation of enterococci. Ethyl violet-azide media may suffice when used by the drop-plate procedure or for confirmation

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of enterococci, but Saraswat et al. (1963) and Burkwall and Hartman (1964) found this medium (A-14) to be low in yield when used for direct plating. Croft ( 1959) obtained unimpressive results in some attempts to use medium A-14 in a millipore filter procedure for water analysis. E. AZIDE PLUSELEVATED INCUBATION TEMPERATURES Use of an incubation temperature of 45°C. in conjunction with azide originated with Hajna and Perry ( 1943), who developed a medium (B22) for presumptive enumeration of streptococci in milk. The elevated incubation temperature, following the work of Sherman (1937b)) was used to limit the type of streptococcus that would grow (see also Allen et al., 1949). Medium B-22, known as SF medium, was modified by Hajna ( 1951) by inclusion of glycerol (medium B-21). The medium also has been modified (B-13, B-14) by reduction of the azide concentration to 0.025%, but Hajna (1951) warned that this reduced the specificity of the medium. As far as is known, incubation temperatures within the ranges discussed would not prevent proliferation of any fecal streptococci, with the possible exception of some strains of S. mitis. Nevertheless, Ferraro and Appleman (1957) demonstrated clearly that erroneously low enterococcus counts were frequently obtained when citrus concentrates were inoculated into medium B-22; preliminary enrichment in phenol red lactose broth resulted in greatly increased counts. Childs and Allen ( 1953) suggested a “subculture” method ( B-14) whereby inoculated tubes are first incubated at 37°C. for 24-48 hours, then positive tubes are subcultured to fresh tubes of the same medium to be incubated at 45°C. There seems to be no reason why the subculture could not be eliminated; the original tubes could be incubated at 37°C. for several hours to permit growth initiation, then removed to a 45°C. incubator for the remainder of the incubation period. This application of 45°C. incubation also has been investigated by Allen et al. ( 1953), Burman ( 1961), and Mead ( 1963, 1964). Interaction between azide concentration and temperature of incubation was mentioned by Splittstoesser et al. ( 1961). An incubation temperature of 45°C. did not reduce counts of enterococci obtained on broth B-6, but did when samples were inoculated into medium B-22. A temperature of 39.5”C., rather than 37”C., was used with Packer’s medium (A-21) by Mossel et al. (1957; medium A-22); increased selectivity, but decreased yields, were obtained. Mossel (1964) seemed to prefer using a 37°C. incubation temperature to obtain greater recovery, combined with confirmation in another medium.

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F. AZIDE PLUSESCULIN The only azide-esculin medium to be proposed (A-24) was described by Morelis and Colobert ( 1958) and was later modified (A-25) by Horie and Saheki (1960). As is discussed in the section on thallium plus other agents, there are certain limitations to using esculin hydrolysis as an indicator of enterococci. In addition, 0.225% sodium azide is a high concentration, which probably accounts for the low yields of enterococci obtained on these two media in comparison with some others (Burkwall and Hartman, 1964). Selectivity of these media, however, is excellent. G. PRECAUTIONS WHEN USINGAZIDE-CONTAINING MEDIA Several precautions are necessary when using azide-containing media. Various investigators have reported that the selectivity may be altered if the azide is sterilized with the basal medium. Mieth (1960), for example, reported that the inhibitory action of a given quantity of azide was diminished when media were autoclaved at 110°C. Guthof and Dammann (1958) also stated that azide was very heat sensitive. Other workers (i.e., Snyder and Lichstein, 1940) did not notice any differences in the selectivity of azide-containing media whether the azide was sterilized separately or with the basal medium. Various investigators have approached this problem in different ways, ranging from a very cautious separate sterilization of the azide by filtration, through separate sterilization by steaming for a short period, to inclusion of azide in the medium before autoclave-sterilization. So many media have been proposed using the latter approach to sterilization that it appears that the method is adequate if the azide concentration is adjusted appropriately to allow for some (as yet unknown) loss. If this is done, the medium should always be sterilized in the same manner. Likewise, prepared media should not be held too long; storage for 1 week at room temperature does not seem to have a deleterious effect (Forget and Fredette, 1962; Pike, 1945a), although Pike (1944) observed a decrease in the inhibitory properties of blood-containing azide media stored for as little as 2 days at room temperature. Therefore, prepared media, especially those containing blood (Pike, 1945a), should be stored in the refrigerator, but not longer than 2 weeks. Care should also be taken in storing dehydrated media (Hall et al., 1963). Loss of azide is accompanied by formation of hydrazoic acid, which is volatile (Gerencser and Weaver, 1959) and probably more toxic (Smuckler and Appleman, 1965) than azide. Transformation of azide to hydrazoic acid is especially rapid in acid media (Gerencser and Weaver, 1959); consequently control of pH of azide-containing media is very

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important. Furthermore, in plates containing much agar the release of hydrazoic acid is less (or reabsorption is greater) than in plates containing small volumes of medium; therefore, azide-containing media may become increasingly inhibitive as the volume of agar per plate is increased ( Gyllenberg and Niemela, 1960). Gerencser and Weaver ( 1959) took advantage of the volatile nature of hydrazoic acid (medium A-26) by incubating cultures in a hydrazoic atmosphere. Azide in acid solution is placed in a separate container in a closed jar, together with petri plates of nonselective medium; quantities of hydrazoic acid absorbed by the medium are determined by the amount of azide used, the size of the container, and the type and possibly the surface area of the culture medium. The hydrazoic acid procedure was used by Gyllenberg and Niemela (1960) in conjunction with the millipore-filter medium of Slanetz and Bartley (1957). Differences in the volume of agar per plate did not influence counts of fecal streptococci or colony colorization when the hydrazoic technique was used; these two factors were altered by volume of agar when the conventional Slanetz and Bartley procedure was used. Although Gerencser and Weaver (1959) reported that hydrazoic acid liberated from azide-containing media may contaminate other media stored in close proximity, Forget and Fredette (1962) and Smuckler and Appleman (1965) did not detect toxicity in media stored beside azidecontaining media or an azide stock solution. Azide apparently exerts its primary function by inhibiting metalloporphyrin enzyme systems, such as catalases and cytochrome c oxidases (see references in Gunsalus and Stanier, 1961, and Nicholls, 1964). Electron transport is interrupted. As mentioned previously, azide penetrates some cells only as the undissociated acid, so the pH of the medium can have a great effect on the selective properties of tce medium.

H. THALLIUM SALTS In 1918, Browning and Gulbransen (1918) reported that certain grampositive cocci were more resistant to thallium acetate (TA) than were other bacteria tested. The selectivity of thallium salts was subsequently examined in detail by McKenzie (1941), Cooper and Linton (1947), Richards et al. (1945a,b), Rantasalo ( 1947), and Sharpe ( 1955); these references indicate the spectrum of organisms inhibited by different concentrations of thallium salts. Kinnear (1931) reported that a broth containing 0.06% thallium nitrate ( T N ) and 0.005% crystal violet was a good selective medium for streptococci and staphylococci, but the thallium salt interfered with hemolysis when incorporated into an agar medium, Interference of hemolysis by 0.1% TA also was noted by Cooper and Linton (1947). McKenzie (1941) developed a 0.1% TA-crystal violet broth

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(B-28) for use in the diagnosis of streptococcal mastitis and noted that TA and TN salts were equally selective. Thallium sulfate (TS) also has been used successfully (Kehler Ellingsen and Hauge, 1953), The selectivity of TA is not affected by minor alterations in pH, although the selectivity of sodium azide is ( McKenzie, 1941). At a level of 0.270 TA, growth of a culture of Streptococcus agalactiae was inhibited, but other streptococci and a culture of S. aureus grew well. Richards et al. (1945a,b) were the first to note that the group D streptococci were not inhibited at all by 0.1% TA, and Rantasalo (1947) recommended the inclusion of 0.087, TA in a medium (A-28) for the isolation of streptococci from the throat. A level of 0.05% TA was used by several investigators (cited by Sharpe, 1955) and by Cooper and Ramadan ( 1955; medium B-26) to isolate streptococci from various sources. The TA medium (B-26) was not as satisfactory for the examination of human feces as was a tellurite medium (B-24), yet Mead ( 1963, 1964) proposed a TA medium (A-33) that was supposedly superior to others for recovery of “human” fecal pollution of water (the level of TA was erroneously listed as 1%in Meads 1963 publication). The efficacy of medium A-33 was questioned by Mundt (1964); it appears to us that positive colonies on medium A-33 are indicative of, but far from specific for, human enterococci. In addition, the medium and culture method is fairly complex, and selectivity is obtained only at sacrifice of yield. Finally, it seems probable that some enterococci from plant sources would produce positive colonies on medium A-33. R. E. 0. Williams and Hirch (1950) obtained growth of greening micrococci and a few other bacteria in addition to the desired streptococci when 0.05% TA was used in a medium to study the distribution of streptococci in the air. A concentration of 0.1% TA was stated to be superior to concentrations of 0.05 or 0.2% (Sharpe, 1955). At a level of 0.1% TA, members of genus Lactobacillus also grew well, as did the two strains of S. aureus examined and an unidentified micrococcus. Two of 23 strains of Lactobacillus and the micrococcus were inhibited by 0.2% TA; however, there was no observable inhibition of the four fecal streptococci examined. Gonococci will grow if the TA concentration is reduced to O.O2C/o (Cooper and Linton, 1947). TA also is a good selective agent for isolation of pleuropneumonia-like organisms ( references are cited in Kleineberger-Nobel, 1962). Thus, different species are inhibited by thallous salts at different levels of thallium-ion concentration. The data of McKenzie ( 1941), Cooper and Linton ( 1947), and Sharpe (1955) indicate that levels of TA somewhat higher than 0.1% might be used successfully if a more selective medium for group D streptococci

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was desired, but this problem has not been investigated further. The mechanism of selectivity exerted by thallium salts is unknown (Cooper and Linton, 1947; Sharpe, 1955).

I. TETRAZOLIUM SALTSAND THALLOUS ACETATE Laxminarayana and Iya (1953) observed that two strains of S. faecalis var. liquefaciens and a strain of “typical” S. faecalis reduced 2,3,5-triphenyltetrazolium bromide more rapidly than an ‘‘atypical” strain of S. faecalis (possibly S . faecium?) or 10 other streptococci belonging to five other species. Barnes (1956a) studied the reducing properties of many strains of fecal streptococci and noted that 2,3,5-triphenyltetrazolium chloride (TTC) was reduced rapidly by S . faecalis and its varieties, but not by s. faecium, s. faecium var. durans, or s. bovis. Earlier, H. G. Neumann (in Goetz and Tsuneishi, 1951) observed that TTC could be incorporated at a level of 0.4% in media containing customarily used inhibitory and selective agents, including azide media for isolation of enterococci, Reinbold et al. ( 1953) reported that 0.0025% ditetrazolium chloride was noninhibitory to enterococci. Further comment on the selectivity of tetrazolium compounds will not be made here, except to mention that Weinberg ( 1953) described toxicity of TTC at concentrations above 0.001% to gram-positive bacteria, and Solberg and Proctor (1960) developed a technique whereby the plate was flooded after incubation to circumvent any possible toxic effects. The differential reducing properties of different species of streptococci were used to advantage in devising two thallous salt-containing media (A-29 and B-27) for selective enumeration and differentiation of S . faecalis and S . faecium (Barnes, 1956a,b, 1959). In Barnes’s original procedure, a basal medium is made, sterilized, and partially cooled, and sterile solutions of TA, T’C, and glucose are added. Plates are poured and allowed to solidify, and the sample is then streaked on the surface of the medium, Streptococcus faecium and its variety durans reduce tetrazolium poorly or not at all at pH 6.0 and produce white colonies (Barnes, 1956a). Some strains of S. bovis grow well on the medium and produce white or pale pink colonies, while other strains grow poorly and produce minute red colonies ( Medrek and Barnes, 1958). Streptococcus faecalis and its varieties grow well1 and reduce tetrazolium strongly (Barnes, 1956a). The medium supposedly is highly selective for group D streptococci (Barnes, 1959), although Sharpe ( 1955) used a similar medium for the selective isolation of lactobacilli. In a survey of bacon factories (Barnes, 1956c; Barnes et al., 1956), only group D and a few group N streptococci were isolated. Lactobacilli apparently caused no difficulty

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in this application when an incubation period of 24 hours was used. According to Barnes (1958), lactobacilli will form colonies when incubated for 48 hours, but Fanelli and Ayres (1959), Hartman (1960), Hartman et al. (1962), and Burmeister et al. (1966) utilized an incubation period of 48 hours and did not encounter excessive overgrowth by unwanted organisms (also see next paragraph). On the other hand, Saraswat et al. (1963) observed that medium A-29 was unsatisfactory for dairy bacteriology because a dense background of lactic bacteria often obscured the results. Analogous difficulties were encountered by Mieth (1960) and Raibaud et al. (1961) when fecal samples were streaked on medium A-29, and Burkwall and Hartman (1964) obtained reduced selectivity as the incubation period was increased when frozen foods were plated. Nevertheless, yields on medium A-29 were greater than on most media for isolation and enumeration of enterococci in frozen foods, and selectivity was fairly good (Burkwall and Hartman, 1964). Modifications of medium A-29 and of procedures for its use have been made by several investigators. Extended incubation periods have already been discussed. In addition, Burkwall and Hartman (1964) used the pour-plate, rather than the streak-plate, method of inoculation. The solidified pour-plate was overlayed with sterile agar; thus, all colonies were subsurface. Use of this overlay may have eliminated growth of some of the unwanted organisms because difficulties were rarely experienced in overgrowth by other bacteria (see also Fry, 1932). Under these conditions, however, group D enterococci could not be differentiated on the basis of tetrazolium reduction; almost all samples of frozen foods (Hartman, 1960; Hartman and Huntsburger, 196l), rumen fluid (Hartman et al., 1962), and ensiled corn (Burmeister et al., 1966) yielded only red subsurface colonies. Another modification of the medium was made by Vera (1961); TA was included in the basal medium, so only sterile TTC had to be added to the basal medium immediately prior to use. McKenzie ( 1941), Cooper and Ramadan ( 1955), and Sharpe ( 1955) also had added TA before sterilization of their media. In some preliminary comparisons of the various modifications of the medium, equivalent results were obtained ( Hartman and Lachica, unpublished data, 1965). Two further modifications of medium A-29 have been made to increase the selectivity. Franklin and Sharpe (1963) used an incubation temperature of 45°C. for 2 days (A-30) with apparent success to adapt the medium for use on dairy products. Lachica and Hartman (1965) examined the combination of 2% citrate ( Reinbold et al., 1953) with medium A-29 and discovered that toxicity of the medium to staphylococci but not to streptococci was increased (medium A-31). These two modifications have not been tested extensively in comparison with other media.

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J. THALLIUM SALTSPLUSOTHERINGREDIENTS Stableforth et al. (1949) and Wilson and Slavin (1950) incorporated TS or TA at a concentration of 0.033% to improve the selectivity of the medium of Edwards (1933) in the diagnosis of mastitis. No recognition was given by these or subsequent authors to the inhibitory effect of TA on hemolysis that was reported by Kinnear (1931), and one must assume that thallous salts do not produce substantial changes in hemolytic reactions until TA concentrations of about 0.1% are attained (Cooper and Linton, 1947). Hauge and Kghler Ellingsen (1953; Kghler Ellingsen and Hauge, 1953; Sandvik and Hauge, 1954 ) incorporated staphylococcus p-toxin and named the resultant medium TKT agar ( Thalliumsulfat-KristallviolettToxinblutagar). The addition of p-toxin arose as a result of studies by Munch-Petersen and Christie (1947 and earlier) on the use of this substance in a blood-containing medium for the identification of s. agalactiae. This medium also was used by Seelemann and Obiger ( 1956), who were probably the first to note specifically that it supported the growth of enterococci. The medium contains 0.5% NaCl, in spite of the fact that McKenzie (1941) mentioned that TA reacts with NaCl to yield insoluble and nonselective thallium chloride. Levels of NaCl supposedly cannot exceed about 0.1% in thallium-containing media ( McKenzie, 1941); however, Barnes (1956c, p. 193) could not obtain evidence that concentrations of NaCl up to 0.5% reduced the inhibitory preperties of TA. Gyllenberg and Koine (1957) used 2% NaCl in a thallium-containing medium, modified after one developed by Garey et al. (1941). Nevertheless, samples containing large quantities of salt may alter the selectivity of TA-containing media. Mieth (1960) used medium A-27 to isolate enterococci in the examination of the fecal fiora of various animals and noted that the 0-toxin was not necessary when the medium was used for this purpose. Better recovery of S. bovis was observed with medium A-27 than with an enrichment procedure using medium B-12, but S. faecium was better detected using the enrichment. Medium A-27 was preferred to medium A-29, because A-27 had no background colonies to interfere with isolation (Mieth, 1960). Judging from the variety of strains of enterococci described by Mieth (1960, 1961, 1962a,b), all group D streptococci grow well on the medium. Yields on a modification of medium A-27 were only one fifth of those obtained on medium A-15 (Burkwall and Hartman, 1964). Although only one of 700 strains of enterococci examined by Mieth ( 1960, 1961, 1962a) was esculin negative, he selected only esculin-positive colonies for further examination. Rochaix (1924) and Swan (1954) concluded earlier that all enterococci fermented esculin (see also Kohler

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Ellingsen and Hauge, 1953, and Seelemann and Obiger, 1956). Esculin had been used for many years in differential culture media (F. C. Harrison and Vanderleck, 1909a,b). Burkwall and Hartman (1964) noted, however, that not all colonies that grew on medium A-27 were positive for hydrolysis of esculin within the recommended incubation period. Similar probdems had been encountered by R. E. 0. Williams and Hirch ( 1950, p. 517). Many of the esculin-negative colonies were enterococci and many were not ( Burkwall and Hartman, 1964). When esculinnegative colonies are excluded from the count, the estimate of the population of fecal streptococci might be low. On the other hand, if esculin-negative colonies are included in the count, some bacteria other than fecal streptococci may be included, Variability in the ability of an organism to ferment esculin was noted years ago (F. C. Harrison and Vanderleck, 1915); some organisms are weakened and give atypical colonies, yet their activity is regained upon repeated culture. Such alteration of apparent fermentative properties by unfavorable environment was noted as early as 1905 ( MacConkey, 1905). Esculin also has been used in media containing sodium azide (media A-24 and A-25) and sodium taurocholate (media A-35 and A-36). At least the first two, and probably all four, of these media have the same shortcoming insofar as colony recognition is concerned. The use of sorbitol fermentation and tyrosine decarboxylase production (medium A-33) as criteria for "specific" biotypes of fecal streptococci would present similar difficulties.

K. CITRATE The use of citrate (2%) as a selective ingredient in media (A-3) for enterococci originated with the studies of Reinbold et al. (1953). Campbell and Gunsalus (1944) and Abd-El-Malek and Gibson (1948) had previously studied citrate utilization by S. faecalis, while MacLeod and Snell (1947) had reported that 2% citrate inhibited the growth of Leuconostoc mesenteroides and all five species of Lactobacillus examined, but had little effect on the growth of S . faecalis (see also Campbell and Gunsalus, 1944). Medium A-3 was modified by Saraswat et al. (1963) by raising the azide concentration to 0.04% to increase the selectivity (medium A-18) for use in dairy bacteriology. Saraswat et al. ( 1963, and unpublished ) found that the increased azide concentration did not lower yields under practical conditions. Burkwall and Hartman (1964) did not encounter organisms other than enterococci when the lower azide concentration (0.01%; Reinbold et al., 1953) was used for plating frozen foods, although yields were rather low, SO it may be that Citrate-Azide agar has certain desirable properties related to selectivity that most

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other media do not possess. This is especially true with samples containing lactobacilli or other organisms that grow on many other commonly used media. The use of citrate in combination with thallous acetate (Lachica and Hartman, 1965) is described in an earlier section. Citrate is inhibitory at concentrations of 1% to such organisms as S. lactis and Streptococcus cremoris, this inhibition being reversed by Ca++ and Mg++ (McDonald, 1957). Low levels of citrate also inhibit certain strains of S , aureus (Rammell, 1962), and the inhibition is reversed completely by Ca+ + and partially by Mg+ +; Mn+ + seems to be an additional element of importance in the reversal of citrate inhibition of Lactobacillus spp. and L. mesenteroides ( MacLeod and Snell, 1947). Citrate metabolism has been discussed recently by Deibel (1964) and and Srere (1965).

L. SODIUMCHLORIDE The addition of 6.5% sodium chloride (Sherman, 1937a) to medium B-22 was used by Ostrolenk and Hunter (1946) to reduce the number of false-positive reactions occurring at 45°C. (medium B-23). The sodium chloride, however, did not appreciably reduce the occurrence of falsenegative reactions. Splittstoesser et al. (1961) used as a confirmatory medium broth containing 6.5% NaCl and incubated at 45°C. (medium B-33). Previously, sodium chloride alone, in concentrations of 6 to 15%, had been shown by Hill and White (1929) to impart selectivity to media for culturing streptococci from clinical specimens. Neter ( 1939) also had used 6.5% NaCl in his studies, while Snyder (1940) devised a 4% NaC1-blood agar ( A-43). Mastromatteo and Baldini (1963) recently described a 5% NaC1-blood agar (see also Kramer and Koch, 1931) for isolation of group A streptococci from the throat and showed that the NaC1-blood agar medium was more selective than an azide-crystal violet medium (Wahl and Meyer, 1957; Pettenela, 1957) for group A streptococci. The NaGl was more selective in agar than in broth (Mastromatteo and Baldini, 1963)) which is of interest because Guthof and Dammann (1958) successfully employed a broth medium (B-W), pH 8.3, containing 3% NaCl and 0.03% sodium azide for presumptive isolation of enterococci from water. An incubation temperature of 45°C. and concentrations of 4.5% NaCl and 0.04% azide in agar were used for confirmation (A-20). It is difficult to reconcile the use of media containing high concentrations of salt with the observations of Mastromatteo and Baldini (1963) and Litsky et al. ( 1953). The latter workers reported that 1.5%salt (in broth) was slightly inhibitory to enterococci. In addition, Mayeux and Colmer (1961) de-

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scribed a medium containing 10% sucrose and only 0.005% sodium azide that was selective for isolation of Leuconostoc spp. from materials that probably also contained large numbers of plant forms of enterococci. These reports would indicate that at least some strains of enterococci are more susceptible than is commonly believed to environments with high osmotic pressures. Therefore, media containing high levels of sodium chloride may give erroneously low recoveries, although these same media might be adequate for confirmation of salt- and temperature-tolerant streptococci ( Splittstoesser et al., 1961).

M. TELLURITE Tellurite was first used as a selective agent in culture media as early

as 1912, when Conradi and Troch (1912) described an improved medium

for isolation of diphtheria bacilli, These authors noted that streptococci and several other groups of microorganisms grew in the presence of 0.02y) potassium tellurite, but the growth of many bacteria commonly found in the throat was inhibited, Other aspects of early studies with tellurite have been reviewed by Gilbert and Humphreys (1926) and Whitley and Damon (1949). Observations similar to those made by Conradi and Troch (1912) were made by Smith ( 1914, 1918); however, Cooper and Ramadan (1955) discovered that a broth (B-25) containing 0.02% POtassium tellurite was quite suitable for isolating streptococci from feces of various animals. An agar medium containing 0.2% tellurite was also proposed (Cooper and Ramadan, 1955). Perry and Petran (1939) had proposed the use of tellurite agar slants specifically for preliminary isolation of hemolytic streptococci from the throat and nose. Fleming (1929) studied the effects of penicillin on different bacteria and observed that this antibiotic exerted a selective action on different species of streptococci. The complementary action of penicillin and potassium tellurite was later used (Fleming, 1932) in devising media for selective isolation of various groups of microorganisms, including streptococci. In a later study (Fleming and Young, 1940), levels of 0.02% to about 0.0002% tellurite were suitable for inhibition of E. coli in fecal material; susceptibility of Protcus spp. was variable ( see also Lichstein and Snyder, 1941). In the same year (Bornstein, 1940) the resistance of enterococci to penicillin and tellurite was confirmed; enterococci and S. (actis were resistant to penicillin, and Streptococcus oiridans strains were susceptible. All enterococci tested were resistant to 0.1% tellurite, while tolerance of other streptococci to tellurite (0.01% ) varied. A tellurite-taurocholate-esculin-sodium chloride agar of high pH (medium A-36) was described by Schafer ( 1953) but apparently was

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never tested further, although recommended by a German commission on methods of examination of water ( Methodenkommission, 1960). A medium for the isolation of S. salivurius was devised by K. D. Rose and Georgi (1941), using 0.03% potassium tellurite and 0.00027, crystal violet. These concentrations are similar to those used earlier by Garrod (1933), but Pike (1945b) reduced the tellurite concentration to 0.01~0 (medium B-24). Chapman ( 1944,1946,1947) proposed a medium (A-38) containing only 0.002% potassium tellurite, but with 0.00008% crystal violet and 0.0075% trypan blue. Sucrose (5% ) was added as a substrate SO S. salivarius would produce polysaccharide and thus be differentiated from S . mitis and most hemolytic streptococci. Enterococci reportedly also produced distinctive colonies on this medium (A-38), known as MitisSalivarius agar. Sucrose ( 5 % ) was similarly used by R. E. 0. Williams and Hirch ( 1950) to examine streptococci from air in a medium (A-37) that contained less tellurite and crystal violet than Chapman’s medium. In addition, trypan blue was omitted. This medium would be expected to be less selective than Mitis-Salivarius agar, but even at these concentrations of- inhibitors growth of some streptococci (none of them S. salivarius) was severely retarded. The mechanism of growth inhibition by tellurite is not fully understood. Of the enterococci, S. faecalis is resistant to 0.05% tellurite; most others are susceptible to 0.05% tellurite (Deibel, 1964; Hartman et al., 1966), although tellurite-resistant strains can be selected from a susceptible population ( McDowell and Hartman, unpublished data, 1964). Fleming and Young (1940) had observed earlier that tellurite-resistant strains of E . coli could be obtained by adapting cultures to increasing concentrations of the inhibitory agent. Tucker et al. (1962) reported that S. faecalis reduces tellurite to pure tellurium metal, and Walper et al. (1962) developed a method for the quantitative determination of tellurite in microbiological media. Tellurite reduction by protoplasts and cell-free preparations has been studied (Thomas et al., 1963), and progress has been made in investigations on the site and nature of tellurite reduction (Tucker et al., 1964; Thomas et al., 1965). Several additional observations are worthy of note in regard to the use of tellurite in culture media. Smith (1918) reported that, “On standing, the tellurate solution gradually loses its antiseptic action, whereas telluric-acid solution, if sterilized by heat, keeps indefinitely.” R. E. 0. Williams and Hirch (1950) reported that storage of tellurite plates for up to 7 days in a refrigerator before incubation had no detectable effect on the streptococcus counts. Fleming and Young (1940) found that, upon extensive incubation (48 hours ), resistant strains could “absorb or render

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inert at least 90 per cent of the tellurite over a distance of 16 mm” from the resistant colony. Tellurite causes hemolysis in blood agar in concentrations as low as 0.002% (Edwards, 1933). Obviously many fecal streptococci are inhibited by relatively low concentrations of tellurite or do not reduce tellurite to produce black colonies. These bacteria would not be included in counts made on tellurite-containing media.

N. PENICILLINAND OTHERANTIBIOTICS J. C. White and Sherman (1944) were the first to combine penicillin with azide for isolation of enterococci (medium A-9). Methylene blue was added in a modification of the medium (A-10) by Winter and Sandholzer (1946a,b), so debris would be stained deep blue whereas colonies were white with a blue center. This permitted better differentiation of colonies from debris. In addition, the penicillin level was doubled, which resulted in better selectivity for fecal streptococci. A penicillincontaining enterococcus confirmation medium ( A-19) was also proposed by Winter and Sandholzer (1946b) for water analysis; enterococci would have to survive a rigorous presumptive test ( B-15a) before encountering the penicillin. M. F. White et al. (1947) omitted penicilhn from the confirmation medium. Yields on these media would probably be low compared with yields attainable on certain other media discussed in previous sections. Other antibiotics have been included in media used to isolate streptococci, and a few of these media have been examined for their application to the isolation of fecal streptococci. Burkwall and Hartman (1964) studied two media (A-40 and A-41), both containing neomycin. These media were not sufficiently selective for enumeration of enterococci in frozen foods; however, changes in formulation of the media might increase the efficacy of these media for such uses. 0. SODIUMTAUROCHOLATE AND BILE SALTS Sodium taurocholate was first used as a selective agent for the isolation of coliform bacteria ( MacConkey, 1905). Application of this selective agent to media for isolation of streptococci was reviewed by Koch (1935), who devised an improved medium (A-35) that apparently possessed good selectivity. Weissenbach (1918; see also Bagger, 1926) preferred ox bile (medium B-30) to the sodium taurocholate preparations available at that time. Mieth (1960) used a taurocholate enrichment broth (B-12) of quite different composition in that sodium azide and sodium chloride were inoluded as additional selective agents. These media, except B-12, probably lack the selectivity necessary for primary isolation of enterococci from foods and other sources. Szita (1957) pro-

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posed a taurocholate-crystal violet-tellurite medium that seemed to function well as a confirmatory agar. Rycroft ( 1956) developed a taurocholate-mannitol broth (B-31) with incubation at 44°C. for use in confirmation of fecal streptococci. Two unusual features daimed of preparations grown in this medium were extensive chaining of cells and prolific production of group D antigen for serological tests. This medium may be superior to that developed by Medrek and Barnes (1962) for group D antigen production by mannitol-fermenting enterococci; however, no one has examined this possibiIity. P. PHENETHYL ALCOHOL A phenethyl alcohol medium (A-42; Lilley and Brewer, 1953; Anonymous, 1956) was found by Burkwall and Hartman (1964) to lack the selectivity necessary for enumeration of fecal streptococci in frozen foods. Nevertheless, phenethyl alcohol is another potentially useful ingredient for increasing the selectivity of existing media. Phenethyl alcohol apparently inhibits growth of gram-negative bacteria (Lilley and Brewer, 1953; Berrah and Konetzka, 1962), except gramnegative anaerobes (Dowel1 et al., 1964). Inhibition probably occurs through blockage of DNA synthesis (Berrah and Konetzka, 1962; Treick and Konetzka, 1964), although the synthesis of messenger RNA and the process of enzyme induction evidently also are affected by phenethyl alcohol (Rosenkranz et al., 1964, 1965).

Q. PH Chesbro and Evans (1959) devised a medium of pH 10.0 for tolerance tests of enterococci. A modification of this medium (pH 9.6) gave low yields when used for direct plating ( Hartman, unpublished data). Yet high pH may stilI be suitable for prdiminary enrichment or for selectivity after preliminary enrichment. Some investigators have devised media with rather high pH values (media A-19, A-20, A-24, A-25, A-35, A-36, B-14, B-15a). Tolerance of enterococci to high pH values is well known (see Chapman and Rawls, 1936))and several workers (Mueller and Whitman, 1931; Stainsby and Nicholls, 1932) inoculated fecal material or pathological specimens into sodium bicarbonate solutions of 0.2 to 1.0% which were incubated for a period prior to plating. This method has also been applied to milk samples (Groesbeck, 1935). The high pH destroys many gram-negative contaminants. Carbonate and bicarbonate have also been used to stimulate growth of fecal streptococci (Burbank, 1929; C. W. Langston, cited in Deibel, 1964; media A-15, A-16, A-17, B-19). The mechanism of stimulation has

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been investigated (see Martin and Niven, 1960; Wright, 1960; Deibel, 1964). Of interest in this regard is the finding of Mickelson (1964) that some strains of S. pyogeries required C02, supplied as NaHCO:,; one COZrequiring strain could be adapted to grow in bicarbonate-free medium. An interesting discovery was reported recently by Sims ( 1964). Streptococcus bovis would grow in media acidified to pH 5.0 and incubated at 37°C. in an atmosphere of 5% C 0 2 in air. Other streptococci cannot grow under these conditions, The potential use of this medium has not been explored. Contaminating lactobacilli could probably be inhibited by 2 F citrate or some other additional ingredient. K. SELENITE

A selenite (about 0,024F )-aniline blue (about 0.071% )-blood platc

w ~ devised s (medium A-34; Guthof, 1952) to differentiate streptococci by modification of a medium originally intended for diagnosis of diphtheria ( Herrmann, 1939). Although not proposed for primary enrichment or plating (which is performed with other media), the selenite-

indicator-plate ( medium A-34) may prove valuable for subsequent differentiation of enterococci (Guthof and Winkler, 1955; Guthof and Dammann, 1958). It could be used to isolate $trains of S. bovis, S. salivarius, or S. mitis, the growth of which might be inhibited on other more commonly used media. S. TETRATHIONATE

Many of the selective media now used for isolation of fecal streptococci were first applied to examination of gram-negative rods of intestinal origin. Thus, it is not surprising that tetrathionate broth, which was used by Cooper et al. (1942) to isolate Salmonella paratyphi from feces, would be applied (medium B-29) to isolate enterococci (Cooper and Ramadan, 1955). Several workers (see Prescott and Baker, 1904) have noted that in these media coliforms grow profusely at first, then are followed, after incubation for 2 or 3 days, by increases in the streptococcal populations. Mallmann and Gelpi (1930) took advantage of this succession of culture types in use of a standard lactose enrichment broth for detecting streptococci. Confirmation was made by microscopic examination. Cooper and Ramadan ( 1955) discovered that tetrathionate broth, though ohten successful with human feces, failed significantly in conccntrating streptococci from sheep and bovine feces. An incubation period of 24 hours was used, however. Perhaps modifications of the constituents of the medium and procedure of isolation (such as use of a longer incubation period ) will enable tetrathionate-based media to be used successfully in food microbiology. The striking differences in

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recovery of streptococci from human, bovine, and sheep sources with tetrathionate versus tellurite and thallium acetate media emphasize the fact that different media do have different selectivities. This fact might be used to good advantage when more is known about the distribution of various fecal streptococci in nature.

Ill. Comparative Studies on Media and Methods So far, we have discussed various media with little regard to the type of sample to be examined (water versus a food versus intestinal contents, etc.), technique to be used for the examination (most probable numbers (MPN) versus pour plate versus surface or smear plate versus membrane filter), or objective of the examination (isolation vs. enumeration). Obviously, no one culture method will be the most suitable for all applications. In like manner, no one medium will be most appropriate for all culture methods or all types of natural materials. Comparative studies on the efficacy of a medium for a specific purpose, therefore, must be interpreted with caution, especially when translating the efficiency of the medium to a different type of material or to a material that has been treated differently. A. GENERALDISCUSSION Many, if not a large majority, of the microorganisms in certain foods and other natural environments are in a different physiological state than similar strains cultivated in the laboratory. This fact had been noted earlier by many investigators (see F. C. Harrison and Vanderleck, 1915; Darby and Mallmann, 1939). Stock cultures may be valuable for initially determining the approximate selectivity of a medium, but frequently these preliminary tests have not been followed by examination of sufficient samples of natural products. With laboratory strains as the test material, continuation of a rapidly growing culture is the usual concern, whereas with bacteria from the natural environment the problem often seems to be not only growth but also growth initiation (see also Ferraro and Appleman, 1957, and Slanetz and Bartley, 1957). The problem of growth initiation has received little attention in research because it is difficult to approach experimentally. Nevertheless, the over-all effect is that a medium usually will be more inhibitory to bacteria in natural products than to rapidly growing laboratory cultures. Because there are so many unknown factors, one studying the complete flora of a material might be well advised to use several media, each with a different selective ingredient or group of selective ingredients (see Cooper and Ramadan, 1955) .

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Another factor that seems to be well known but usually is equally well ignored is that various media have different selectivities. Too often the method of defining a group of indicator organisms, such as “coliforms” or “enterococci,” has been to determine the flora that will grow on a medium used to detect the group, rather than to define the group first and then obtain media with appropriate selectivity. These factors become of paramount importance when natural materials, such as foods, are to be examined. As improved classifications of the fecal streptococci become available (Deibel et al., 1963; Deibel, 1964; Hartman et d., 1966), comparative studies will be facilitated. Meanwhile, it might be well to point out that organisms within a culture vary in their ability to grow under one set of conditions (see Whittenbury, 1963), and no single medium or set of conditions is likely to result in selective recovery of all of the fecal streptococci in a sample containing quantities of other, sometimes closely related bacteria. Preliminary enrichment may overcome some of these difficulties. The value of preliminary enrichment in sugar broth had been described as early as 1952 by Allen and co-workers (cited in Childs and Allen, 1953) and was emphasized again recently by R. E. Rose and Litsky (1965). Other workers have chosen to inoculate “presumptive” media (i.e., B-13 or B-16), transferring later to more selective “confirmatory” media (i.e., A-19 or B-15). Still other workers have preferred to use very highly selective media (i-e., A-3, A-8, A-18, A-24, A-25, A-27) with some sacrifice in yield. A list of all publications in which each of the various media and methods has been used for specific applications would mean little. Most workers have compared their new medium with only one or two other media. These studies, although they may be excellent, are so difficult to correlate with each other that adequate conclusions cannot be reached. On the other hand, there have been some rather comprehensive comparisons involving several or many types of media and methods simultaneously. In only two of these studies (Saraswat et al., 1963; Burkwall and Hartman, 1964) have different pour-plate media been compared extensively. Some key comparative studies have been selected to illustrate the present status of media and methods for the determination of enterococci. IN WATER B. ENTEROCOCCI

The anallysis of enterococci in water was given impetus by the work of Litsky et al. ( 1953), who found that a most probable number (MPN) procedure involving selective enrichment in B-6 followed by confirmation in B-17 gave much greater yields than use of media B-15a or B-22. The B-6, B-17 combination has been widely used in water bacteriology

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and seems better than many other media, such as a B-21, B-17 combination (Croft, 1959) and B-21 alone (Kenner et al., 1961). Slanetz and Bartley (1957) devised a membrane filter procedure (A-13) that gave higher yields from water than the B-6, B-17 combination. Kenner et al. (1961) also reported that A-13 was superior to the B-6, B-17 combination, although Croft (1959) found the two methods equivalent. New media (A-17, B-19) were developed by Kenner et al. (1961) for MPN, membrane filter, and direct plating techniques. The MPN procedure gave highest yields; direct plating in A-17 was probably intermediate and slightly higher in yield than the membrane filter method; all were superior to A-13. The membrane filter and direct plating methods were recommended where applicable because colonies could be confirmed without additional plating. Mallmann and Kereluk ( 1957) reported that considerably greater numbers of enterococci were recovered by the drop-plate method using medium A-14 than by an MPN method using B-6, B-17, but these observations have not been applied to other media. MPN versus direct plating methods are discusssed in the next section. Although many combinations of media and methods await extensive testing, the three most logical selections for use in water bacteriology at present are the B-6, B-17 combination for use with the MPN procedure, A-13 for the membrane filter procedure, and A-17 and B-19 for MPN, membrane filter, or direct plating. The method chosen would be dictated by the circumstances of analysis. Regardless of the procedure selected, isolates should be confirmed, at least in preliminary experiments. Confirmation, especially from filter membranes, shouId probably be preceded by enrichment on a nonselective medium (Croft, 1959). Such a practice also might be necessary for confirmation in medium B-17, because this medium inhibits many strains of S . bovis and S. equinus (Kenner et al., 1960). The value of preenrichment for MPN or membrane filter techniques has already been discussed.

C. ENTEROCOCCI IN NONDAIRY FOODS The first comprehensive comparative study of methods for enumerating enterococci in foods was done by Zaborowski et al. (1958). Medium B-15 was the best of four MPN presumptive media examined; medium B-6 gave higher counts but was not as selective. Medium B-17 was satisfactory for confirmation. Thus, the optimum procedure developed by Zaborowski et al. (1958) would be use of B-6 or B-15a with confirmation in B-17. Fanelli and Ayres (1959) also found the B-6, B-17 combination good for MPN determinations, and Splittstoesser et al. (1961) reported that medium B-6 gave higher counts than three other presumptive MPN media. Hall et al. (1963), however, preferred medium B-21 to the B-6,

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B-17 combination. The latter workers reported that B-21 was equivalent to B-19, a finding in contrast to previous reports on the inadequacy of medium B-21 for food analyses. In summary, the best of the MPN procedures so far developed is use of the B-6, B-17 combination, or possibly B-19. Hall et al. (1963) reported that B-19, a broth, was slightly more inhibitory than an agar medium of the same formulation (A-17) to some, but not all, species of enterococci, Fanelli and Ayres (1959) also obtained highest yields by using an agar medium (A-29) and direct platins procedures, rather than MPN techniques. Medium A-29 was found by Burkwall and Hartman (1964) to be the best of 15 direct plating media examined. Medium A-17 also was satisfactory, as was a new medium ( A-15, improved formula, A-16). These media, although they are high in yield, lack selectivity and need at least some confirmation. This has been emphasized by Mossel ( 1964) and Saleh et al. (1966). Some agar media apparently possess sufficient selectivity that confirmation is unnecessary. The best of these, as determined by Burkwall and Hartman ( 1964), was medium A-3. ( A modification of this medium has been proposed recently, medium A-18.) Lower yields might be obtained on this medium than on the media discussed in the preceding paragraph. On the other hand, one might wish to use a very high-yielding medium, such as A-42, but there may be many false-positive results (Burkwall and Hartman, 1964), so confirmation would probably be essential. D. ENTEROCOCCI IN DAIRY PRODUCTS

Of ten media examined, only medium A-18 met the rigid requirements established by Saraswat et al. (1963) for enumeration of enterococci in dairy products. Media A-30 and A-31 also may be suitable, but their efficacy in relation to medium A-18 has not yet been well established.

E. ENTEROCOCCI IN INTESTINAL CONTENTS There is such divergence of opinion with regard to the best medium for isolation and enurneration of fecal streptococci from intestinal ingesta that we only refer readers to the publications of Kenner et al. (1960), Mieth ( 1960), and Raibaud et al. ( 1961) as examples of what some investigators think of other investigators’ media. F. FUTURE NEEDS

A comprehensive comparison of media and methods representative of all of those presently available is urgently needed. These media and methods should be utilized simultaneously, using the same samples or

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subsamples. Subsamples would probably be necessary because the study should include interlaboratory or, preferably, intercontinental collaboration. Isolation and quantification of fecal streptococci in natural materials of all types should be examined. This would be a gigantic undertaking because confirmation of positive colonies and presumptive tests would be necessary. Likewise, negative presumptive tubes (colonies) should also be examined ( a matter that few investigators have considered). Before such a large screening program is undertaken, there would have to be agreement on what bacterial species or strains are desired and what are not, and then procedures would have to be developed to evaluate the eficacy of the media. Only when such experiments are completed can optimum procedures be selected for specific applications with a reasonable degree of confidence.

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