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Bacteriological Examination of Unbottled Soft DrinksF. M. RAMADAN AND H. A. ABD-ELNABY

Water Pollution Unit, National Research Centre, Dokki, Cairo, United Arab Reputblic

Received for publication January 25, 1962

ABSTRACT

RAMADAN, F. M. (National Research Centre, Cairo,United Arab Republic) AND H. A. ABD-ELNABY. Bacteri-ological examination of unbottled soft drinks. Appl.Microbiol. 10:311-320. 1962. A study of 300 samples,representing 14 different unbottled drinks, indicated thatthere are three vitally important criteria pertaining totheir bacteriological examination. First, the total viablecounts may be better accomplished by the pour-platemethod, using enriched media, with incubation at either30 or 37 C. Second, a comparative study of the coli-aerogenes group and the enterococci as indices of pollutionunquestionably favors the latter as the reliable indicator,owing to false interpretations of the presumptive test andto lack of accurate definition of fecal and nonfecal coli-forms recovered from positive cases. The use of enterococci,however, did not provide as reliable an indicator as thepour-plate method. Third, the results with enterococci, indefining the probable source of pollution, are more precise.Experiments judiciously selected and simultaneouslyconducted revealed that the heat and heat-telluriteresistance tests, and the tetrazolium-reduction test,matched in relating 98.9 % of available enterococci toan animal source. Negligible but vital discrepancies wereobtained with the two odd strains which qualified ashuman-derived according to the heat and heat-telluriteresistance tests. The differential criterion of Skadhaugeand Barnes, based on the failure of animal-derived entero-cocci to grow in the presence of a low concentration ofpotassium tellurite, did not apply to the other twomethods, since 99.5 %o of the recovered strains were foundtolerant to the specified tellurite concentration.

At the present time, the processing of Egyptian un-bottled drinks is not controlled by designated standardsor adequate specifications. Formulae employed in themaking up of such drinks are quite varied and rest mainlyon the so-called "individual art." Bacterial contaminationreaching these feeds may be derived from the raw materials(usually of plant origin), water, sugar, ice, flavoringmatter, coloring matter, utensils, hands of the producer,and surroundings (e.g., tables, flies, and dust).The main object of any bacteriological standards pro-

posed for such foods is to secure three basic criteria,namely, their freedom from pathogens, toxinogenicbacteria, and high bacterial counts. Because the chiefsource of enteric pathogens of man is human fecal matter,

and because it would be tedious and impractical to lookfor all pathogens during the routine examination of suchfoods, it is a common concept to search for certain indicesof pollution. Accordingly, a growing mass of knowledgehas developed on the use and merits of each indicator.

Little sanitary significance is attributed to the pollutionindex of Clostridium perfringens, since most of strainsgaining access to foods were found to originate from soil(Beerens and Delcourte, 19.58; Buttiaux, 1959).Appleman (1955) reviewed the merits of coliform

organisms and fecal streptococci as indices of pollution incitrus concentrates, and though unable to give preferenceto either indicator, he favored reliance on the indicatorgroup with the greatest longevity in these foods. A decisionmay be reached, however, when the sanitary significanceof coliforms other than Escherichia coli, and the reasonsfor which enterococci are present on sound citrus fruits,have been established.The marked superiority of enterococci over coliforms as

indices of pollution in frozen foods has been suggested byseveral investigators (Berry, 1946; Ostrolenk, Kramer, andCleverdon, 1947; Burton, 1949; Kaplan and Appleman,1952; Larkin, Litsky, and Fuller, 1955a, b, c), who recog-nized their marked resistance to storage conditions and theease with which they can be isolated and identified. Burton(1949), however, adds that in unfrozen foods coliformswould be the more reliable indicator.

Buttiaux (1959) discussed the recent belittling of thesignificance of the coliform tests, e.g., the role of anti-biotics in disturbing the balance between members of thecoli-aerogenes group in the human intestines, and thediversity of opinion as to their probable origin whendetected in foods, and concluded that reliance should bebased primarily on fecal streptococci and secondarily onthe coliforms. The simultaneous recovery of enterococciand a coli-aerogenes strain from food justifies in his opiniona verdict of fecal contamination.

Investigations carried out by Larkin et al. (1955a) onfrozen vegetables and citrus concentrates revealed thegreat variations which may be obtained by the presump-tive and confirmed -coliform procedures on the same foodproduct. They also stated that the most probable ntumber(MPN) of enterococci was generally higher than the MPNfor coliform organisms. In their studies on lemon juice,lemonade, grapefruit juice, and orangeade concentrates,thev observed that no coliforms or enterococci could berecovered even after the acidity was neutralized.

Cooper and Ramadan (19.55) investigated the possi-311

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RAMIADAN- AND ABD-ELN\-ABY

bility of tracing the source of human or animal pollutionby means of fecal streptococci, and found that human-derived enterococci stand by themselves as a class readilydifferentiated from those of the cow and sheep. FIurtherinformation (unpublished data) on the classification andsignificance of enterococci from other animals tends tostupport the hypothesis of Cooper and Ramadan. Ramadan(1960) reported that enterococci would give a betterindication than coliforms in the detection of pollution andits source in water, milk, and ice cream.The present investigation compares the merits of entero-

cocci and coliforms as indices of pollution in variousunbottled drinks. The total viable counts recovered fromsuch foods, and possibilities of adopting the membrane-filter techni(ue in their examination, have received con-sideration.

MIATERIALS AND AMETHODS

A total of .300 samples, representing 14 different drinks,was collected aseptically in sterile containers from shopsand street dealers in the different quarters of Cairo city.The designation and composition of such drinks may besummed up as follows. Sugar-cane juice: stem of sugarcane (Saccharum spp.), trimmed and decoated with aknife and then mechanically pressed several times betweenwooden cylinders; ice and sometimes water are added.Licorice water: dried roots of licorice (Glycerhyza spp.) areput inside a small cloth bag and soaked in water for about3 hr, and then mascerated by hand with frequent additionof water; ice is then added. Tamarind syrup: dried fruitsof Tamarindus indica are soaked in water for a few hoursand then emulsified to produce a thick paste; water, sugar,coloring matter, and ice are added. Carob syrup: driedfruits of Ceratonia siliqua, ground into a fine powder andthen soaked in a small amount of water for several hours;water, sugar, coloring matter, and ice are added. Barleysyrup: wheat of barley (Hordeum spp.) is wetted withwater to form a thin paste and left overnight to ripen;sugared water and ice are then added to bring the neces-sary dilution. Rice syrup: wheat of rice (Oryza sativa)prepared in the same manner as barley, but the resultingsyrup is thick in consistency; usually refrigerated beforebeing served. Orange juice: whole orange juice; usuallyrefrigerated. Orange syrup: orange juice, water, sugar,coloring matter, and ice. Lemon juice: whole lemon juiceplus concentrated sugar solution and ice. Lemon syrup:citric acid, sugared water, coloring matter, and iee.Strawberry syrup: fresh strawberry fruits (Fragaria spp.),squeezed with sugar-water and ice added. Mango syrup:whole juice of mango (Alagnifera indica), either refriger-ated or diluted with sugar-water and iced. Pomegranatesyrup: pomegranate fruit (Punica granatum), peeled, theseeds squeezed, and the resulting juice refrigerated; insome cases the juice is diluted with sugar-water and iced.Carrot juice: washed and peeled roots of carrots (Daucuscarota var. sativa) are pressed for their juice, which is

The pH levrels of the samples were measured electri-cally, and their sugar concentrations recorded in terms ofpercentages by means of a Zeiss food refractometer(Tropenmodell I). The following bacteriological proce-

dures were carried out on each sample after thoroughmixing for 2 min.

Plate counts. Serial tenfold dilutions ranging from 1:10to 1:1,000 were prepared and 1-ml inocula were used forpouring nutrient agar plates (1.57% agar). A set of fourplates was made. Another set of four plates was pouredusing glucose-yeast-agar medium (0.25)%G glucose, 0.25 %/yeast extract, and nutrient agar). Two plates from eachset were incubated at 37 C and the other two at 30 C.Readings of plates were recorded after 48 hr.

Coliform tests. The presumptive coliform test was

conducted by transferring 1-ml inocula from the originalsample to two tubes of MacConkey broth (single strength)and incubating at 37 C. Examination for acid and gas

production was carried out after 24 and 48 hr. Tubesshowing positive reaction were used for subculturing on

eosin-methylene blue plates and further incubation at 37 C.From the resulting 24-hr growth, a colony with a metallicsheen was transferred to a tube of 1 %O peptone-water andleft to incubate at 37 C for 16 hr. Typing of the coliformstrain in question was carried out on the resulting growthby testing its IMViC-reactions plus gas production inMacConkey broth at 44 C and gelatin liquefaction (Reportof the Coliform Sub-Committee, 1949). The nomenclatureof available strains is in accordance with the Report ofthe Coli-Aerogenes Sub-Committee (1956).

Enterococci tests. Samples (1 ml) were inoculated intothe following three selective media: potassium telluritebroth, 1: 5,000 (modification of Cooper and Ramadan, 1955);sodium azide broth, 1:4,000 (1 %/c peptone, 0.3 %o yeastrel,0.5 % glucose, 0.5 % NaCl, 0.5 % K2HPO4, 0.5% K112PO4,0.025 NaN3, pH 7.4); thallium acetate broth, 1:2,000(Cooper and Linton, 1947). Incubations were carried outat 37 C for 24 hr. Subcultures were made from all tubeson MacConkey agar plates; from the resulting 24-hrgrowth, an enterococcus colony was picked and sown on a

chocolate-agar plate to ensure purity of growth. A colonyfrom the resulting 24-hr growth was transferred to a tubeof Bacto-Peptone broth and left to incubate at 37 C for16 hr. The following tests were then carried out from theresulting growth: testing resistance to heat at 63 C for 30min; testing heat and tellurite (0.04 %) resistance;testing the rate of reduction of Janus Green in milk.Technical procedures for performing and translatingresults of these three tests were fully described by Cooperand Ramadan (1955) and by Ramadan (1960). It may berecorded, however, that additional Janus Green batcheswere standardized during the course of this work, andthat the dye designated "diethylsafranin azodiethylanilin"prepared by the British Drug Houses, when added to milkat a final concentration of 1:8,000, has constantly repro-

duced differential results between human and animal-then iced.

312 [VOL. 10

derived enterococei. Also, one batch of Janus Green


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BACTERIOLOGY OF SOFT DRINKS

prepared by Merck & Co., Inc. (batch no. 620282) wastested and found suitable when brought to a final concen-tration of 1: 10,00.

Tetrazolium reduction. Platings were made (Barnes,1956) on tetrazolium agar plates (1 % peptone, 1 %

Lab-lemco, 0.5% NaCl, 1 % glucose, 0.001 % 2,3 ,5-tri-phenyltetrazolium chloride, 2.5 % agar, pH 6). Plates wereincubated at 37 C for 24 hr, after which the rate of tetra-zolium reduction was recorded.

Growth on 0.04 % tellurite agar plates. One stroke from

TABLE 1. Evaluation of total viable counts in unbottled drinks

Distribution of samples on:*Designation of sample Total no. Sugar range pH range Bacteria g GY agar N agar GY agar thogesain

37C 37C 30C 30 C

Rice syrup 10 20-22 4.5-5

Carob syrup

Sugar-cane juice

Barley syrup

Carrot juice

Mango syrup

Licorice water

Orange juice

Strawberry syrup

Lemon syrup

Pomegranate syrup

Orange syrup

Tamarind syrup

Lemon juice

24 2-10

45 14-22

20 15-18

11 8-10

25 9-22

36 1-2

22 11-12

13 17-20

17 14-20

5 15-17

28 3-18

31 16-20

13 18-20

5-5.5

4.4-5.5

7-7.5

6-6.5

4.7-6.5

8-9.2

3.5-4

3-3.5

3-6.5

3.5-3.7

2.8-6.5

2.5-3.5

2.5-3

107 4 4 3 4106 4 5 6 5105 1104 1 1 1 1

107 3 4 3 4106 6 7 6 6105 7 5 6 7104 6 6 8 7103 2 2 1 -

107 1 2 1 2106 17 20 16 17105 21 18 22 22104 6 5 6 4

106 9 10 8 10105 6 4 6 5104 5 6 6 5

106 3 3 2 410' 8 8 9 7

106 5 4 5 6105 11 12 10 10104 5 5 6 5103 4 4 4 4

106 5 6 5 610' 21 20 19 21104 9 9 11 810' 1 1 1 1

106 2 2 2 210' 2 2 2 2104 10 11 9 9103 7 6 8 8102 1 1 1 1

106 1 1 1 1105 1 1 2104 8 8 7 7103 2 1 1 1102 2 2 3 2

105 9 9 9 9104 5 5 6 6103 3 3 2 2

10' 2 2 2 2104 3 3 3 3

105 8 9 9 9104 10 9 10 910' 9 9 7 9102 1 1 2 1

105 1 - 1104 5 8 8 8103 9 6 7 8102 12 13 12 1010' 4 4 4 4

104 1 1 1 2103 2 3 3 4102 7 4 6 5101 3 5 3 2

Averages in thousands.585.21 646.57 519.86 652.49

* N agar = nutrient agar; GY agar = glucose-yeast agar.

4,253.50

1,747.75

769.50

504.25

345.00

245.25

211.75

104.50

90.50

56.50

46.00

37.00

4.25

1.21

1962] 313


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RAMADAN AND ABD-ELNABY

the mother culture was plated on a tellurite agar medium(1 % peptone, 1 % Lab-lemco, 0.3 % yeastrel, 1 % glucose,2.5 % agar, and potassium tellurite to a final concentrationof 1:2,500). Plates were left for 48 hr at 37 C and thenexamined for growth.

Application of the membrane-filter technique. A bacteria-filtration apparatus (Coli .5) was employed. Pilot experi-ments were conducted for the fixation of dilutions andinocula suitable for obtaining readable membranes. Theyrevealed that 10-ml inocula from dilutions between 1:100and 1:500 were most suitable for all of the investigateddrinks with the exception of sugar-cane juice. In thelatter case, dilutions ranging from 1:5,000 to 1:10,000were found necessary to overcome the blocking of mem-branes by the hanging fibers to be found in that drink.

After passing 10 ml from the dilution of choice into thesterile apparatus and membrane, the membrane wastransferred to a plate of Endo-agar (1 % peptone, 0.5%beef extract, 1 % lactose, 0.1 % alcoholic basic fuchsin,and 0.125% of a freshly prepared solution of anhydroussodium sulfite; pH 7.4). Incubation was carried out at37 C for 24 hr.

RESULTS

Determination of total viable counts. Bacterial countsmade from 300 samples representing 14 different drinksyielded variable results according to medium and incuba-tion temperature (Table 1). Statistical analysis to ascer-tain the variance for different methods of recovery andfor different drinks (Table 2) revealed a number of inter-esting points.

First, the adequacy of medium and incubation tempera-ture were in favor of the glucose yeast agar mediumincubated at 30 C or (second best) 37 C. There was littledemonstrable difference between the two incubationtemperatures. On plain nutrient agar medium, the leastreproducible colony counts were demonstrable at 30 C.Incubation at 37 C reproduced better counts but not upto the standard obtainable by the more enriched medium.

Second, the bacterial counts between different drinks

TABLE 2. Analysis of variance for bacterial counts (inthousands) according to methods of recovery*

F (tab-Degreesultda)

Sources of variation of Sum of squares Mean of F (cal- ufreedom squares culated)

0.05 0.01

Among methods 3 161,856.0 53,952.0 3.04t 2.84 4.31of recovery

Among drinks 13 68,742,131.0 5,287,856.0 298.31 t 1.95 2.56Residual 39 691,329.0 17,726.0Total 55 69,595,316.0

* SD = 133.14; SE for methods of recovery = 35.56; SE fordrinks = 66.57; LSD between methods of recovery at 0.05 =98.55 X 103; LSD between drinks = 184.49 X 103.

t Significant at 0.05 level.I Significant at 0.01 level.

varied markedly. The drinks are listed in a descendingorder of bacterial count in Table 1.The available information indicates that sugar concen-

tration bears little or no marked influence on the growthof bacterial populations in any of the investigated drinks.In attempting to group drinks according to the incidenceof bacteria they usually harbor, they tend to fall into threedistinct categories. One category would include lemonjuice, tamarind syrup, orange syrup, pomegranate syrup,lemon syrup, strawberry syrup, and orange juice; thisgroup may be regarded as generally having a low bacterial

TABLE 3. Indication of pollution by coliforms and enterococci

Coliform results Streptococcal index

Designation of Total Hu-sample no. No. of No. of Hu a n Sourcepos. & E.cl types pos. & man mal~ doubt-

ne. ypes t neg. de- de- funeg..

neg- ~rived rived fu

Sugar-canej uice

Licorice water

Tamarindsyrup

Carob syrup

Barley syrup

Rice syrup

Orange juice

Orange syrup

Lemon juice

Lemon syrup

Strawberrysyrup

Mango syrup

Carrot juice

Pomegranatesyrup

Total

45

36

31

25

20

10

22

28

16

13

13

25

-11

5

300

+41-4

+36-0

+8-23

+22-3

+19-1

+10-0

+7-15

+23-5

+2-14

+12-1

+9-4

+22-3

+10-1

+5-0

+226-74

26

7

8

8

11

8

2

11

2

4

4

5:

6

3

105

15

29

14

8

2

5

12

8

5

17

4

2

121

+40-5

+34-2

+13-18

+18-7

+20-0

+8-2

+10-12

+26-2

+3-13

+13-0

+5-8

+24-1

+11-0

+5-0

+230-70

1

1

2

18

3

6

6

7

7

4

15

2

5

2

4

6

2

87

22

31

6

12

12

1

6

11

1

8

3

20

5

3

141

314 [VOL. 10


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TABLE 4. Correlation between coliform typesand enterococcal source

Streptococcal source

Designation of Coliform results Human Animal Doubt- Strepto-drink HmnAia ob-Srpo

de- de- ful cocci 'drived rived source negative 0

.

E. coli typesNonfecal typesNegatives














1

1

171

1

3

3

3

3

12

6

61

1

3

762

2

311

1

1

1

3

411

2

7132

624

4

2

39

471

1

32

4

6

1

1

7

2

1

2

14

3

2

1

2

212

2

1

17

241

2

2

10

2

2

11

332

2

population. Members of this group are usually high inacidity.A second category would include licorice water, mango

syrup, carrot juice, and barley syrup; this group is denotedby intermediate bacterial counts. The pH values in thisgroup of drinks are not as highly acidic as in the firstgroup; the values tend towards neutrality. One drink,licorice water, maintains conspicuous alkalinity.The third category includes drinks with high bacterial

content, such as rice syrup, carob syrup, and sugar-canejuice. They show remarkable variations in the density ofbacterial population. They usually have pH values between4.4 and 5.5.

Indication of pollution by coliforms and enterococci. Thedata in Table 3 reveal that fewer polluted samples areindicated by either the coliforms or the enterococci inmost of the investigated drinks. Marked variations weredisplayed by the results obtained from tamarind, carob,and strawberry syrups.

Over-all, the coliform index indicates that 75.3% ofthe samples carried these organisms. Typical E. colistrains (types I, II, and III) constituted 46.4% of thetotal coliform bacteria recovered. The other organismswere distributed among Klebsiella aerogenes (Aerobacteraerogenes), Citrobacter freundii (Escherichia freundii), andirregular forms. The precise designation and incidenceof the coliform flora recovered from all drinks was asfollows: E. coli I, 60 (26.5%); E. coli II, 19 (8.4%); E.coli III, 26 (11.5%); K. aerogenes I, 19 (8.4%); K. aero-genes II, 14 (6.2%); C. freundii I, 13 (5.7%); C. freundiiII, 23 (10.2%); irregular coliforms (9 spp.), 52 (23%).The indication of pollution by the use of enterococci

showed 76.6% of the samples to be polluted. The sourceof pollution was traced to a human source in two samples(0.8%). Of the other enterococci, 37.8% were traced to ananimal source, and 61.3% were of doubtful origin.A correlation of the results obtained by the two indices

of pollution is presented in Table 4. Discrepancies, interms of samples failing to yield coliforms but showingthe presence of enterococci, amounted to 29 (9.6%). Onthe other hand, discrepancies denoted by enterococcibeing absent in the presence of coliforms amounted to 27samples (9%). Of these samples, 14 (4.6%) yielded E. colitypes.

Different interpretations are possible for the entero-cocci in the 121 samples showing the presence of coliformorganisms other than E. coli, i.e., the organisms mostprobably of nonfecal origin. Only 13 of these samples(10.7 %) were reported free from fecal pollution. The rest(108 samples) presented enterococci of animal origin in18 (16.6%) and of doubtful origin in 90 (83.3%).The two samples which presented enterococci from a

human source were reported as free from coliforms in onesample and as harboring E. coli in the other.

Differential methods for the identity of enterococci. Table5 shows the correlation between three different methods

Sugar-canejuice

Licoricewater

Tamarindsyrup

Carob syrup

Barley syrup

Rice syrup

Orange juice

Orange syrup

Lemon juice

Lemon syrup

Strawberrysyrup

Mango syrup

Carrot juice

Pomegranatesyrup

1962] 315


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recommended by different workers for the differentiationof Streptococcus faecalis and other members of group Dstreptococci. A close correlation exists between the methodof Cooper and Ramadan (1955) and that of Barnes (1956,1959), which depends on the rate of tetrazolium reduction.Thus, the 145 strains identified as animal-derived entero-cocci were found for the most part (97.9 %) to satisfy thecriteria given for S. faecium or other enterococci reportedto give similar reactions but not those for S. faecalis

and its variants, which are accepted as dominant in theintestines of man. The remaining strains (about 2 %) werepartial reducers, i.e., variables, a picture associated withS. bovis.

Enterococci of doubtful origin (276 strains) were foundto belong exclusively to the biochemical group VI ofCooper and Ramadan (1955), in which the benefit of thedoubt is given to an animal origin rather than to a humansource. Once more, the Barnes tetrazolium test confirmed

TABLE 5. Comparison of three differential methods for the identification of enterococci

Streptococcal technique Tetrazolium reductionDrink No. of strains Janus Green Heat Tellurite Source* Pink Variabie Pale Growth on 1:2,500lK2TeO3

milk resistance resistance Source* Pink Variahle Pale

Sugar-cane juice

Licorice water

Tamarind syrup

Carob syrup

Barley syrup

Rice syrup

Orange juice

Orange syrup

Lemon juice

Lemon syrup

Strawberry syrup

Mango syrup

Carrot juice

Pomegranate syrup

(4268

t26

69 o0

01522 6

(1

34 f2212

02235 t1

L12

19 f14

15 fio15

49 f27122

4 2

27 16

14 f{ls36 f2917

22 {

9 6

(276 +Total 423 g 145

(2 +

* H = human-derived; A = animal-derived; D =_

+_ - D 0 7

- - - A 0 1

+ - - D 1 4- - - A 0 0

+

35

25

559

- - D 0 3 12- - A 0 1 5+ + H 0 0 1

+ - - D 0 4 18- - - A 0 0 12

+

+

- - D 0 4 18+ + H 0 1 0- - A 0 0 12

- - A 0 1 13- - D 0 0 5

- - D 0 1 9- - A 0 1 4

+ - - D 1 6 20- - - A 0 0 22

+ - - D 0 0 2- - A 0 0 2

+ - - D 1 2- - - A 0 0

+ - - D 0 0- - - A 0 0

1311

95

+ - - D 0 2 27- - - A 0 0 7

+ - - D 0 0 11- - A 0 0 11

+ - - D 0 1 5- - - A 0 0 3

- - D 3 35 238- - A 0 3 142+ + H 0 1 1

doubtful source.

+40-2+26

+60+9

+15+6+1

+22+12

+22+1+12

+14+5

+10+5

+27+22

+2+2

+16+11

+9+5

+29+7

+11+11

+6+3

+274 (-2)+145+2

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BACTERIOLOGY OF SOF1,T DRINKS

stuch a hypothesis by reporting 98.9 c of these strainsas behaving like S. faecium and S. bovis. Only three strainswere capable of reducing tetrazolium to formazan, thutsindicating enterococci of huiman origin.

l'or the two human-derived strains, the tetrazoliumtest gave discordant results; one strain was a nonreducerand the other was variable in reaction.The third method, that of testing the tolerance of

enterococci to 1: 2,500 potassium tellurite, showed that99.5 'Xc of the recovered strains grew well in the presenceof that concentration; consequently, the results coincidedwith the results obtained by neither the streptococcaltechnique nor the tetrazolium-reduction test.

Selectivity of media for isolation of enterococci. MIediaused for concentrating fecal streptococci from soft drinksrevealed that potassium tellurite broth was the leastsulccessful, and that both sodium azide broth and thalliumacetate broth were about equally effective. From the 230samples harboring enterococci, the potassium telluritebroth succeeded in detecting only 115 (50 %G) of thesesamples. Sodium azide broth and thallium acetate brothpermitted recovery of enterococci from 168 (73 %) and164 (72%), respectively.

It should be noted that the tellurite broth was immedi-ately turned black (owing to reduction of tellurite totelluirium) on the addition of 1-ml inocula from most ofthe investigated drinks, especially those which wereheaxvily contaminated. Sugar concentrations and possiblyother organic ingredients present in such drinks mighthave had some effect in that respect.An additional problem concerns yeast survival in the

three selective media, especially the tellurite broth. OniAIacConkey agar, the yeast cells produce colonies which

TABLE 6. Correlation between the membrane-filter techniqueand the coliform tests

Membrane filterColiform typing results Colonies with No growth with Total

metallic sheen metallic sheen

E. coli types 19 1 20Nonfecal types 16 4 20Coliform negatives 11 24 35Total 46 29 75

TABLE 7. Discrepancies shown by the membrane-filter,coliform, and streptococcal techniques

Coliform results Streptococcal results*Membrane- No. offilter results samples Presump- E. coli Other

tive test types types H A D Neg

Positive 11 +5 - - - 3 3 5-6

Negative 5 +5 1 4 - 2 2 1

* H = human-derived; A = animal-derived; D doubtfulsource; Neg = negative.

may be easily confused with enterococci unless carefullyexamined (e.g., with a lens) to differentiate between thefaint pale pink growth of yeast and the more pink orbrick-red colonies of enterococci.

Results with the membrane-filter technique. The resultsobtained from the examination of 75 samples (five or sixfrom each of the 14 drinks) indicate that the total viablecounts revealed by the membrane-filter (MF') techniqueand the pour-plate method may be compared in thefollowing manner: counts on MF equal to counts onpoured plate, 32; counts on MIF less than counts on pouredplate, 26; counts on MFIF more than counts on pouredplate, 9; uncountable growth on M\IF, 8. The MAIF techniqueshould be regarded as useless when the number of bacteriain the original sample exceeds 104 per ml.The search for coliforms or colonies showing a metallic

sheen on the "Coli 5." membrane filter incriminated 46samples (61.3 %o) as carrying coliform organisms. This testis correlated with the coliform tests, run simultaneouslyon the same samples, in Table 6. Full agreement betweenthe two techniques was shown in 35 samples. I)iscrepanciesappeared with 16 samples. Correlation of these resultswith the streptococcal technique is shown in Table 7.The five samples showing no colonies with metallic sheen

on the MIF yielded E. coli from one sample and nonfecalcoliforms from the rest. The streptococcal techniquedetected four of these samples as carrying fecal strepto-cocci. This would show an agreement of 87.5%o by the MFtechnique and the coliform tests in establishing the pres-ence of an actual coliform strain from the polluted samples.

Failure of the MF technique and the coliform tests toprovide a conclusion for 11 samples, when verified by theresults of the streptococcal technique, instills equal doubtas to the accuracy of both techniques. Although the MFtechnique falsely reported five samples as polluted, thecoliform test allowed six samples to pass erroneously asunpolluted.

DISCUSSIONThe primary object of this study was to find a simple

technique for the examination of native unbottled drinks.A number of interesting criteria emerged from this investi-gation.

Estimations of bacterial counts were facilitated by usingan enriched nutrient agar medium rather than plainnutrient agar for pour plates. The temperature, whether 30or 37 C, did not seem to have a material influence oncounts obtained after incubation for 48 hr.The ranges of bacterial populations varied quite

markedly in each of the investigated drinks. This opensthe way to investigation of the raw materials, the process-ing procedures, and the general hygienic conditions underwhich a particular drink is prepared and maintained. Weshould stress the fact that drinks served in the juice formare usually prepared on order and commonly consumed assoon as they leave the blendor or pressing machine. Syrupdrinks, on the other hand, take several hours from prepa-

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ration to final consumption, although during that periodthey are usually retained at low temperatures (normallybelow 10 C). Low temperature-tolerant bacteria andyeasts would be exempted from this hypothesis. Broadlyspeaking, such marked changes in bacterial contentamong different samples of a drink may be largely at-tributed to the nature of the raw materials, e.g., thedonor fruits (Faville and Hill, 1931; Patrick, 1951), andto processing conditions.

In attempting to classify the investigated drinksaccording to the density of their bacterial populations, adifferential criterion that may be employed is acidity.Sugar variation does not serve as a useful criterion inthis respect.The relationship of coliforms and enterococci seems to

agree, although allowing for different interpretation ofpollution source. True-positive presumptive coliformtests were reported by 75.3 % of the total samples, andenterococci were detected in 76.3 %/ of cases by means ofthree selective media. Samples yielding enterococci but nocoliforms amounted to 9.6 %. On the other hand, dis-crepancies in samples presenting coliforms only reached9 %; of these, 4.6 % were E. coli types.The indication given by enterococci in regard to samples

harboring K. aerogenes, C. freundii, and irregular coliformsqualified 10.7 %l as pollution-free and incriminated theother 89.3 %o as bearing fecal pollution. These findings arein agreement with reports from other laboratories withregard to the doubtful status of the so-called fecal andnonfecal members of the coli-aerogenes group (Clarket al., 1957; Madecka-Borkowsky, 1957; Masinova andCledova, 1957; Lieb, and Anschau, 1959; Ramadan andMoussa, 1960).None of the three media used for recovering fecal

streptococci was absolutely selective for concentratingthese organisms from 1-ml inocula of soft drinks, althoughsodium azide and thallium acetate broths secured over75 % success. Potassium tellurite broth gave only 50%osuccess, but recovered a number of strains which escapedboth the azide and thallous acetate media. Until a failure-proof selective medium or method is attained, more thanone selective medium should be used in concentratingenterococci from soft drinks and possibly milk and otherfoods. Barnes (1959) realized this with reference to sodiuimazide and thallous acetate media.The relation between three of the recommended methods

for tracing the source of fecal streptococci may be arguedfrom a number of distinct and interesting points of view.The Cooper and Ramadan technique absolutely supportedthe Barnes tetrazolium reduction criterion in all entero-coccal strains designated as animal-derived. On the otherhand, the tetrazolium test identified 98.9 %/O of the doubtful-origin strains of Cooper and Ramadan as strains which areknown to be derived mainly from animal intestines.Putting the question of apportionment of credit aside and

looking at the matter more broadly, one finds completeagreement between the two techniques in tracing animalorigin, when the Janus Green reaction is omitted from theCooper and Ramadan technique. A quantitative differ-ential between human and animal enterococci would bereached only with freshly isolated strains, e.g., from feces;otherwise a marked change in the redox potentials takesplace, especially with weak reducers (or animal-derivedstrains) when grown for some time on artificial media(Ramadan, 1951; Cooper and Ramadan, 1955).

Nevertheless, discrepancies between the two techniquesshowed up in the definition of source of five strains (1.2 O4,).These strains were either strong tetrazolium reducers(three strains) but not heat resistant or, conversely, wereheat and heat-and-tellurite-resistant (two strains) butshowed weak or only variable tetrazolium reduction.There are several vital reasons why one should giveserious consideration to the recovery of heat-resistantand heat-and-tellurite-resistant enterococci from water orfoods. The foremost reason is that S. faecalis, which is thedominant streptococcus in the human intestine, is uni-versally acknowledged as possessing these two characters.One can further say that the stability of the reductivepower of dyes for most animal-derived enterococci isdebatable; this necessitates reliance on the heat andtellurite tests for reaching a differential criterion betweenanimal- and human-derived pollution in foods. Langston,Crutierrez, and Bouma (1960) reported such instabilitieswith tetrazolium reduction and S. faecium strains re-covered from silage.The confidence displayed by Skadhauge (1950) and

adopted by others, e.g., Barnes (1956, 1959), regardingthe lone tolerance of enterococci other than the animal-derived types for growth in the presence of potassiuimtellurite has not been found justifiable with our entero-coccal strains which were typed as animal-derived strains.Langston et al. (1960) reported the same observation and,most specifically, with S. faecium strains.The substitution of the MF technique for the pour-

plate method in the determination of bacterial counts inunbottled drinks does not seem feasible, since the availableresults record only 42.6% reliability. Such findings coincidewith those of Marshall and Edmondson (1960) on milk.The determination of coliforms with the MF on Endo-

medium revealed that not all coliform colonies produce ametallic sheen. Actually, 12.5 % of the coliform collectionfailed to show the sheen. On the other hand, 31.4 % of theorganisms other than coliforms showed up with themetallic sheen. Such organisms were mainly yeasts andstreptococci.The identity of coliforms "with no metallic sheen" was

confirmed by the presumptive and typing tests. Thestreptococcal technique supported the inference of pollu-tion in all five samples yielding such strains.With "metallic sheen" organisms other than coliforms,

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the presumptive test discarded some and falsely reportedothers as being polluted. No pollution case could beestablished, however, by the typing tests. The strepto-coccal results, on the other hand, incriminated more thanhalf of these samples as being polluted. This may be amatter of resistance or tolerance by enterococci in samplesovergrown with yeasts or other organisms capable ofenforcing false-positive presumptive coliform results.

It would seem essential from the public health stand-point to set up control measures for the distribution ofnative drinks. In many instances, evidence of food poison-ing mav be limited or mild and pass unrecorded, but onefamous epidemic involving hundreds of victims originatedin 1959 from a shop dealing with rice syrup which waslater found to have been contaminated with Staphylococcusaureus derived from a skin lesion on the hand of one ofthe workers.The standards recommended by Buttiaux and M\iossel

for the examination of fresh foods not obtained by micro-bial fermentation (Symposium on Problems in the Mlicro-biological Analysis of Foods, 1957) would serve as an ade-qtuate measure for most drinks for the time being. How-ever, critical revision of processing procedures ought toreduce the total bacterial counts and improve the qualityof the drinks.The detection of fecal pollution in drinks would be more

reliable through the use of enterococci as a pollution index.Also, the recovery of a heat-and-tellurite-resistant strainshouild be considered proof of human origin. Enterococcinot resistant to heat can be assumed to be from an animalsource. No or weak tetrazolium reduction by an entero-coccus can be relied upon as indicating animal-derivedstrains, primarily strains not resistant to heat. The reduc-tion of tetrazolium to formazan by some species of theenterococcus group should not be taken as an absoluteindication of S. faecalis or its variants, since this reactionmay also be obtained with many atypical strains presentin both human and animal intestines.Although the MF technique fails to give an accurate

determination of the viable counts, it can serve as anadded index of fecal pollution. It is needless to add thatserious consideration should be given to drinks showinghuman-derived pollution. Verification of pollution froman animal source should be related to such parameters asmanture, wind, insects, birds, and rain.

ACKNOWLEDGMENT

The assistance furnished by AI. S. Ibrahim in conductingthe analysis of variance is sincerely appreciated.

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BARNES, E. M. 1959. Differential and selective media for the faecalstreptococci. J. Sci. Food & Agr. 10:656-662.

BEERENS, H., AND F. DELCOIURTE. 1958. Procde' de diff6nciationentre Clostridiumn perfringens d'origine fecale et tellurique.Abstr. 6th Intern. Congr. Microbiol, p. 435.

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BURTON, M. 0. 1949. Comparison of coliform and enterococcusorganisms as indices of pollution in frozen foods. FoodResearch 14:434-438.

BUTTIAUX, R. 1959. The value of the association Escherichieae.-group D streptococei in the diagnosis of contamination infoods. J. Appl. Bacteriol. 22:153-158.

CLARK, H. F., E. E. GELDREICH, P. W. KABLER, R. H. BORENDER,AND C. B. HUFF. 1957. The coliform group. I. The boric acidlactose broth reaction of coliform IMViC types. Appl. Micro-biol. 5:396-400.

COOPER, K. E., AND A. H. LINTON. 1947. The value of thalliumacetate for the isolation of gonococci and streptococci.Monthly Bull. Ministry Health. Lab. Service 6:204.

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FAVILLE, L. W., AND E. C. HILL. 1951. Incidence and significanceof micro-organisms in concentrated orange juice. FoodTechnol. 5:423-425.

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LANGSTON, C. W., J. CRUTIERREZ, AND C. BOUMA. 1960. Motileenterococei (Streptococcus faecium var. motilis var N)isolated from silage. J. Bacteriol. 80:714-718.

LARKIN, E. P., W. LITSKY, AND J. FULLER. 1955a. Fecal strepto-cocci in frozen foods. I. A bacteriological survey of somecommercially frozen foods. Appl. Microbiol. 3:98-101.

LARKIN, E. P., W. LITSKY, AND J. FULLER. 1955b. Fecal strepto-cocci in frozen foods. II. Effect of freezing storage on Escher-ichia coli and fecal streptococei inoculated onto green beans.Appl. Microbiol. 3:102-104.

LARKIN, E. P., W. LITSKY, AND J. FULLER. 1955c. Fecal strepto-cocei in frozen foods. III. Effect of freezing storage on Escher-ichia coli, Streptococcus faecalis and Streptococcus liquifaciensinoculated orange concentrate. Appl. Microbiol. 3:104-106.

LIEB, F., AND H. ANSCHAU. 1959. tber einige Erfahrungen mitden IMVC-Reaktionen. Arch. Hyg. Bakteriol. 143:429-441.

MADECKA-BORKOWSKY, I. 1957. Studies on the variability of E.coli induced by iodoacetate. Bull. Acad. Polon. Sci. Ser. Biol.5:375-378.

MARSHALL, R. T., AND J. E. EDMONDSON. 1960. Substituting theM. F. for the standard plate count in the bacteriologicalexamination of milk. Dairy Science Abstr. 22:12.

MASINOVA, L., AND V. CLEDOVA. 1957. Evaluation of the positivetest for coliform bacteria in the sanitary tests of water andsoil. Inst. Hyg. Prague Rept., p. 13-14.

OSTROLENK, M., N. A. KRAMER, AND R. C. CLEVERDON. 1947.Comparative studies of enterococci and Escherichia coli asindices of pollution. J. Bacteriol. 53:197-203.

PATRICK, R. 1951. Sources of coliform bacteria in citrus juice forconcentrates. Proe Florida State Hort. Soc. 64:178-181.

RAMADAN, F. M. 1951. Studies in the detection of pollution; withspecial reference to distinguishing human and animal pollu-tion. Ph.D. Thesis, University of Bristol, England.

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