TESTS FOR THE ADULTERATION OF HUMAN MILKBY

R. A. MILLER and R. W. B. ELLISFrom the Royal Hospital for Sick Children, Edinburgh, and the Department of Child Life and Health, University of

Edinburgh

(R.EcEIvm FOR PUBUICATION JANuARY 26, 1953)

The necessity for testing human milk destined formilk banks in order to eliminate specimens whichhave been adulterated with water or cow's milk hasbeen widely recognized since the introduction ofsuch banks in 1910 (Mackintosh, 1947). In somecentres, however, no tests for adulteration seem tobe used, in spite of the fact that the mothers pro-viding milk for these banks are not supervisedbetween the time they express the milk and deliverit to the milk bank or milk bank official.

Various tests have been used to detect adultera-tion, and there is to this day no agreement on whichare most reliable. The selection of tests in the pasthas depended partly on the efficiency of a test butalso on other factors such as the speed and easewith which a test can be performed, the simplicityof the apparatus, the ease with whih reagents andsera can be obtained, and financial considerations.It would appear therefore that the ideal test orcombination of tests used in the detection of adul-teration of human milk with water or cow's milkshould be quick and easy to perform and preferablyshould not involve compliated or expensiveapparatus. The test must be reliable and able todetect minor degrees of adulteration. With thesecriteria in mind the many tests recommended for thedetection of adulteration have been reviewed andthose which best fulfil the conditions selected fordiscussion. In addition an investigation has beenmade into the freezing point of women's milk; thevalue of assessing the freezing point or the elctricalconductivity of women's milk for the detection ofadulteration of the milk has been explored.

Review of the LieratweSince last century many methods of detecting the

adulteration of women's milk have been devised.Usually one test appears no better than another andthe tests are neither sufficiently sensitive nor reliable.This is probably because the physical and chemicalproperties of milk vary from case to case and evenin a given individual.

At least six simple tests have been advocated forthe detection of water added to women's milk Twoare based on the determination of a physical pro-perty of the milk, and three depend on its chemialcomposition. The sixth is a combination of the twotests, one involving a physical property and one achemical property of milk. Of these tests based onphysical properties, the first necessitates the esti-mation of the refractive index of the milk specimenin question. The interpretation of the results mustbe made cautiously because they vary for genuinemilk, particularly if the sugar or solids-not-fat con-tained in the milk alter either under physiologicalconditions or on account of disease. Gnosspelius(1940) when critimng the test says that it is neces-sary to obtain a control sample of milk reliablyobtaied from the woman under test. The refractiveindex of both the given milk sample and the controlsample is made and the results compared. By thismethod 10% or more of added water can bedetd(Kappeller and Gottfried, 1920; Zaribnicky, 1942).In the dairy world the refractive index is not areliable test for detecting adulteration, and it there-fore appears that the results of the refractometrictest are unlikely to be reliable for the detection ofadulterated human milk. The second test involvesmeasurements of the specific gravity of the milkspecimen. This is an unsatisfactory test because it isonly suitable for detecting gross adulteration.Of the three tests based on the chemical composi-

tion of milk, the first involves the estimation of itschloride content. From the results it is usuallypossible to detect large amounts of added water.The test, however, is not reliable or of practicalvalue (Miller, 1949). In the second test both chlorideand lactose content of the specimen are determined.From the results the chloride-lactose ratio is calcu-lated. In the case of genuine human milk it is0-44-0-015. The ratio may be greater and approxi-mate to that of cow's milk, 2-23+0 082, if the milkis from women in the first month of lactation or fromwomen with inadequate lactation. It is highly

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ARCHIVES OF DISEASE IN CHILDHOODunlikely, however, that such women would be donorsto a milk bank (Keller and Mai, 1930). Milk adul-terated with large amounts of water has a chloride-lactose ratio less than 0-425. The third test isdependent on the relatively high lactose and lowalbuniin content of the milk to detect adulteration.From the tint which originates by a reduction of acopper salt solution by the lactose in the milk-serumand by the amount of oxide of iron necessary for aquantitative precipitation of albumin, Edelstein(1921) was able to ascertain whether or not thehuman milk had been mixed with 20 to 30'O ormore of water. This test, however, is not reliable.The final test for the detection of added water

involves the estimation of the specific gravity of themilk and its fat content. The data is then used togive a point on the chart devised by Strom (1939) orfor a formula. From the position of the point on thechart or from the result obtained by a specialformula it is possible to detect 30'/ or more ofwater added to the specimen.

It has been shown that it is difficult to detect milksamples to which water has been added. Probablythe least unsatisfactory method is to measure therefractive index of the milk. Other methods whichmay be adopted involve the estimation of the specificgravity and the percentage of milk fat, or thechloride and lactose content of milk, or the lactoseand albumin content of milk.Many quick and easy tests for the detection of

human milk adulterated with cow's milk are avail-able. These tests have been placed in one of fourgroups for the purpose of description and discussion.Group 1 includes tests dependent on a physicalproperty of the milk, Group 2 includes those testswhich necessitate investigation of one or two con-stituents of the milk, tests in Group 3 require theestimation of the hydrogen ion concentration of themilk in question, while those in Group 4 entail abiological investigation. In Group I (physicalproperties) the ultra-violet light test has been widelyused because it is possible to detect the addition ofsmall amounts of cow's milk in this way. Humanmilk normally gives a blue and cow's milk a yellowluminescence. Unfortunately both false positive andfalse negative results are sometimes obtained by thismethod (Griebel, 1937; Kayser, 1938; Smith, 1942).Another test, involving the estimation of therefractive index of the suspected milk detects anadulteration with cow's milk of 2000 or more pro-vided a reliable sample of milk from the woman whois being investigated can be similarly tested and thetwo results compared (Kappeller and Gottfried,1920; Zaribnicky, 1942).The second group of tests necessitates either the

qualitative or quantitative analysis of some con-stituent of the milk. In one test both the chlorideand lactose are estimated (Keller and Mai, 1930;Moruzzi and Tanzi, 1931). From the results thechloride-lactose ratio is calculated; when the ratioexceeds 0-455 adulteration must be suspected.Twenty-five per cent. or more of cow's milk addedto the sample is usually detected. Edelstein in 1921introduced the lactose-albumin test which detects20 o or more of added cow's milk, but the authorstates that the results are unreliable. Umikoff in1896 devised a method of detecting adulterated milkby adding ammonia to the sample and heating it to60- C. The colour of the heated mixture dependedon the amount of lactose and citric acid present inthe milk. In this way the presence of 2000 or moreof cow's milk could be detected. The end-point ofthis test is sometimes indefinite and difficult tointerpret. The xanthine-oxidase test (Rodkey andBall, 1946) is a simple test and is based on the factthat cow's milk is rich in the enzyme, xanthine,while human milk has so little xanthine it is un-detected by this test. Milk adulterated with 1000 ofcow s milk or more can be detected. Unfortunatelythe test is useless if the milk is boiled because theenzyme, xanthine oxidase, is destroyed.

In the third group of tests the hydrogen ion con-centration of the milk under test is estimated by theaddition of some indicator such as neutral red(Moro, 1912) nile-blue sulphate (Bauer, 1913;Sole, 1935) or haematoxylin (Smith, 1942). By thesemethods 20 to 30% or more of added cow's milkcan be detected. On some occasions, however, theinterpretation of the colour reaction can be extremelydifficult, while in others fallacious results may beobtained because the milk has been kept too longbefore testing. Soxhlet-Henkel's test for titratableacidity results which are more reliable and informa-tive (Eldson and Walker, 1942). By their methodacidity over 2 degrees indicates adulteration withcow's milk provided the test is made on fresh milk(Kayser, 1935).

In the fourth group, tests involving the use of theanaphylactic response have proved impractical andthe technical procedure of the complement fixationtest far too complicated (Gnosspelius, 1940). Theprecipitation test, however, using specially preparedsera is very sensitive and reliable and can be quicklyand easily performed. By means of this test 200 ormore of added cow's milk can be detected. There islittle doubt that this is the ideal test for the investi-gation of adulteration of human milk with animal'smilk (Kayser, 1938; Urbach, 1938; Brakhage andRaettig, 1940; Gnosspelius, 1940). Unfortunately,at the present time the sera required for this test are

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TESTS FOR THE ADULTERATION OF HUMAN MILK

not available in Great Britain. Once they can beobtained it is unlikely that there will be any necessityto resort to any alternative procedure. Until then,however, it will be necessary to select at least one ofthe tests already mentioned for the detection ofadulteration of human milk with cow's milk. Itshould be remembered, however, that no test exceptthe precipitation test gives conclusive evidence ofthe type of animal milk which has been added to thehuman milk.

Investigation of Freezing Point of Women's MilkMethods. A few comments about the apparatus

and mfethod for measuring the freezing point,electrical conductivity and chloride content of milkare necessary.The cryoscope devised by Hortvet in 1921 is the

generally accepted apparatus for determining thefreezingpoint ofcow's milk (Lyons andO'Shea, 1950).This apparatus was used for measuring the freezingpoint of women's milk. The method of standardiza-tion of the thermometers was that suggested byAschaffenburg and King (1951) and the apparatus wasoperated as described by the Association of OfficialAgricultural Chemists (1930). A more rigid method ofstirring the milk under test, tapping the thermometerand reading the depression of freezing point recom-mended by Aschaffenburg and King (1951) wasadopted. Throughout the investigation, which lastedfrom April to August, 1952, the temperature of theroom was noted to vary by 9C C. The freezing pointofeach specimen was recorded in degrees Centigrade.In no case was any allowance made for this variationor for the degree of acidity of the milk.The electrical conductivity was measured with a

commercial Wheatstone bridge; a U-shaped cell forholding the milk to be tested and two platinumelectrodes were used in the circuit. The specificconductivity was calculated from the resistance fora given sample of milk and the cell constant for theapparatus. The results were all corrected to give thespecific conductivity of the milk at 18' C. Fulldetails of this type of investigation are given byKermack and Miller (1951).The chloride content of the milk was determined

by the method described by Patterson (1928). Tenmillilitres instead of 2 ml. milk were used for thetitration because this gave more accurate results.The chloride content of the milk was calculated interms of milligrams of sodium chloride per 100 ml.of milk.

MateriaL The investigation was made on milksamples obtained from 215 women who wereapparently healthy, at some stage of lactationbetween the fourth day and twenty-fourth week.

One hundred and seventy of these women providedone sample of miLk each. Twenty-six women suppliedone sample from each breast and each of thesewomen gave her two samples on the same day.Nineteen women gave two samples each but thesewere taken at different stages of lactation. Thus atotal of 260 samples of milk were obtained andexamined. Almost invariably the milk was expressedinto a specially prepared screw-capped jar after thebaby had been fed. The size of each sample wasapproximately 30 ml. and it was stored in a refri-gerator until tested. The test was performed within24 hours of the time the milk was expressed. Thefreezing point was determined for each of the 260samples and the electrical conductivity and chlorideestimations were made on 245 samples.

TO SHW THE PERCENTAGE OF WEN WHO NNE MLKWITH A GIVEN DEPRESSION OF FREEZING POINT (A)

50-

to

40-~~~~~~~4-

20-> 50 700~~~~~~~1

A 0C Of MILKFK;.10.

Results. The results of the investigation into thefreezing point of huiman niiLk are given in Fig. 1.In this figure the percentage of the 260 samples ofnmiLk with a depression of freezing point between 0- 531and O 540, 0- 541 and 0 550, 0 551 and 0- 560, 0 561and O-570, O- 571 and -580 - 581 and0 -590' C.has been adopted. The greatest number of resultsin any one group is 46 6% in the group ranging fromn561 to 0 570, while thespntaglest number of results,2 - 3 % is in the group ranging from O 531 to O- 540'C.The minimum andd iaximum depressions of freezingpoint obtained in this investigation are 053616C.and 0 586' C. respectively and the mean depressionof the freezing point for the 260 observations is0- 564' C. These figures are of importance whenusing this test to detect the addition of water tomilk.

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164 ARCHIVES OF DISEASE IN CHILDHOODTABLE I

AVERAGE DEPRESSION OF FREEZING POINT OF MILK (A) TAKEN FROM WOMEN AT DIFFERENT STAGES OF LACTATION

Week of Lactation

1 2 3 4 5 9-12 13-16 17-20 21-24

Average A:'C. ... 0-563 0-566 0-570 0-563 0-563 0-563 0-569 0-567 0-566

No. ofsamples tested .. .. 90 85 12 4 36 16 6 6 5

No. ofwomen tested .. .. 76 74 12 4 34 15 6 6 6

If the freezing point test is to be used to detectadulterated miLk it is important to know whetherthe mean depression of the freezing point varies withthe stage of lactation. Table I has been constructedto elucidate this point. In this table are the meandepressions of freezing point for milk samples takenfrom women in each of the following periods oflactation: the second, third, fourth, fifth to eighth,ninth to twelfth, thirteenth to sixteenth, seventeenthto twentieth and twenty-first to twenty-fourth weeksafter childbirth. Set out with each mean are both thenumber of observations and the number of womentested. The means do not differ markedly from themean of 0 564° C. for the entire investigation. Inthe two groups whose means differ most from thecommon mean the values have been based onrelatively few observations and are, therefore, moreliable to deviation. It appears, therefore, that thereis no tendency for the depression of freezing pointto alter significantly as lactation advances from thefirst to the twenty-fourth week after childbirth.

TABLE 2COMPARISON OF DEPRESSION OF FREEZING POINTS (9)OBTAINED FOR SAMPLES OF MILK TAKEN FROM THE

% TWO BREASTS

A° C. Milk

Case No. Right Breast Left Breast

1 0-550 0-5602 0-540 0-5403 0-541 0-5494 0-550 0-5515 05-43 0 5436 0-549 0-5447 0-550 0- 5508 0-560 0-5609 0-555 0-55110 0 553 0-55011 0- 550 0-56012 0 555 0-55813 0 -568 0-56014 0-552 0-6015 0-549 0-54316 0-550 0-55117 0-540 0-54818 0-530 0- 53919 0-562 0-55220 0- 542 0-55021 0-55A 0-550

0-560 0-6023 0-549 0-55124 0-561 0-55525 0-549 0- 54526 0-530 0-530

In two further investigations the freezing point ofmilk samples taken first from the two breasts wascompared, and second a comparison was madebetween two samples obtained from the same womanat different stages of lactation. Two series of womenwere investigated; the first comprised 26 women eachof whom expressed milk from both breasts keepingeach specimen separate. The freezing point of themilk from each breast was detenmined and the resultsrecorded in Table 2. A difference between the tworesults for a given woman was generally noted. Itwas usually no greater than 0004W C. which couldbe due to either a slight difference in the compositionof the two samples or to experimental error. In 11cases, however, the difference exceeded 0-004 C.an amount which could not entirely be accountedfor by experimental error. Thus some variation inthe composition of the two samples would seem tobe the most likely explanation for the difference inthe freezing points of milk from the same woman.A series of 19 women were selected for the investi-

gation into the effect of state of lactation upon thefreezing point of their milk. These women providedtwo samples of milk each taken at a different periodof lactation. The freezing points of the samples arerecorded in Table 3 where it can be seen that the

TABIE 3COMPARISON OF DEPRESSION OF FREEZING POINTS (_)FOR TWO SAMPLES OF MILK OBTAINED FROM THE SAME

WOMAN AT DIFFERENT STAGES OF LACTATION

Week of Week ofCase No. Lactation C. Lactation C.

I 1 0-565 2 0 5822 1 0-554 2 0-5653 1 0-565 2 0-5754 1 0-561 2 0-5655 1 0-549 2 0- 5766 1 0-543 2 0-5557 1 0-563 2 0-5628 1 0-565 0 5679 1 0 -56 1 6 0-56410 1 0-66 6 0-55511 2 0-564 2 0-56512 2 0-585 3 0-57013 2 0565 3 0-55414 2 0- 575 7 0- 56515 3 0- 563 6 0-56516 3 0-555 7 0-55017 3 0- 565 13 0-57418 6 0-563 10 0-55619 10 0-575 17 0-572

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TESTS FOR THE ADULTERATION OF HUMAN MILK

0-53-

0-54-

0-55--J

j 0-56-

, 0-57

058

* *... * ........

.

..-

20 40 60 80 100 120 1CHLORIDE CONTENT OF MLKmq. PERCEI

FJG. 2.

value obtained for the second sample may be eithergreater or smaller than that obtained for the firstsample. The difference between the freezing pointsfor the two samples from a given woman in somecases amounts to at least 0-005c C., an amountwhich can be explained by a change in the com-position of the miLkAn investigation was made into the freezing point,

chloride content and electrical conductivity ofsamples of human milk to determine whether anyrelationship existed between the freezing point andthe other physico-chemical properties. Two hundredand forty-five milk samples were examined, on eachof which the freezing point, chloride content andelecrical conductivity were estimated. The resultsare summarized in Tables 4and 5 where it is apparent that 0-53the average freezing point ofmilk samples with a low 0-54chloride content does not differappreciably from that for milksamples with a high chloride ~ 055content. The scatter graph Z .(Fig. 2) shows that there is no O-56 .relationship between individual , *values and depression of freez-ing point. From Table 5 it can 4 0-57 *also be seen that the average :

freezing point for milk of a low 0 8eletrical conductivity does not .differ appreciably from that formilk of a high electrical con-ductivity. The scatter graph 160 180(Fig. 3) shows that there is no ELEC1relationship between individual

160 180

electrical conductivity valuesand the depression of the freez-ing point. It is, therefore,apparent that there is norelationship between thedepression of freezing point ofmilk and its chloride content orits electrical conductivity.Freezing Point Test The addi-

tion of water to cow's milk canbe detected quickly and easilyby determining the freezingpoint of the given sample. Ifwater has been added the freez-ing point is higher than that ofunadulterated milk. This resultcan be used to estimate the

TABLE 4AVERAGE VALUE FOR THE DEPRESSION OF FREEZINGPOINT (A) FOR MILK OF DIFFERENT CHLORIDE CONTENT

Chloride Content(mg- ) No. of Specimens AveragA C.

1-50 64 0-56351-100 105 0-567101-150 58 0-564151-200 18 0-562

TABLE 5AVERAGE VALUE FOR THE DEPRESSION OF FREEZINGPOINT (A) FOR MILK OF DIFFERENT ELECTRICAL

CONDUCTIVITY

Elcrical Conduc(10-50hm-1) No, of Specimens Average A C.

l5l-210 85 0-566201-250 94 0-566251-300 53 0-562301-350 13 0-563

200 220 240 260 280 300 320

*TRICAL CONDUCTIVITY OF MILK x tO 505oms- cm-.FIG. 3.

340

*...~~~~~~00

165

00

0

0

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approximate amount of water added to the specimenby means of the formula

100 (T-Tr) O

W T

recommended by the American Association ofOfficial Agricultural Chemists where W is theamount of water added, T' is the freezing pointdepression of the sample, and T is the freezing pointdepression of genuine milk (or an average forgenuine milk), and though the results obtained bythe formula are a slight over-estimation of thepercentage of added water, they are sufficientlyaccurate for practical purposes (Elsdon and Walker,1942).A series of observations were made on human

milk, to which had been added varying amounts ofwater, to determine whether the above formula wasof value in the estimation of adulteration of women'smilk with water. A quantity of women's milk wasdivided into four parts, and each part was adulter-ated with 3-3, 16 6, 33-3 and 66-6% of waterrespectively. The freezing point of each specimen (x)was estimated by the method already described andrecorded in Fig. 4. The theoretical freezing point (-)of each specimen was calculated from the formulaand lay on a straight line connecting the depressionof freezing point of unadulterated milk with thefreezing point of water (see Fig. 4). The comparisonof the experimental-theoretical values for a givendilution of milk showed that they were either thesame or the experimental value was slightly higherthan the theoretical value. It was also apparent fromFig. 4 that if the amount of added water werecalclated from the theoretical value it might be asmuch as 20% in excess of the correct amount. Thisdifference is not of practical importance. Therefore,the suggested formula, based on the principle thatthe raising of the freezing point of milk by theaddition of water is proportional to the amount ofadded water, is of practical value in calculating thedegree of adulteration of women's milk. Similarresults have been obtained for dilution of cow'smilk and have been explained by Elsdon and Walker(1942).The sensitivity of the freezing point test is partly

dependent upon the range of freezing point depres-sion for genuine human milk; this range is 0-531 to0- 586- C. Since the addition of water to milk raisesthe freezing point, the percentage of water whichmust be added to milk with the lowest freezing point,0- 586c C., to raise it above the highest freezing point,obtained for normal unadulterated milk is a measureof the test's sensitivity. This value is 9 75 %. There-fore, the freezing point test will detect 100% or more

of water added to women's milk. If the test is usedfor milk which has a higher freezing point than0 586°C. less than 10% adulteration can be detected.

The Electrl Conductivity TestThe electrical conductivity test for detecting

adulteration of women's milk has not hitherto beendescribed. The measurement of electrical conducti-vity is simple (Kermack and Miller, 1951) but theinterpretation of the results, together with the

0-00

010

o 20-

-I

o 0.30/

0-40

0-50/

20 40 60 80PERCENTAGE ADDED WATER TO MILK

FiG. 4.-To show the changes in the depression of freezing point ofmilk ^ which occurs when it was watered.

(X) represents the depression of freezing point of mixtures basedon practical observ-ations while ( ) represents the depression of

freezing point based on theoretical calCulations.

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TESTS FOR THE ADULTERATION OF HUMAN MILK

sensitivity of the test and its fallacies require carefulconsideration. It is, therefore, necessary to considerthe test as a method for the detection of both addedwater and added cow's miLk. The addition of waterto human milk reduces its electrical conductivity.The reduction is almost proportional to the amountof added water. Usually the experimental value for agiven dilution of milk is 1-9% less than the theoreti-cal electrical conductivity value (Fig. 5). The differ-ence between the measured and the theoretical values

ADULTERATION OF NORMALWOMENS MILK WITH WER

fi x EXJ'ERXEMNTAL MUMES_20b THEORETICAL VALUES

2:

in

x 1501--

1--

zoC.)

-J5c50--

-Jm"a20 40 60

% ADDED WATERFIG. 5.

is partly due to the change in the viscmilk and to the increased dissociationelectrolytes in the diluted milk.The amount of added water which can

by the electrical conductivity test depeinitial electrical conductivity value of th4ranges from 210 to 150x l04ohm4lcm.-lactating adequately in the second to fifllactation (Miller, 1951). Since the valuewater is added, milk with the highest elkductivity value, 210 x lO`ohm'cm.:', willmaximum amount of water (330) to lo,ductivity below the minimum value oohm-'cm.-'. Therefore it follows that by n

test at least 330 added water can bemilk from mothers with adequate lactzsecond to fifth month of lactation. Occaelectrical conductivity test fails to detadded water because the genuine milkductivity value greater than 210x 10-This, however, is a relatively rare occurre

such milk is usually obtained from m

inadequate lactation and they would therefore notbe donors to a milk bank.By using the electrical conductivity test it is

possible to detect human milk which has beenadulterated with cow's milk. It has been shown thatthe addition of cow's milk raises the conductivity ofhuman milk, and that it is most unusual for theelectrical conductivity of milk obtained from womenlactating adequately in the second to fifth month oflactation to exceed 210x I04ohm-lcm.-I. If, there-fore, a value greater than this is obtained, the possi-bility of inadequate lactation and of adulteration ofthe milk must be considered. To obtain a con-ductivity value greater than 210 x lOohmlcm:.l asmuch as 23% of cow's milk may require to be addedto human milk (Miller, 1949). To prove, however,that the increased conductivity value was due toadulteration of the human milk with cow's milkrather than to inadequate lactation would entailfurther investigation and probably involve test-weighing the mother's infant over a period of 24hours.

Comments\\ ~~The practical value of the depression of the

freezing point as a test for the adulteration ofwomen's milk with water depends not only on theease with which it is estimated, but also on the

80 accuracy of the result and to possible variationsarising from mother to mother and the stage oflactation. Unfortunately previous research on thissubject (Winter, 18%; Winter and Parmentier,1904; Wardlaw and Dart, 1926) has been made with

osity of the different types of apparatus (e.g. Beckman's thermo-of certain meter) and technical procedures which influence the

freezing point of a given milk sample. These resultsbe detected are not strictly comparable with those obtained in,nds on the the present investigation and no conclusions can bemilk. This drawn as to the accuracy of the results. It has beenfor women stated, however, by Wardlaw and Dart (1926) that

:h month of the individual values in their series varied froms fall when -0- 545 to -0 622A C. This is a greater variation thanoctrical con- that of -0- 531 to 0- 586- C. obtained in the presentrequire the series of observations. This smaller variation in-wer its con- creases the value of the freezing point test as a meansif 150 x I0O of detecting adulteration since the amount of waterneans of the which must be added to alter normal women's milkdetected in of the lowest freezing point above that of the highestLtion in the normal value is less if the variation is small. Wardlawsionally the and Dart (1926) noted a tendency for the freezing!ct 330% of point to rise as lactation advanced, but the meanhas a con- values for various stages of lactation differed veryohm-'cm.-'. little from the mean value for all the observations.nce because The depression of freezing point in the presentromen with series of observations was not significantly affected

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by the stage of Laction. The test, therefore, shouldprove equally satisfactory at whatever stage oflactation it is used. The same authors stated thatmilk from women aged 23 to 26 had vahls whichon the average were slightly hir than that foryounger or older women. It is unlikely that thedifference between the two groups of women issignint.

Before any definite statement as to the value of thetest as a means of detecting adulteration of humanmilk with water is made a further series of obser-vations made by the same method is desirable. Itappears, however, that the test is sensitive and thetechnique easy and not too time consuming. Itwould appear equally sensitive at whatever stageof lactation it is performed and not markedly affectedby the variations normally found in milk of appar-ently healthy mothers. The only other test used forthe detection of water added to human milk whichhas comParable sensitivity is the refxactometric test.This test, however, requires two specimens from thewoman under investigation, one of which must beknown to be unadulterated. This latter sample maynot be easily obtained. The fact that the freezingpoint test is a quantitative one and can be done withone sample of milk means that it is preferable to therefractometric test.The electrical conductivity test as a detector of

added water to human milk is probably as efficientas most tests used for this purpose, but it is onlysuitable for detecting 33% or more of added waterto milk obtained from a woman who has passed herpuerperium and is lactating adequately. The test is,however, more rapidly and easily performed thaneither the specific gravity test or the chloride-lactosetest, both of which detect similar degrees of adultera-tion. The refractometric test is possibly moreefficient but more time consuming.The electrical conductivity test is of little practical

value for the detection ofhuman milk to which cow'smilk has been added. This is because it may givefalse positive results and to eliminate the possibleerror would involve much work. Many other testsfor adulteration with cow's milk also give unreliableresults. The preapitation test, however, is reliableand most satisfactory. It is capable of detecting 2%of adultetion. The refratometric test is anotherreliable test but only for the detection of 20% ormore of added cow's milk.The most accurate method of investigating the

possible adulteration of women's milk would appearto be the testng of the milk for acidity along thelines advocated by Soxhlet-Henkel (SH). Sampleswith an acidity not greater than 2 degrees (SH) areregarded as fresh. The freezing point of these

samples should be estimated to detect adulterationwith water. Samples with an acidity exeding 2degrees (SH) are either sour or contain cow's milk.These should be subjeted to the precipitation test.Application of the freezing point test to samples ofmilk with an acidity greater than 2 degrees (SH)reqiires further investigation.

The depression of freezing point of human milkhas been investigated. Estimations have been madeon 260 samples of milk taken from 215 apparentlyhealthy women between the fourth day to the sixthmonth of lactation. The results showed that thedepression of freezing point ranged from 0-531 to0-586° C. The mean vahle for all the observationswas 0 564° C. The freezing point for samples takenfrom the two breasts frequently differed, as did thefreezing point for samples taken at different timesfrom the same woman. The mean values for samplestaken from women at various stages of lactation didnot differ appreciably and certainly did not suggestthat the freezing point of milk tended either to riseor fall as lactation progressed.There was no relationship between the depression

of freezing point of women's milk and the chloridecontent or the electrical conductivity of the milk.

It has been shown that it is possible to detect theaddition of 10% or more of water to human milkby using the freezing point test From the result it isalso possible to state the approximate degree ofadulteration. The reliability of these conclusionsdepends upon the milk being fresh.The use of theelel conductity test to detect

adulteration of humn milk with either water orcow's milk has been described. The test is recom-mended for the investigation of possible adulterationwith water should both the freezing point and therefractometric tests be impracticable. The elctricalconductivity test does not appear to be of practicalvalue for the detection of adulteration of humanmilk with cow's milk, there being at least three moresuitable tests for this purpose.A scheme for testing human milk involving the

freezing test and precipitation test along with aquantitative estimation of acidity has been suggested.

We wish to express our thanks to Dr. A. Comrie, ofthe Agricltural Colege, Edinburgh, for his advice onhow to iwasure the depression of freezing point of milk.We are also gratefuil to the nursing staff of the SimpsonMemorial Maternity Pavilion, Royal Infirmary, Edin-burgh, for their atanc in collecting many of the milksamples, and to the Board of Management, EdinburghCentral Hospitals, for a grant to cover the cost of theapparatus.

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TESTS FOR THE ADULTERATION OF HUMAN MILK 169REFERENCES

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