investigations into the euglena for the assay...

J. clin. Path. (1964), 17, 14

Investigations into the Euglena method for the assay

of the vitamin B12 in serum

BARBARA B. ANDERSON

From the Department ofHaematology, Postgraduate Medical School ofLondon

SYNOPSiS Details are given of a modified Euglena method for the assay of B12 in serum. Serum invery small quantities is usually inhibitory to the growth of the Euglena. In general, this inhibition ismost marked when the organism is growing prolifically. For estimating serum B12 values, a standardcurve is used from B12 standards containing 0.01 ml. of serum of low B12 content, so that growth intest serum solutions is compared with growth in serum-containing standards. The recovery of B12added to serum, incomplete when estimated from aqueous standards, is now complete and the vari-ation in results from batch to batch is markedly reduced.

Various factors-such as medium, light, and inoculum-can affect growth in aqueous and serum

solutions differently and are an important cause of variations between batches and laboratories.Optimal conditions were studied and they are essential for the most sensitive and accurate assay,

but if the results are estimated from serum-containing standards accurate values can be obtainedeven under less than optimal conditions.Subnormal serum B12 concentrations can be detected visually as early as 24 hours after the start of

assay. For diagnostic purposes the assay can be read at three days, provided that appropriatedilutions are used, conditions are optimal, and growth is measured in a 1 cm. cell.

Microbiological assay with Euglena gracilis has beenwidely used to measure the vitamin B12 concentra-tion of serum. Initially, Euglenagracilis var. bacillariswas used as the test organism (Ross, 1950, 1952) butsubsequently Hutner, Bach, and Ross (1956) intro-duced the z strain. Using this strain and an improvedmedium, much more growth is obtained, the incuba-tion period is shorter, and the assay is more sensitive.The Euglena method has several advantages for

assaying B12 in serum. It has a high specificity and isvery sensitive. Because whole serum is assayed, pre-liminary extraction is unnecessary and directmeasurements can be made of both bound and freeB12. However, growth conditions need careful con-trol and the results often vary widely in differentlaboratories (Table I). It is also general experiencethat considerable variation may occur betweenresults in different assay batches. In addition, in myexperience, it is rarely possible to obtain full recoveryof B, added to serum.

In the assay the serum B12 concentration isusually determined by comparing the growth of theorganism in serum with its growth in aqueousReceived for publication 3 May 1963.

14

standards of B12. A possible explanation for theshortcomings of the method is that the Euglenareacts differently in aqueous and serum solutions.The work described in this paper was designed to

investigate this point and to establish the optimalgrowth conditions necessary for the most sensitiveassay.

MATERIALS AND METHODS

The method is basically that of Hutner, Bach, and Ross(1956) but modifications based on the work described inthis paper are included.

Glassware used, and its rigid cleaning and preparation,was essentially that recommended by Hutner et al. (1956).A detergent, Diversey Pyroneg (Deosan Ltd.), however,was used for cleaning and was shown not to affect theassay. Glass-distilled water produced by the Lough-borough all-glass still was used throughout. The assaymedium was that recommended by Hutner et al. (1956).

THE ASSAY ORGANISM The z strain of Euglena gracilis wasused'. It was maintained by weekly subculture into the

'The organism was obtained from the Culture Collection of Algae andProtozoa, Botany School, Downing Street, Cambridge.

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Investigations into the Euglena methodfor the assay of the vitamin B12 in serum

TABLE ISERUM VITAMIN B,2 CONCENTRATIONS IN NORMAL SUBJECTS USING EUGLENA GRACILIS

AS TEST ORGANISM

Year Strain Used No. ofSubjects

Serum B12 Type of SubjectConcentration (Al±g./ml.)

Range Mean

Mollin and RossKillanderHeinrich and LahannLear et al.Pitney and BeardMollin and RossDixit et al.Dixit et al.KillanderRaccuglia and SacksMillerNicholas and PitneyShintonCooperCooperBanerjee et al.Davis and KellyAnderson and Pope

195219531954195419541957195619561957b195719581 958195919591959196019621962

BacillarisBacillarisBacillarisBacillarisBacillarisBacillarisBacillarisBacillarisBacillarisBacillarisBacillarisBacillaris and z

z

Bacillarisz

Bacillarisz

z

6556

2056

223377815618022

218120322751100149

100-720100-72062-460

292-85686-460100-90092-80072-2 160130-1200144-1080270-1270190-875'130-750

98-940160-875163-925

358360238532212356259592501387640456390499305282398472

Normal'Normal

NormalNormalNormalandcontrol'Indian normalsIndian soldiersNormal and controlNormalNormal and controlNormalNormalControlControlNormalNormalNormal

'Normal = healthy hospital staff or blood donors.2Control = patients convalescing from minor disorders.3These were the average of the values obtained by the bacillaris and z methods.

liquid stock culture medium recommended by Hutner etal. (1956).

VITAMIN B12 STANDARDS Cytamen cyanocobalamin(Glaxo Laboratories Ltd.) of a concentration of 50 ,ug./ml.was used for the B12 standards. This was diluted to 40pug./ml., from which the following range of standardswas usually set up:-0, 0-5, 1, 2, 4, 6, 8, 10, and 12LuLg./ml. final concentration. In some experiments 15 and19 p,,g./ml. standards were included. Three to six tubeswere set up for each concentration.Aqueous vitamin B12 standards These contained the

amounts of the standard concentrations of B12 made upto 2 ml. with distilled water together with 2 ml. of double-strength medium.

Serum-containing vitamin B12 standards These con-tained in addition small amounts of serum of B12concentration less than 30 ptg./ml. The amounts addedvaried in some experiments, but routinely 0-1 ml. of a1 in 10 dilution of serum in water was included in the B12standards. The B12 content of the added serum wasnegligible.

COLLECTION OF BLOOD New and sterile glassware andsyringes and needles washed, prepared, and sterilized,and known to be B.2 free, were used. Latterly, disposablesyringes and needles were used and are recommended.

PREPARATION OF SERUM FOR ASSAY All sera were assayedin two or three assay batches. Routinely, unless the serumB12 concentration was expected to be abnormally high orlow, each serum was assayed initially at a 1 in 80 dilution.If the growth proved to be too high or too low in the firstassay, a more suitable dilution was used in a subsequent

assay chosen from the following range: 1 in 20, 1 in 40, 1in 80, 1 in 200, 1 in 400, or higher if needed. Dilutions lessthan 1 in 20 are inadvisable due to the precipitation ofproteins interfering with the assay. The dilution used wasselected to give a reading equivalent to between 2 and 10pjsg./ml. on the standard curve.Three tubes were set up for each serum and contained

the appropriate amount of serum made up with distilledwater to 2 ml. together with 2 ml. of double-strengthmedium.

CONTROL SERA Control sera of known B12 concentra-tions, with and without added B12, were included in eachbatch at three different dilutions for each serum. Thedilution agreement and completeness of recovery ofaddedB12 served as checks on the accuracy of the assay. Thesera used were as follows:-

1(a) = Pooled serum with a B12 concentration less than60 ppug./ml.; l(b) = serum l(a) with the equivalent of200 ,uj,g./ml. of added B12; 2(a) = pooled normal serumwith a B12 concentration between 400 and 500 ,usg./ml.;2(b) = serum 2(a) with the equivalent of 2,000 ,upg./ml. ofadded B12.To prepare these, 200 ml. of suitable sera was pooled.

To 100 ml. was added sufficient B12 to give the requiredconcentration of added B12. The sera, with and withoutadded B12, were divided into aliquots of about 2 ml.,sufficient for assay in two batches, and were stored at - 12to -17°C.

MEASUREMENT OF TOTAL VITAMIN B12 IN SERUM The tubescontaining standards and solutions for assay were heatedin a boiling water bath for 15 minutes. This released theB12 bound to serum protein and also served to sterilizethe assay tubes adequately.

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Barbara B. Anderson

MEASUREMENT OF UNCOMBINED VITAMIN B12 IN SERUMThe solutions of serum for assay were either not heated orheated at 50°C. for half an hour which sterilized theassay tubes but freed very little B12 from the protein.

INOCULUM A seven-day-old culture was used. Theinoculum was washed and prepared as recommended byHutner et al. (1956) and was finally suspended in 20 ml. ofsterile single-strength medium. The preparation of theinoculum was varied where stated.

INOCULATION After heating, the tubes were distributedat random in the perspex racks, in which they wereinoculated and incubated. Three drops of inoculum weredelivered into the stock culture tubes and thereafter onedrop was added to each assay tube using a sterile Pasteurpipette calibrated to deliver 50 drops per millilitre.

WATER BATH FOR INCUBATION Figure 1 is the diagram ofthe water bath, a modification of that used by Ross (1952),and was designed and assembled by the PostgraduateMedical School. It consists of a perspex tank mounted onan aluminium cabinet. The two rows of five perspex racksholding 54 tubes each are supported 2-7 inches above thebottom of the tank on ledges along the sides and downthe centre of the tank. The metal cabinet houses lights,motors, and a fan and has removable panels on the sidesand perforated ends to allow a free flow of air.The light and temperature requirements of the Euglena

are supplied and controlled in the following way:Light There are four 3 ft. 30 watt 'warm white'

fluorescent strip lights in the cabinet beneath the tank.The light is diffused by means of one or two layers ofCinemoid filter placed between sheets of opal glass justbeneath the tank. The lights are controlled by a Variactransformer and it is possible to vary the amount oflight over a range of 25 to 250 foot-candles, measuredwith a Weston Master III Universal exposure meter heldat the level of the top of the tank.

PERSPEX TANKI1'5

LEN6ES

2.

TUERNUSAT_

PERFIRATER PANEL

L STRIP LI6HTS

ALMINIUMN CANINE --

WITN FRUOT PANELSREMUV

A black cloth is placed over the perspex tank to cut outall outside light, so that the assay tubes receive light onlyfrom the strip lights below. There is slightly less light atthe edges but this only affects very large batches of up to540 tubes. With smaller batches the periphery of the bathis left free.

Temperature There is no heating element. Thermo-stats regulating the heating element occasionally fail andas a rise of 2 or 3°C. is critical, large batches may beruined.The temperature is maintained by the heat given out

by the lights enclosed in the cabinet. It is also dependenton the temperature of the room and the amount ofcovering on top of the bath. In order to prevent thetemperature from rising above that required, a thermo-stat in the tank controls a fan in one end of the cabinet.This causes air to pass through the cabinet beneath thetank, and the water can be maintained at the temperaturerequired provided that temperature changes in the roomare not too great.The mixing of the water by a stirrer at one end of the

tank is facilitated by the fact that there is 2-7 inches ofwater in the tank beneath the racks, as well as the watersurrounding the assay tubes. This ensures that the temper-ature of the water is uniform in all parts of the tank.

INCUBATION Each assay batch was incubated in thewater bath for four and three-quarter days, but a shorterincubation period is recommended. The optimal amountof light, measured as previously mentioned, was 50 foot-candles. The optimal temperature was 28-5°C. Theseconditions were varied where stated in some of theexperiments.

MEASUREMENT OF GROWTH A homogeneous suspensionof Euglena was obtained by shaking and growth wasmeasured in a Unicam colorimeter SP 300 using a redfilter (Ilford 204). When using the range of B12 standards0 to 12 pg./ml. a more accurate curve is obtained by

SHE SF TENPEISPEueK

I.lstimmi. -I ___I _I IU

.- -\~q; STIB."^.r@ U N ILglT

T~~~~~a FAA-t- NOTI1~~~~~~~ .TME Ni!D-s"X"

-SEPARATE FUSES

STIRRER SWITl

FIG. 1. Water bath for the incubation of the Euglena assay.

A--iqr,I)

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measuring growth in a 0 5 cm. cell, but for the sake ofuniformity in this paper all growth was measured ina 025 cm. cell unless otherwise stated. Occasionallycorrections had to be made for turbidity of the superna-tant due to slight protein precipitation. In this case theoptical density (O.D.) of the supernatant after centrifuga-tion was measured and subtracted from the reading of thewhole suspension.The O.D. readings were averaged and the standard

curve was plotted on linear graph paper as O.D. x 100against the final B12 concentration in usg./ml.The growth curves of the aqueous and serum-contain-

ing B12 standards have been designated 'aqueous curve'and 'serum curve' respectively.

ESTIMATION OF SERUM B12 CONCENTRATION The threeO.D. readings for each serum were averaged and the B12concentration was estimated from the standard curveallowing for the dilution at which it was assayed. In thispaper comparisons have been made between the serumB12 concentrations estimated from the aqueous curveand the serum curve. The present results suggest thatestimations from the serum curve represent the true value.

RESULTS

RECOVERY OF VITAMIN B12 ADDED TO SERUM Recov-

ery experiments were carried out in which amountsof 150, 200, and 2,000 ,u,ug./ml. of B12 (cyanoco-balamin) respectively were added to three differentsera. The sera, with and without added B12, wereassayed in repeated and consecutive batches and theresults were estimated from aqueous B,2 standardsas in the method of Hutner et al. (1956). The recoveryoften varied in individual assay batches and wasrarely complete (Table II). The mean recovery for thedifferent sera ranged from 82 to 87 %, but in individ-ual batches the recovery varied from 65 to 107%.

EFFECT OF KCN ON RECOVERY It has been shown thatthe addition of cyanide considerably increases theyield of B12 in the supernatant when serum proteinsare precipitated by heat (Spray, 1955; Killander,1957a; Girdwood, 1960; Matthews, 1962).Using the Euglena method, where whole serum is

assayed, the recovery of cyanocobalamin added toserum was usually very slightly higher than that ofhydroxocobalamin, when 200 and 2,000 ,u)g./ml. of

No. of Assays B,, Added(pLi.g./ml. serum)

either was added to each of two sera. The addition ofKCN (5 ,ug. per assay tube) to the medium did notimprove the recovery of cyanocobalamin, whereas itdid seem to improve slightly the recovery of hydroxo-cobalamin. The recovery of both forms of B12 whenKCN was added was therefore similar, but far fromcomplete, and in this particular experiment was only68%.

EFFECT OF HEAT ON RECOVERY It is possible thatheating at 100°C. for 15 minutes does not completelyliberate all the B,2 from the serum protein or thatheating destroys some B12. The effect of heating wastherefore investigated.Aqueous B,2 standards, and control sera, with and

without KCN (5 ,ug. per assay tube), were heated at100°C. for three, 10, 15, or 30 minutes and a furtherset autoclaved at 1180C. for five minutes. Recoverywas incomplete and similar for each procedure.

EFFECT OF ADDITION OF SERUM TO VITAMIN B12STANDARDS ON GROWTH AND RECOVERY The effectof adding serum to the B12 standards was studied inorder to determine whether the inadequate recoveryof B12 added to serum was due to a depressing effectof serum on the growth.

Effect of addition of small amounts of serum ongrowth The effect on growth of varying amounts ofserum, ranging from trace amounts to the amountsusually assayed, was studied by adding 0-00001,0-0001, 0 0002, 0 001, 0-01, 0 05, 0-1, and 0 2 ml. ofpooled serum (B12 concentration 18 ,up,g./ml.) toeight different sets of B,2 standards respectively. Thegrowth at each concentration of B,2 was comparedto that in standards to which no serum had beenadded (Fig. 2). When more than 0 01 ml. of serumhad been added corrections were made to the O.D.readings for the B12 content of the added serumand for turbidity of the supernatant (see Materialsand Methods)2.At B12 concentrations of more than I 0 ,qu,g./ml.

growth was less in all the serum-containing standards.

2The true concentrations of the standards containing added serumwere considered to be the sum of the crystalline B12 in the standardsand the assayed concentration of B,, in the added serum.

3LE IIRECOVERY OF VITAMIN B12 ADDED TO SERUM

Estimatedfrom Aqueous Curves Estimated from Seruin Curves

B12 Concentration % Recovery ofAdded B,, B12 Concentration %,Recovery of Added B,,ofSerum (plsg./ml.) ofSerum ( g.I/ml.)

Range Mean ± S.E. Range Mean ± S.E.

75-91 82 ± 165-99 87 ± 371-107 85 ± 1

8448

451

95-117 104 ± 1

90-104 98 ± 192-110 100 ± 1

17I542

150200

2000

7046

387

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Barbara B. Anderson

50

40

Ppq/m1.

_ s - .o ~~~~~~~~12

o * * ~~~~106

_ 6~~~~~

__4

@2

0-".

. , , @_

00001 -0001 *001 *01 01 1.0

Serum added (ml.)

a

, 30

3-

0_in

a

o0 k . . I

0 2 4 6 8 10 12 14 16 18 20

Cyanocobelemin (pgq. per ml.)

FIG. 2. The effect on growth in vitamin B12 standards ofthe addition ofsmall amounts ofpooled serumofpooled serum B12 concentration 18 ,Iqg./ml. o o aqueous standards * * serum-containingstandards.FIG. 3. Growth in aqueous vitamin B12 standards 0 o and in similar standards to which 0 01 ml.ofserum oflow B12 content (less than 30 p.ug./ml.) has been added * * . Each curve is the mean of40 consecutive assays.

The addition of 0-2 ml. of serum did not depressgrowth to any greater extent than did 0 001 ml., andthere was even some depression of growth when as

little as 0-00001 ml. of serum was added.Effect of addition of serum on growth and recovery

in repeated assays The effect of the addition ofamounts of 0 01 ml. of serum of low B12 content(less than 30,tug./ml.) to B,2 standards was studiedin 40 consecutive assay batches. The difference ingrowth between aqueous and serum-containingstandards often varied considerably from batch to

0

0

x

-

c

w

0

so

40

30

20

10

batch. The mean curve of growth of the serum-containing standards was significantly lower thanthat of the aqueous standards above 10 ,u,ug./ml.B12 (P<0 001) (Fig. 3). The recovery of 200,u,ug./ml.of B12 added to serum of low B12 content and 2,000,u,ug./ml. added to normal serum was incomplete andvariable when estimated from the aqueous curvesbut complete when estimated from the serum curves.The mean recovery estimated from the aqueouscurves was 86-1 % at the 200,u,ug./ml. level and 85-4%at the 2,000 ,u,g./ml. level and estimated from the

FIG. 4. The variation in growthin individual assays in aqueous

0Io o and serum-containingvitamin B12 standards *0 * .

Each curve is the mean of the40 consecutive assays and thelines through the mean readings ateach concentration of B12represent ± 2 x S.D.

Cyanocobalamin C upg. / ml.)

40

c>30

., 20A

g lo

0

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TABLE I1IVITAMIN B12 CONCENTRATION OF 11 CONTROL SERA ASSAYED REPEATEDLY AND ESTIMATED FROM AQUEOUS

AND SERUM-CONTAINING STANDARDSNo. ofAssays

2

3 = 2 + 150145 = 4 + 20067 = 6 + 20089 = 8 + 2,0001011

1131 ILI -/Ml-

4013171821151599

4242

Estimatedfrom Aqueous Curves

Bl, Concentration ( lig./ml.)

Range Mean S. D.

100-15855-82167-21241-60187-25538-56167-245418-487

1,950-2,470319-463

1,735-2,560

1317019351

22246

219457

2,150387

2,085

13-57-4

12-16-6

16-95-4

23-020-316637-8199

Estimatedfrom Serum Curves

CoefficientofVariation(%)

10-310-66-313-07-611-710-54-57-79-89-5

B12 Concentration (lpgg./ml.)

Range Mean S.D.

135-16569-93223-25550-68

241-28345-55231-254518-592

2,445-2,750390-490

2,300-2,635

15284

24057

26348

243550

2,600451

2,440

7-05.9

10-15-711-03-47-519-2

11921-883-3

CoefficientofVariation(%)

4-67-04-210-04-27-03-13-54-64-83-4

serum curves was 102-2 and 99-6% respectively (alsosee Table II).

EFFECT OF ADDITION OF SERUM TO AQUEOUS VITAMIN

B12 STANDARDS ON REPRODUCIBILITY OF RESULTS

Figure 4 compares the variation in growth in stan-dards containing 0 01 ml. of serum with that in theaqueous standards in the same 40 individual assaybatches. The addition of serum had the effect ofstabilizing growth, for there was less variation frombatch to batch in the serum-containing standards.The greater accuracy is reflected by the smaller

variation in the mean recovery per batch estimatedfrom the serum curves (95 to 107%, S.D. 3 8%)compared with recovery estimated from the aqueouscurves (74 to 101 % S.D. 6-9%).The combined coefficient of variation for 11 con-

trol sera assayed at three dilutions per assay inreplicate and consecutive assays was 5 1 % whenestimated from the serum curves, compared with9-2% when estimated from the aqueous curves(Table III)3. In the triplicate assays on 769 test seracarried out in each instance in three different assaybatches at one dilution per batch, the combinedcoefficient of variation was 5-3 % for the serumcurves compared with 8-0% for the aqueous curves.The variation within a batch was small for either

method of estimation. When the same serum was

assayed at three dilutions, six or seven times withinthe same batch, the combined coefficient of variationat the various dilutions, assessed within six suchbatches, was 3-7% for either method.There was fairly good agreement within a particu-

lar method of estimation between values of a serum

'All results reported in this paper were obtained using approximatelyoptimal and constant conditions. In previous work, when conditionswere not as well controlled, variation in results estimated from theaqueous curve was much greater.

assayed at different dilutions. However, the agree-ment is probably better for the results calculatedfrom the serum curves, for the combined coefficientof variation between the dilution values was greater(7 5% compared with 5-5 %) for the values estimatedfrom the aqueous curves assessed from the 11 con-trol sera assayed at three dilutions in repeatedbatches.

EFFECT OF DIFFERENT CONDITIONS ON GROWTH AND

RECOVERY IN AQUEOUS AND SERUM SOLUTIONS The

foregoing results indicate, first, that the presence oftraces of serum usually depresses the growth of theEuglena and, secondly, that there is less variation inthe results from batch to batch when estimated fromthe serum curves. It seems that the growth ofEuglena is affected differently in aqueous and serumsolutions by small variations in growth conditions.The purpose of this section is to study the growthconditions and their effect on the growth of Euglenain aqueous and serum solutions.

TABLE IVCOMPARISON OF GROWTH AND RECOVERY USING TWO

SELECTED DIFFERENT MEDIA IN SAME ASSAY BATCH

B12(ffiwg./ml.)

2-0406-08-010-012-0

% Recovery ofB12 added toserum

Medium A

Aqueous Serum-containing

9.4118-325-029-333-837-7

7-815-220-324-927-330-0

73

Medium B


6-312-618-022-325-930-3

7-312-917-420-523-627-0

97 91 95

'Growth measured as O.D. units x 100.A = medium supporting good growthB =- medium supporting poor growth.

Sera

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Barbara B. Anderson

50

40

00x

a

c.cL0

50

20

10

Cyanocobalomin Cju,g. / ml.)

Basal medium The effect on growth and recovery offive different lots of media was studied comparingaqueous and serum-containing standards in six toeight assay batches for each lot of medium. Thesame culture of Euglena was used and conditionswere constant throughout. There was less variationin the mean growth in the serum-containing stand-ards between the different lots of media. Meanrecovery was constant and complete (99 to 103%mean 100-6 %) when estimated from the serumcurves, but was rarely complete (81 to 95% mean85 8 %) and the variation was greater when estimatedfrom the aqueous curves. It is noteworthy thatrecovery in the latter was lowest with the mediumthat stimulated greatest growth. This was moreobvious when growth and recovery were comparedin the same assay batch using two selected media, onesupporting good growth and the other poor growth(Table IV). Ammonium succinate was the only

FIG. 5. The effect of differentintensities of light on the growthin serum-containing vitaminB12 standards.Curve I in either diagram is themean growth in six assays at theoptimal light intensity of 50foot-candles. Curves 2, 3, 4,and S represent growth in theindividual assays at lightintensities of 250, 200, 100, and75 foot-candles respectively,and curves 6 and 7 at lightintensities of37 5 and 25 foot-candles respectively.

14 16 18 20

ingredient which was different in the media and wasobtained from different manufacturers.

It is probable that unexplained differences inbatches of other chemicals may also produce thiseffect.

Effect of light The optimal light intensity forgrowth of the Euglena was first determined approxi-mately by comparing growth in standards at lightintensities from 25 to 250 foot-candles and was foundto be about 50 foot-candles. The effect of differentintensities of light was then studied more precisely insix assays by comparing the growth in aqueous andserum-containing standards at intensities of 250, 200,100, 75, 37 5, and 25 foot-candles respectively withthe growth at 50 foot-candles in each case. The sixassays were carried out in two baths with all con-ditions identical except the light intensity. The samelot of medium and size and age of inoculum was usedthroughout the series and the maximum variation in

so ,

40 AA

1-050

20

0I6 I 1 S I

2 4 6 10 12 14 16 18 0 2 4 6 10 12 14 16 15 20

Cyonocobolcmin C pug./ml.)FIG.6. The effect ofoptimal and excessive amounts oflight on the growth in the aqueous o0 --0o and serum-containing*----- - vitamin B12 standards. The upper pair ofcurves in A, B, and C are the mean ofsix assays at the optimal lightintensity of50Sfoot-candles. The lower pairs ofcurves in A, B, and C are each the mean of two assays at light intensitiesof 250, 200, and 150 foot-candles respectively.

00x

:0

to

c

.0

20


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temperature from one bath to another or from oneassay batch to another was 1°C. and was usually lessthan 0-5°C. Figure 5 compares the growth in theserum-containing standards at the different lightintensities.4The most satisfactory growth curve was again

obtained at a light intensity of 50 foot-candles. Whenthe light intensity was either decreased or increasedthere was less growth, the only exception being aslight increase in growth from 0 to 2 ,uqg./ml. B12when the light was decreased. In the aqueous stand-ards growth was usually considerably higher than inthe serum-containing standards, the exception beingat 150 foot-candles. There was an increase in growthin the aqueous standards at the higher concentra-tions of B,2 at light intensities of 200 and 250 foot-candles (Fig. 6)5, and for this reason the recovery ofB,2 added to serum and estimated from the upperportion of the aqueous curve at a light intensity of250 foot-candles was 15% lower than at 50 foot-candles, whereas estimated from the serum curvewas approximately 100% at either light intensity.

4There were small variations between the six sets of control curves at50 foot-candles and therefore for a true comparison small correctionshad to be made to the readings of the other curves. To do this therespective 50 foot-candles curves were compared with the average ofthe six sets of curves and the readings at the 250, 200, 100, 75, 37 5,and 25 foot-candles were corrected accordingly."Small corrections were made to the readings of the growth curves at250, 200, and 150 foot-candles as in Figure 5.

Effect of temperature Small changes in tempera-ture had considerable effect on the growth of theEuglena. Preliminary experiments suggested that theoptimal temperature for growth was 28 5°C. Todetermine this more precisely the growth in standardsat temperatures of 23 5°C., 26°C., 31°C., and 31° to33°C. respectively, was compared with the growth at28 5°C. in four assays. These four assays werecarried out in two baths with all conditions similarexcept for temperature. The temperatures were theaverage for the four and three-quarter days' incuba-tion and were recorded twice daily using a maximumand minimum thermometer. The variation was notgreater than ±0 5'C. except when the temperaturerange 31°C. - 33°C. was used. Figure 7 compares theserum curves at the different temperatures6.As the temperature fell below 28 5°C. the growth

progressively decreased at all concentrations. At31°C. growth at lower concentrations of B12 wasmuch the same as at 28 50C. but was markedlydepressed at higher concentrations. When thetemperature rose to 330C. the depression of growthstarted at an even lower concentration of B12 andwas greater. Where growth was profuse there wasinterference with chlorophyll production and the

TABLE VCOMPARISON OF GROWTH AND RECOVERY US1NG

WASHED AND UNWASHED INOCULA

v-

0 2 4 6 8 10 12 14 16 18 20

Cyonocobelamin (pp'q. per ml.)

FIG. 7. The effect of temperature on the growth in theserum-containing vitamin B12 standards.

The curve at the optimal temperature of 28-5'C. is themean of four assays. The curves *- * and -------

represent growth at temperatures lower and higher than theo;2fimal temperature respectively.

°c

285 2-0406-0

26 8-0,31 10-0

12-0% Recovery of

31-33 Bi, added to123-5

9.2316-021-826-429-632-8

7-213-017-721*225-128-2

Inoctulum B2


6-214 519 524930-133 1

488 912 516720523-9

71 94 59 94

'A = washed and suspended in 20 ml.2B = unwashed and made up to 20 ml. with culture supernatant.3Mean growth in two experiments measured as O.D. units x 100.Inocula prepared from seven-day cultures.

organism became yellow. There was more growththroughout in the aqueous standards but the patternwas similar to that of the serum curves at the differ-ent temperatures.

Effect of washing the inoculum An unwashedculture was used as inoculum until Kristensen (1955,1956) recommended washing the culture to removethe B12-binding substance in the supematant. Theeffect on growth and recovery of a washed and an

"As for the light curves, small corrections were made to the readings ofthe curves at 23 5'C., 26'C., 31'C., and 31' Lo 33'C. for truecomparisonwith each other and the mean curve at 285'C.

50

40

C>

c:>

20111

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B1, Inoculum Al(,LzAg./Ml.)


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50

40

00

c30

0

20

0

I0

0

2

0 2 4 6 8 10 12Cyonocobalomin (piq. per ml)

FIG. 8. The effect of size of inoculumon growth in aqueous o o andserum-containing * * vitamin B12standards. Curves 1, 2, and 3 representgrowth when using inocula preparedfroma seven-day washed culture diluted in 20,10, and 5 ml. respectively. Each curve isthe mean oftwo experiments.

unwashed inoculum was studied by comparingaqueous and serum-containing standards. Thedepression of growth using the unwashed inoculumwas far greater in the serum-containing solutionswith the result that recovery of B12 added to serumand estimated from the aqueous curve was only 59%compared with 71 % using the washed inoculum(Table V).

Effect of density of inoculum The effect of vary-ing inoculum density on the growth of the Euglena inaqueous and serum-containing standards was studiedby using three different inocula of density equivalentto a 1 in 20, 1 in 10, and 1 in 5 dilution of the originalculture respectively (Fig. 8.)The more dense the inoculum, the greater was the

growth at a particular concentration of B12 and lessobvious the inhibitory effect of serum. In fact growthwas greater in the serum-containing standards usingthe heaviest inoculum (1 in 5) in low concentrationsof B12 (less than 2 ,u,ug./ml.).

Therefore recovery of B12 added to serum andestimated from the aqueous curve was greatest usingthe densest inoculum, but, except when readings wereequivalent to very low concentrations of B12, therecovery was still not complete. Recovery was

approximately 100% for all inocula when estimated

from the serum curves. Readings were more regularand over several experiments the differentiation ofgrowth at very low concentrations of B12 was betterwhen the lightest inoculum (1 in 20) was used.

Effect of length of incubation This was studiedby comparing the growth in aqueous and serum-containing standards after different lengths of incu-bation. Sets of standards were incubated for two,three, four, four and three-quarter, and six days.Four control sera were also set up and incubated forthe same lengths oftime and the values were estimatedfor each incubation time.Though growth was greater in the aqueous stand-

ards, the pattern of growth for the different lengths ofincubation was essentially similar for the aqueousand serum curves. The serum curves are compared inFigure 9. Provided that sera were set up at theappropriate dilutions, and growth was measured in a1 cm. cell instead of the usual 0-25 cm. cell, there issufficient differentiation of growth even at two orthree days at the lower concentrations of B12 for theserum B12 to be accurately estimated, and there wasa good agreement between the values of a serumestimated from two days onwards. Figure 10 shows apair of aqueous and serum curves after three days'incubation when growth was measured in a 1 cm.cell. Further, a low serum B12 can be detected asearly as 24 hours, for the Euglena, in response tosmall amounts of B12, quickly ceases to grow andsettles within 24 hours in a button at the bottom ofthe assay tube.

NATURE OF MATERIAL IN SERUM AFFECTING GROWTHOF EUGLENA Growth of the Euglena in aqueous B12standards was compared with that in standardscontaining trace amounts of (1) the supernatant afterthe extraction of proteins from serum, (2) serum, (3)albumin, (4) gamma globulin, (5) Tween 807, and (6)serum + Tween 80. Serum and the individualproteins caused a similar marked inhibition ofgrowth, but there was only a slight inhibitory effectby the protein-free supernatant. It seems likely thatprotein is responsible for this inhibition. Tween 80caused an inhibitory effect very similar to that ofserum and proteins, but when Tween 80 was addedto the serum-containing standards there was nofurther inhibition.

SERUM VITAMIN B12 CONCENTRATIONS IN CONTROLSThe serum vitamin B12 concentrations were assayedof 149 healthy control subjects, who were membersof the hospital staff aged 20 to 50 years. The frequen-cy distribution of the normal values is shown in7The effect of Tween 80 was compared because it had also been foundto exert an inhibitory effect on growth. It had been added to themedium in an attempt to diminish clumping and sticking of theorganism to the bottom of the assay tube.

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60 we1s6

50

4¾3/

40

4

30

CL20

3

l0

2

0

0 2 4 6 8 10 12 14 16 18 20Cyanocobalamin (ppq. per ml.)

HIG. 9. The effect ofdifferent lengths of incubation on thegrowth in serum-containing vitamin B12 standards.The growth was measured in a 0-25 cm. cell.

Figure 11. The range of values determined from theaqueous curves was 125 to 765 (mean 402, S.D. 129,uqg./ml.) and of these only five (3 4%) were less than200; determined from the serum curves the range

was 163 to 925 (mean 472, S.D. 151 pug./ml.) andonly three (2-4%) were less than 200.

40

30

20

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anw

0

z

I0

0

Range: 12

A | Mean: 4(

A

100

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25-765 ppg./ml.W0 ,u,ug. /ml.

240 580 520 660 800

Range: 163-925 uppg./ml.0

| Mean: 472 uug./nml.

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50

40

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2-

20.2_CLC>

10

0

20

10

',I

/7'"",X

0 2 4 6 8 10 12

Cyunocobalamin (ppq. per ml)

HG. 10. Growth curves of aqueous O O and serum-

containing 0 0 vitamin B12 standards after three days'incubation. The growth was measured in a I cm. cell.4

o 240 380 520 660 Soo

Serum Vitamin B12 Cpupg. ml.)

944-I

10

FIG. 11. Thefrequency ofthe serum B,, concentrations of149 normal subjects estimated from aqueous vitamin B12standards (A) and serum-containing vitamin B12 standards(B).

Duplicate sera were taken from 125 of thesenormal subjects, mostly at intervals of two weeks,and were assayed together in three batches at onedilution in each batch. Assessed over six batches, theS.D. of estimations on aliquots of the same serum,assayed in the same batch under conditions similarto those used for the duplicate sera, was 3-1 % of themean. Hence the S.E. of the difference between a

pair was V/2 x S.D. or 4-4% of the mean. Twenty-nine per cent of the duplicate sera varied from eachother by more than twice the S.E. (8-8 % of the mean)and therefore were significantly different at the 95%

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level of significance. These pairs of sera varied fromeach other by 9-2 to 29-6% of the mean, average14-4%.

SERUM VITAMIN B12 CONCENTRATIONS IN PERNICIOUSANAEMIA PATIENTS The serum vitamin B12 con-centrations were assayed of 59 patients with perni-cious anaemia whose haemoglobin levels varied from3 to 15-2 g./100 ml. The values ranged from 7 to 96(mean 32, S.D. 20,u,ug./ml.) when estimated from theaqueous curves and 9 to 113 (mean 38, S.D. 23,uq.g./ml.) when estimated from the serum curves. Inthe latter range all but one were less than 95 ,u/g./ml.

DISCUSSION

The observations reported in this paper show thatthere can be a considerable variation from batch tobatch in the assay results of sera estimated from anaqueous curve. Furthermore, recovery of B12 addedto serum was rarely complete and there was often awide variation in recovery obtained in differentbatches, on occasions being as low as 60%. Otherworkers have also reported a wide variation in theresults of repeated assays of the same serum indifferent batches (Ross, Hutner, and Bach, 1957;Killander, 1957a; Nicholas and Pitney, 1958;Shinton, 1959), but most workers have reported a'satisfactory' mean recovery when B12 was added toserum. The exception was Pitney, Beard, and VanLoon (1954) who at no time could obtain a recoverymuch greater than 60%. The considerable variationin recovery which may occur from batch to batch hasnot been reported.The wide variation in results can be considerably

reduced by using standard and optimal conditionsbut even under these conditions the recovery is notcomplete. Variation is more markedly reduced andrecovery is complete if results are estimated fromB12 standards to which small amounts of serumhave been added.The presence of serum had an inhibitory effect on

the growth of the Euglena and within limits this wasindependent of the amount of serum present (Figs. 2and 3). Because of this inhibition the recovery ofB12 was poor when the growth in serum-containingsolutions was compared to that in aqueous standards.The addition of trace amounts of serum to theaqueous standards corrected this by ensuring thatgrowth in the standards was inhibited to the samedegree as in the test solutions. In addition, thepresence of serum in the standards caused theorganism to react to small alterations in the assayconditions in a similar manner in both standard andtest solutions. This reduced the variation in resultsfrom one batch to another, even when less thanoptimal conditions were used.

The depression of growth by serum was generallymost obvious in assay batches in which the organismgrew most prolifically and the aqueous standardcurve was steepest (Table IV), and this fact wasclosely associated with the batch of medium used. Itis interesting that Nicholas and Pitney (1958) alsoreported that their serum assay results were lowwhen the aqueous standard curve was steepest.

However, in three observed instances the degree ofinhibition was not related to the amount of growth.First, the presence of serum was less inhibitory ifprofuse growth was the result of a large inoculum.When a large inoculum was used, growth at very lowconcentrations of B12 was in fact higher in theserum-containing standards than in the aqueousstandards, so that recovery was greater than 100%(Fig. 8). Serum still inhibited growth at other con-centrations of B,2 and recovery of B12 added toserum was still usually incomplete if estimated fromthe upper portion of the aqueous curve. Secondly,there was markedly less growth in the serum-contain-ing standards when poor growth was due to excessivelight during incubation (Fig. 6) and recovery of B12added to serum was abnormally low when estimatedfrom the aqueous curve. Thirdly, serum causedmarked inhibition when poor growth was due to theuse of an unwashed inoculum and recovery of B12added to serum was less than 60% when estimatedfrom the aqueous curve (Table V). In all these threeinstances, recoveries estimated from serum curveswere approximately 100%.

NATURE OF MATERIAL CAUSING INHIBITION OF GROWTHThe substance causing inhibition of growth of theEuglena was fully active in amounts as little as 0 001ml. ofserum. The nature of the substance is uncertainbut it is probably protein, for a protein-free superna-tant had only a slight inhibitory effect and theaddition of albumin or globulin at a concentrationeven less than in 0-01 ml. serum, had an inhibitoryeffect similar to that ofserum. The material apparent-ly acts by interfering with the growth of the Euglena.How it does this is uncertain but it may act bylowering the surface tension of the aqueous fluid.The addition of serum to aqueous solutions mini-mizes clumping and sticking of the organisms.Tween 80, an agent known to lower surface tension,which is sometimes added to prevent clumping oforganisms, also had an inhibitory effect on thegrowth of the organism similar to that of serum.

EFFECT OF CYANIDE The addition of cyanidemarkedly improves the yield of B12 in an extractionprocedure (Spray, 1955; Killander, 1957a; Gird-wood, 1960; Matthews, 1962), apparently by prevent-ing the adsorption of B12 onto the denatured protein

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(Matthews, 1962). Hydroxocobalamin is known tobind more strongly to proteins than cyanocobalamin(Bauriedel, Picken, and Underkofler, 1956; Skeggs,Hanus, McCauley, and Rizzo, 1960), and it isprobable that cyanide, by converting any hydroxo-cobalamin present in serum to cyanocobalamin,prevents the adsorption and precipitation of thehydroxocobalamin onto the denatured proteinduring extraction, for it has been shown that whereasa large proportion of hydroxocobalamin added todenatured protein adheres and is not recoverable inthe supernatant, only a small proportion of addedcyanocobalamin is not recoverable in the superna-tant (Anderson and Pope, unpublished observations).The Euglena method does not involve an extrac-

tion procedure and the results of previous workerssuggested that the addition of cyanide to the mediumdid not improve the levels of B12 in the serum(Hutner et al. 1956; Shinton, 1959). This wouldsuggest that B12 in the presence of the denaturedprotein in the assay tube is available to the Euglena.However, in spite of the fact that the Euglenareacted equally to aqueous solutions of cyanoco-balamin and hydroxocobalamin, the recovery ofhydroxocobalamin added to serum was slightlylower than that of cyanocobalamin, and was slightlybut significantly improved by the addition of KCN.Furthermore, in my experience although the additionof cyanide did not have the striking effect on theyield of B12 that it has in an extraction procedure, itdid usually cause a very slight improvement in theserum B12 concentration. The improvement was toosmall to justify including cyanide routinely in themedium.

SERUM CONCENTRATIONS OF VITAMIN B12 WITH THEEUGLENA ASSAY Relatively small differences in assayconditions, for example, differences in medium, sizeof inoculum, amount of light, and the addition ofTween 80 to the medium, have been shown to affectthe serum B12 values when estimated from theaqueous curve. It is not surprising, therefore, thatthere is a great variation in the normal rangesreported from different laboratories. Reported meanconcentrations have varied from 212 to 640Q,uug./ml.(Table I).

In addition there are other factors which contribu-ted to this variation. Before 1955, when Kristensenintroduced the use of a washed inoculum, mostworkers used an unwashed inoculum. This in itselfwould have led to falsely low serum values becauseunder these conditions serum had a greater inhibi-tory effect on growth (Table V). But also it isprobable that the use of two strains of Euglenaemploying two different media contributed to thiswide range of values reported from different labora-

tories. In detailed comparisons of the two strains,using a washed or diluted inoculum, the serum valuesobtained with the bacillaris were reported to bemarkedly higher than with the z strain (Nicholas andPitney, 1958; Cooper, 1959). It was suggested thatthe higher values by the bacillaris were due to the factthat serum itself provided a growth factor notpresent in the incomplete bacillaris medium(Nicholas and Pitney, 1958; Cooper, 1959). But thiscould also be explained by the fact that the inhibitoryeffect of serum was likely to be less obvious in thebacillaris method where growth was considerablyless than in the z strain method.

CONCLUSION

Although some variation is inherent in all micro-biological assay work, the variations in the Euglenamethod as previously used were particularly seriousbecause of the unsuspected and variable inhibitoryeffect of whole serum, and because small changes ingrowth conditions produced unexpected differenteffects on growth in aqueous and serum solutions.These difficulties could be overcome if an extractionprocedure were used, but this would remove a greatadvantage of the Euglena method, namely, thatbecause no extraction procedure is involved largenumbers of sera can easily be assayed. The studies inthis paper indicate that the variations can be greatlyreduced, partly by establishing constant and optimalassay conditions, and especially by using serum-containing standards for estimation of B12 inserum, which also ensures full recovery of B, 2 addedto serum. It is of interest that the new normal rangereported in this paper is similar to that reported bySpray and Witts (1958) and Matthews (1962) assayedby the Lactobacillus leichmanii.

This work was supported by a grant from the MedicalResearch Council to Dr. D. L. Mollin.

I am grateful to Dr. D. L. Mollin for assistance andadvice in the preparation of this paper. I would also liketo thank Professor J. V. Dacie, Dr. G. I. M. Ross, andDr. A. H. Waters for their useful criticism of the manu-script, and Mr. W. J. Dempster for providing laboratoryfacilities for a part of this work. Mr. C. Lordan and Mr.S. A. Roberts were responsible for the design and con-struction of the water bath, and Mr. D. Banks for thediagram. I am grateful to Professor I. D. P. Wootton andDr. E. H. Belcher for statistical advice.

REFERENCES

Banerjee, D. K., Ghose, S., Ghosh, S. K., and Chatterjea, J. B. (1960).Blood, 15, 630.

Bauriedel, W. R., Picken, J. C. Jr., and Underkofler, L. A. (1956).Proc. Soc. exp. Biol. (N. Y.), 91, 377.

Cooper, B. A. (1959). J. clin. Path., 12, 153.

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Davis, R. E., and Kelly, A. (1962). Aust. J. exp. Biol. med. Sci., 40,437.

Dixit, C. H., Mody, B. M., Jhala, H. 1., Parekh, J. G., and Ramasarma,G. B. (1956). Indian J. med. Sci., 10, 419.

Girdwood, R. H. (1960). Scot. med. J., 5, 10.Heinrich, H. C., and Lahann, H. (1954). Z. Vitamin-, Hormon-u.

Fermentforsch., 6, 126.Hutner, S. H., Bach, M. K., and Ross, G. I. M. (1956). J. Protozool.,

3, 101.Killander, A. (1953). Svenska Ldk.-Tidn., 50, 2260.

(1957a). Acta. Soc. Med. upsalien., 62, 39.(1957b). Acta paediat. (Uppsala), 46, 585.

Kristensen, H. P. 0. (1955). Acta physiol. scand., 33, 232.(1956). Ibid., 37, 8.

Lear, A. A., Harris, J. W., Castle, W. B., and Fleming, E. M. (1954).J. Lab. clin. Med., 44, 715.

Matthews, D. M. (1962). Clin. Sci., 22, 101.Miller, A. (1958). J. clin. Invest., 37, 556.

Mollin, D. L., and Ross, G. I. M. (1952). J. clin. Path., 5, 129.- (1957). In Vitamin B, und Intrinsic Factor. 1. EuropaischesSymposium, Hamburg, 1956, edited by H. C. Heinrich, p. 413.Enke, Stuttgart.

Nicholas, D. R., and Pitney, W. R. (1958). Aust. J. exp. Biol. med. Sci.,36, 603.

Pitney, W. R., and Beard, M. F. (1954). J. clin. Nuttr., 2, 89.-,I -, and Van Loon, E. J. (1954). J. biol. Chem., 207, 143.Raccuglia, G., and Sacks, M. S. (1957). J. Lab. clin. Med., 50, 69.Ross, G. I. M. (1950). Nature (Lond.), 166, 270.

(1952). J. clin. Path., 5, 250.Hutner, S. H., and Bach, M. K. (1957). In Vitamin B12 und

Intrinsic Factor. I Europaisches Symposium, Hamburg, 1956,p. 305. Enke, Stuttgart.

Shinton, N. K. (1959). Clin. Sci., 18, 389.Skeggs, H. R., Hanus, E. J., McCauley, A. B., and Rizzo, V. J. (1960).

Proc. Soc. exp. Biol. (N. Y.), 105, 518.Spray, G. H. (1955). Clin. Sci., 14, 661.

, and Witts, L. J. (1958). Brit. med. J., 1, 295.

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