KLEYN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO.

5, 2001 1499
FOOD COMPOSITION AND ADDITIVES

Determination of Fat in Raw and Processed Milks by the Gerber

1
Method: Collaborative Study
DICK H. KLEYN2
Rutgers University, Cook College, New Jersey Agricultural Experiment Station, Department of Food Science, New
Brunswick, NJ 08903
JOANNA M. LYNCH3 and DAVID M. BARBANO
Cornell University, Department of Food Science, Northeast Dairy Foods Research Center, Stocking Hall, Ithaca, NY 14853
M. JEFFREY BLOOM
Weber Scientific, 2732 Kuser Rd, Hamilton, NJ 08691
MARTIN W. MITCHELL
Certified Laboratories, Inc., 200 Express St, Plainview, NY 11803
Collaborators: L.S. Cooper; E. Cusak; M. Fick; T. Hanks; M.K. Hesen; J. Johnson; D.H. Kleyn; F. Mercer; D. Monahan;
B. Peat; M. Petit

The Gerber method is used worldwide as a simple

and rapid method for determining fat in raw and
processed milks. However, the volume of the test
portion used in the method has not been internationally agreed upon. A collaborative study was
conducted to evaluate performance of the Gerber
method using either a weighed test portion
(11.13 g) or by a 10.77 mL test portion delivered by
pipet. For each method, laboratories received
10 test samples: 5 raw and 5 pasteurized homogenized milks, 2 of which were blind duplicate pairs.
Eleven and 10 laboratories participated in the evaluation of aliquot addition by weight and pipet, respectively. Mojonnier ether extraction (Method
989.05) was used as the reference method.
Interlaboratory study statistics were similar between methods of test portion addition and between raw and processed materials; therefore,
summary interlaboratory study statistics were
pooled. The fat content of milk samples ranged
from 0.96 to 5.48%. Absolute reproducibility and
repeatability were not affected by fat level, and
pooled statistical performance (invalid and outlier
data removed) was (g fat/100 g milk) sr = 0.026, sR =
0.047, r = 0.074, and R = 0.132. Relative standard
deviations increased with decreasing fat content,
Submitted for publication November 2000.
The recommendation was approved by the Methods Committee on
Commodity Foods and Commodity Products and was adopted by the
Official Methods Board of AOAC INTERNATIONAL. See Official
Methods Board Actions, (2000) Inside Laboratory Management, August
issue.
1
This research was presented as a poster at the AOAC
INTERNATIONAL Annual Meeting, 2000, Abstract J-1202, p. 102.
2
Deceased.
3
Author to whom correspondence should be addressed.

and were summarized by fat level: 12% fat milk,

mean = 1.437, RSDr = 1.809%, RSDR = 3.271%;
26% fat milk, mean = 4.156, RSDr = 0.626%,
RSDR = 1.131%. Compared with ether extraction,
test results by the Gerber method were slightly
lower (0.02% fat) using a weighed test portion and
significantly lower (0.06% fat) using a 10.77 mL
volume addition by pipet. A trend toward underestimating fat content at lower fat concentrations
(12% fat) was observed with the weighed test portion but not when a pipet was used. The Associate
Referee recommends that the Gerber method using a weighed test portion be adopted as First Action with applicability limited to whole milk.

he Gerber test is a simple, rapid, and inexpensive

method for determination of the fat content of raw and
processed milks. The method is used worldwide in a
variety of applications including payment testing and process
standardization. Briefly, a test portion of milk is pipetted into a
Gerber milk butyrometer containing sulfuric acid. Isoamyl alcohol is added, and the contents of the butyrometer are mixed
to dissolve the curd and release the fat. The released fat is isolated in the neck of the butyrometer by centrifugation. The
percentage of fat in milk (g/100 g) is determined by reading
the calibrated scale on the neck of the butyrometer. The
Gerber method shares many similarities with the Babcock
method for determination of milk fat content (1).
A proposal submitted by Associate Referee Dick H. Kleyn
to conduct a collaborative study of the Gerber method for determination of fat in milk was approved by AOAC INTERNATIONAL in 1993, and the study was conducted and completed that same year. Sadly, Dr. Kleyn passed away in 1993
and a final report was never submitted until now.

1500 KLEYN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 5, 2001

The Gerber test is described in International Organization

for Standardization (ISO) International Standard 2446 (2) and
International Dairy Federation (IDF) Provisional Standard
152:1991 (3). In both standards, the volume or weight of the
milk test portion is not specified. Instead, the analyst is instructed to periodically compare results of the Gerber method
with those from a reference method (e.g., ether extraction),
and adjust the Gerber test portion accordingly. Because most
laboratories using the Gerber method do not have the equipment or training to conduct ether extraction reference testing,
this approach is not practical.
In 1988, Kleyn et al. published results of an in-house comparison of 3 different procedures for adding test portions for
the Gerber method with the Mojonnier ether extraction
method (4). The 3 portions were delivered with either an
11.07 mL TD (to deliver) pipet, a 10.77 mL TD pipet, or by
directly weighing 11.125 g milk. The 11.07 mL TD pipet represented the specifications described in Standard Methods for
the Examination of Dairy Products, although it is incorrectly
referred to as a TC (to contain) pipet (5). The 10.77 mL TD
pipet and 11.125 g weighed portion, respectively, were selected based on work done in the Netherlands (6) and New
Jersey (D. Levowitz, personal communication to Dr. Kleyn,
1959), indicating close agreement with Roese-Gottlieb. In this
study, 51 raw milk materials were tested in a single laboratory
by the Gerber method using the 3 different test portions, and
the results were compared with those from Mojonnier ether
extraction (4). There were no significant differences in results
using the 10.77 mL pipet, the weighed test portion, and the
Mojonnier ether extraction. Test results using the Gerber
11.07 mL pipet were significantly greater than all other methods. The actual amount of milk delivered by the 10.77 mL
pipet was 11.068 g (average of 44 samples) and was closer to
the 11.125 g added by weighing than the weighed amount delivered by the 11.07 mL pipet (11.334 g, average of the same
44 samples). Authors recommended that the 10.77 mL pipet
replace the 11.07 mL pipet in the method, as weighing the test
portion was time consuming (4).
The objective of the collaborative study was to determine
method performance of the Gerber method using both the
10.77 mL pipet and weighing the test portion, and to compare
the test results with Mojonnier ether extraction.
Collaborative Study
Separate studies were conducted for the Gerber by weight
method (11 laboratories) and the pipet method (10 laboratories). For the Gerber by pipet method, collaborators were supplied with calibrated, certified 10.77 mL milk pipets (Gerber
Instruments AG, Effretikon, Sweden). For each study, each
laboratory received 10 milk samples consisting of 5 raw and
5 pasteurized homogenized milks. Two of the raw milks and 2
of the pasteurized, homogenized milks represented blind duplicate pairs. Thus, 8 individual test materials were analyzed
in each collaborative study. Samples were coded with random

3-digit numbers, and analysis order was randomized within

laboratories.
For both studies, raw milks were obtained from the Rutgers
University herd and cooled to 4C immediately after collection. Pasteurized, homogenized milks were obtained from the
New Jersey Department of Corrections Dairy Plant (Bureau of
State Farms, Trenton, NJ). All milks were cold-split on the day
of collection. To cold-split, each milk (ca 4 L) was mixed by
pouring from one container to another several times and
subsampling into 60 mL plastic flip-top vials. These test samples were sealed and refrigerated immediately after splitting.
Splitting uniformity was verified by testing 3 randomly selected
vials from each subsampled lot for fat by the Gerber test.
Two practice milk samples (one raw and one pasteurized
homogenized) of known fat contents were also prepared and
sent to collaborators with the test samples in each study. Laboratories were instructed to analyze the practice samples before
proceeding with the test samples.
Test samples and practice samples were shipped by overnight delivery to each laboratory in insulated boxes containing
frozen refrigerant packs. Collaborators were instructed to analyze the samples within 24 h of receipt. All laboratories received copies of the methods along with forms to record test
results and verify temperature of samples upon arrival.

Reference Testing
The Barbano Laboratory at Cornell University (Ithaca,
NY) conducted the Mojonnier ether extraction reference tests
for both studies using AOAC Method 989.05 (7). Ether extraction analyses were conducted in duplicate within 24 h of
receiving the test samples.

Statistical Analysis
In the original collaborative study protocol, laboratories
were instructed to analyze each test sample in duplicate by the
Gerber method. However, current AOAC guidelines for collaborative studies state that each test sample be analyzed once
and that blind duplicate test samples be used to determine
method repeatability parameters (8). To conform to current
AOAC guidelines, only the first result (uncensored) from the
duplicate analyses was used in the statistical analyses. Repeatability parameters were determined from the 2 materials (one
raw and one pasteurized homogenized) tested in blind duplicate in each study.
Using data from the first test of the duplicate analyses,
interlaboratory study results were determined by AOAC
guidelines (8). Test results from the Gerber method were
compared with ether extraction results using paired t-tests.
The significance level for outlier identification was set at =
0.025. The significance level for all other statistical analyses
was set at = 0.05.

KLEYN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 5, 2001 1501

AOAC Official Method 2000.18

Fat Content of Raw and Pasteurized Whole Milk
Gerber Method by Weight (Method I)
First Action 2000

(Applicable to raw, batch pasteurized, high temperature

short time [HTST] pasteurized, and homogenized whole
milk.)
Caution: See Appendix B (Official Methods of Analysis of
AOAC INTERNATIONAL) safety notes on centrifuges, sulfuric acid, mercury, and organic solvents. Dispose of waste solvents according to
applicable environmental rules and regulations.
See Table 2000.18A for the results of the interlaboratory
study supporting acceptance of the method.

A. Principle
Milk is weighed into Gerber butyrometer containing
H2SO4. Isoamyl alcohol is added and butyrometer contents
are vigorously mixed to dissolve curd and release fat. Fat is
isolated by centrifugation and quantified in the graduated portion of the Gerber butyrometer.

B. Reagents
(a) Sulfuric acid.Specific gravity 1.8201.825 at
15.5C. Technical grade.
(b) Isoamyl alcohol.Specific gravity 0.8140.816 at
15.5EC, bp 128 to 133EC, free of water, acid, fat, and furfural.
(Caution: Vapors are poisonous.)

C. Apparatus
(a) Standard Gerber milk-test butyrometer (graduated
08%) with lock stopper and key.(See Figure 2000.18.)
Clear transparent, colorless, resistant borosilicate glass, annealed and free from defects; neck, large bulb, flat tube, and
small bulb on a straight median axis, joined smoothly to permit free flow of liquid; wall thickness adequate to provide sufficient strength but not less than 0.9 mm at any point. Total
length 190 2.5 mm.
(1) Small bulb.Tapered, with matte surface on side
above graduations for sample identification; #15.0 mm od;
capacity, measured between top graduation line and internal
end of bulb $1.5 mL.
(2) Large bulb.#25.0 mm od; capacity, 21.5 0.4 mL.

(3) Neck.Cylindrical and plain; length, 15 1.0 mm;

11.5 0.5 mm id; rim, optionally reinforced; total thickness of
external rim, #2.5 mm diameter.
(4) Flat tube.Uniform in cross-section graduated
length, $70 mm; uniform cross-section beyond each terminal
graduation, $3.0 mm; external width, $10.0 mm. Graduated
with permanent, clean lines of 0.100.20 mm width and permanently fused color contrasting sharply with color of milk
fat, perpendicular to flat tube axis. Graduated volume in flat
tube at 20EC is 1.000 mL (13.546 g Hg), divided into 80 equal
parts, each equivalent in volume to 0.0125 mL. Error of total
calibrated length is not to exceed of smallest graduation.
Graduation lines are uniformly centered on flat tube face, with
0.1% lines not less than 3 mm long, the 0.5% lines extending
1 mm on each side beyond the 0.1% lines, and the 1.0% lines
extending not less than 1 mm on each side beyond the 0.5%
lines or extending across the entire flat face. Each whole percent graduation is identified by pigmented numbers running
serially from 0 to 8, at the right and just above the
whole-percent lines, with the zero measurement nearest the
large bulb. Bottles are identified Milk, with the name or
symbol of the manufacturer or distributor permanently inscribed on the body and a symbol applied after recalibration
where required to attest conformance with regulatory specifications.
(5) Testing.Accuracy of each bottle should be determined before use (usually by manufacturer or calibration laboratory). Mercury is added to the butyrometer by means of a
special volumetric apparatus. Testing is conducted at ambient
temperature at 20 2EC. The small bulb is opened to prevent
trapped air bubbles. The apparatus has 2 volumetric settings.
The first setting delivers a variable volume that permits filling
mercury into the butyrometer until the meniscus reaches the
0 graduation line. The second setting dispenses 1.000 mL
mercury, corresponding to 8% on the graduated scale. The difference between the expected and observed scale readings
must not exceed division of a graduation line ( 0.05%
fat). The accuracy of the testing apparatus is regularly verified
with officially calibrated glassware.
(6) Lock stopper and key.Lock stopper is of reagent-resisting rubber, of standardized dimensions to fit in
neck of butyrometer, molded to provide a seat for the ball or
plug, and with a channel for the key. Rim and channel are rein-

Table 2000.18A. Interlaboratory study results for percent fat in milk (g/100 g) determined by Gerber by weight
methoda
Sample ID

Mean

No. of labs

sr

sR

RSDr, %

RSDR, %

Raw

4.431

1011

0.023

0.053

0.52

1.20

0.065

0.148

Pasteurized, homogenized

3.677

1011

0.029

0.040

0.78

1.08

0.080

0.111

Mean based on analysis of 7 raw whole milk and 4 pasteurized homogenized whole milk, respectively. Standard deviations (sr, sR) and
prediction values (r, R) based on analyses of 8 raw milks (2 in blind duplicates) and 8 pasteurized homogenized milks (2 in blind duplicates),
for a total of 200 tests. Of the total of 200 tests, statistical outliers were identified for 5 raw milk tests and 5 pasteurized homogenized milk
tests. Relative standard deviations (RSDr, RSDR) were calculated as (sr/mean) 100 and (sR/mean) 100, respectively.

1502 KLEYN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 5, 2001

Figure 2000.18. 8% Gerber milk-test butyrometer.

forced by metal, plastic, or other firm binding insert. Key is of

noncorrodible material suitable for inserting into the stopper.
(b) Butyrometer support rack.To hold butyrometers in
a secure, vertical position.
(c) Dispensers.To deliver 10 mL H2SO4 and 1 mL
isoamyl alcohol.
(d) Centrifuge.For butyrometers, preferably provided
with a speed indicator to assess 1150 70 rpm. If not equipped
with speed indicator, check operation speed of Gerber centrifuges under full load regularly with a tachometer. Design of
the centrifuge shall ensure that temperature of butyrometer
contents after centrifuging is between 30 and 50EC.
When loaded, the centrifuge must produce, within 2 min, a
relative centrifugal acceleration of 350 50 g at the outer
end of the butyrometer stopper. This acceleration is produced
by centrifuges with the following effective radius (horizontal
distance between the center of the centrifuge spindle and the
outer end of the butyrometer stopper) operated at the speed indicated (Table 2000.18B).
(e) Water bath for test butyrometers.With thermometer,
device to maintain temperature of fat column at 6063EC, and
providing intermittent or constant agitation. Depth of water
bath must permit vertical immersion of butyrometer to terminal (small) bulb.
(f) Water bath for tempering milk samples prior to weighing.With thermometer and device to maintain temperature
of milk at 39 1EC.
(g) Analytical balance.Weighing to nearest 0.01 g.
Check accuracy periodically and whenever balance is moved
or cleaned. Keep record of balance calibration checks.
(h) Calibration weights.Class S, standard calibration
weights to verify balance accuracy within weight range to be
used for weighing empty milk butyrometers and butyrometers
containing test portions.

Table 2000.18B. Centrifuge speed

Effective radius, mm

Revolutions per min, 70 rpm

240

1140

250

1120

260

1100

270

1080

300

1020

(i) Reading light.As background when measuring fat

columns. Light should be diffused (soft white) and provide illumination from angles above and below level of fat column
and at eye level. Magnification device to aid reading is useful.

D. Determination
(a) Milk sample preparation.Place milk test sample in
H2O bath maintained at 39 1EC. Level of H2O should be at
or above milk level. Mix milk 10 by inversion. If fat line remains on inside surface of container, run hot tap H2O (ca
5060EC) over outside surface for 1520 s. Mix thoroughly
by inversion and weigh test portion immediately. Do not allow
milk to remain in H2O bath more than 15 min after reaching
38EC.
(b) Testing.Add 10.0 0.2 mL of H2SO4 at 1521EC to
butyrometer. Tare butyrometer containing H2SO4 on analytical balance. Weigh 11.13 0.03 g tempered milk test sample,
D(a), into butyrometer, adding milk slowly at first to prevent
charring and violent reaction with acid. Add 1 0.05 mL
isoamyl alcohol to butyrometer containing test portion. Insert
lock stopper securely using hand-held key. Wearing insulated
gloves, grasp butyrometer at graduated neck with stoppered
end up. Without allowing small bulb to empty, shake until all
traces of curd disappear. Holding butyrometer by both
stoppered end and graduated neck, invert at least 4 times to
mix acid remaining in the small bulb and graduated neck with
the contents of larger bulb.
Place butyrometers in centrifuge, small bulb pointing up,
and counterbalance. Centrifuge 4 min after proper speed is
reached. Transfer butyrometers to H2O bath maintained at
6063EC and immerse leaving only small bulb exposed. Let
fat column equilibrate for $5 min.
Remove one butyrometer from water bath and wipe dry.
Apply gentle pressure to lock stopper to bring bottom line of
fat column upward to coincide with nearest whole percentage
graduation mark. Promptly read scale at bottom of upper meniscus to nearest 0.05%.

E. Repeat Analysis
Repeat analysis if fat column is turbid or dark in color, or if
there is white or black material at bottom of fat column. Ac-

KLEYN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 5, 2001 1503
Table 1. Fat in milk (g/100 g) determined by Gerber by weight method
Laboratory
Typea

Replicate

Raw

3.65

3.65

3.70

3.70

3.70

3.65

3.65

3.60

3.65

3.65

3.65

Raw

3.85

3.75

3.80

3.85

3.85

3.80

3.80

3.75

3.80

3.75

3.80

3.80

3.75

3.80

3.80

3.85

3.70

3.80

3.75

3.75

3.75

3.80

Raw

5.25

4.80b

5.30

5.25

5.25

5.10

5.15

5.20

5.15

5.20

5.15

Raw

5.55

5.30

5.50

5.55

5.60

5.50

5.40

5.40

5.45

5.50

5.50

PH

0.95

1.00

0.95

0.95

1.05

0.90

0.95

0.95

0.95

0.90

1.00

PH

1.95

1.95

1.90

2.00

2.00

1.95

1.95

1.90

2.00

1.95

1.90

PH

3.25

3.20

3.15

3.20

3.30

3.20

3.20

3.00b

3.20

3.15

3.20

Material

8
a
b
c

PH

3.20

3.20

3.20

3.25

3.25

3.20

3.20

3.10

3.20

3.20

3.25

4.30

4.30

4.30

4.25

4.30

4.25

3.20b

4.10c

4.25

4.20

4.00c

Raw = raw milk; PH = pasteurized homogenized milk.

Statistical outlier (single Grubbs test).
Statistical outlier (double Grubbs test).

Fat in Milk

ceptable fat columns are pale to strong yellow and uniform

throughout with no light or dark particles.

Gerber Method by Pipet (Method II)

(Method II, although collaboratively studied, was not adopted)

(Applicable to raw, batch pasteurized, HTST pasteurized,

and homogenized whole milk.)

F. Calculations
Calculate % (w/w) fat content in milk as follows:

Caution: See Appendix B (Official Methods of Analysis of

AOAC INTERNATIONAL) safety notes on centrifuges, sulfuric acid, and organic solvents. Dispose of waste solvents according to applicable
environmental rules and regulations.

Fat, % =
reading at upper meniscus reading at lower meniscus
Ref.: J. AOAC Int. 84, XXXXXX(2001)

Table 2. Fat in milk (g/100 g) determined by Gerber by pipet method

Laboratory
Material
9

10

Typea

Replicate

Raw

1.35

1.45

1.40

1.40

Raw

1.45b

1.35

1.40

1.35

1.40

1.40

1.35

1.45

1.35

1.40

1.35

1.35

1.35

1.35

1.40

1.40

3.45

3.45

3.45

3.50

3.50

3.40

3.45

3.50

3.40

3.45

11

Raw

4.55

4.50

4.50

4.55

4.60

4.50

4.45

4.05

4.50

4.50

12

Raw

5.00

4.90

5.00

4.90

4.95

4.90

5.35c

4.85

4.95

4.80

13

PH

0.95

1.00

0.95

0.95

1.05

0.95

0.95

0.95

0.95

0.95

14

PH

1.95

1.95

1.90

1.95

2.00

1.95

1.85

1.90

1.95

1.90

15

PH

3.25

3.25

3.20

3.20

3.25

3.20

3.20

3.20

3.20

3.25

3.25

3.20

3.20

3.25

3.25

3.25

3.20

3.20

3.20

3.15

4.10

4.00

4.00

4.00

4.10

4.00

3.95

3.95

4.00

4.00

16
a
b
c

PH

Raw = raw milk; PH = pasteurized homogenized milk.

Statistical outlier (Cochran test).
Statistical outlier (single Grubbs test).

1504 KLEYN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 5, 2001

Note: Accuracy (as defined by agreement with Mojonnier

ether extraction, Method 989.05) is dependent on volume capacity of the pipet used to deliver the test portion.

A. Principle
See Method I.

irregularities that may hinder free flow of liquid. Delivery end

is reinforced and tip ground at a 45E angle with axis of pipet.
The time of outflow shall be 58 s. Capacity is determined at
20EC and 1 bar atmospheric pressure using 20EC water with
an assumed density of 0.9982. Measurement is made with bottom of meniscus on graduation mark in the horizontal plane.

D. Determination

B. Reagents
See Method I.

C. Equipment
See Method I, and in addition:
(j) PipetTo deliver 10.77 mL at 20EC. Made with clear,
transparent, colorless, resistant borosilicate glass, well annealed and free from defects. The suction tube shall be cylindrical. The graduation mark shall be finely etched, of uniform
width #0.25 mm, durable, and carried completely around the
suction tube. The graduation mark shall be at right angles to
the axis of the pipet. The end of the suction tube shall be
firepolished and be at right angles to the axis of the pipet. Bulb
is cylindrical and evenly drawn out in both directions, with no

(a) Milk sample preparation.See Method I.

(b) Testing.Add 10.0 0.2 mL H2SO4 at 1521EC to
butyrometer. Cool tempered milk, (a), to 2025EC. Mix test
sample by pouring from one container to another 4. Withdraw test portion, (a), using pipet, aligning top of meniscus
with graduation line of pipet. Touch off any part-drop remaining at tip. Pipet test portion into butyrometer, adding milk
slowly at first to prevent charring and violent reaction with
acid. Wait 3 s after flow stops and blow out the last drop. Continue as described in Method I, D(b).

E. Calculation
See Method I.

Table 3. AOAC statistical parameters by test material for fat in milk (g/100 g) determined by Gerber by weight
method
Statistic
Material

Typea

No. of labs

No. of tests

Mean, %

sr

sR

RSDr, %

RSDR, %

No outliers removed
1

Raw

11

11

3.659

0.030

Raw

11

22

3.789

Raw

11

11

5.164

0.134

2.602

0.376

Raw

11

11

5.477

0.085

1.547

0.237

PH

11

11

0.959

0.044

4.556

0.122

PH

11

11

1.950

PH

11

22

3.196

PH

11

11

0.028

0.041

0.824
0.744

0.039
0.034

4.132

0.061

1.086

0.084
0.079

1.986
1.055

0.323

1.896

0.115

0.108
0.094

7.829

0.170
0.906

Statistical outliers removed

1

Raw

11

11

3.659

Raw

11

22

3.789

Raw

10

10

5.200

0.062

1.199

0.175

Raw

11

11

5.477

0.085

1.547

0.237

4.531

Summary of raw materials

5

PH

11

11

0.959

PH

11

11

1.950

PH

10

20

3.210

PH

Summary of Gerber by weight (raw and PH materials)

0.028

0.041

0.058

0.824
0.744

0.744

0.044

0.035

2.597

0.027

3.564

0.028

1.086

1.290

0.084
0.079

0.079

4.556

0.039
0.027

4.269

Summary of PH materials

0.030
0.028

1.095

0.039

0.853

0.050

0.780

0.037

0.165
0.122

1.986
0.853

0.115

0.108
0.077

0.098

1.499

0.077

0.110

1.393

0.079

0.140

0.871

0.104

Raw = raw milk; PH = pasteurized homogenized milk.

Summaries were calculated from individual material performance statistics with outliers removed. Summary means were calculated by
averaging. All other statistical summaries were derived from variance averaging.

KLEYN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 5, 2001 1505
Table 4. AOAC statistical parameters by test material for fat in milk (g/100 g) determined by Gerber by pipet method
Statistic
Material

Typea

No. of labs

No. of tests

Mean, %

sr

sR

RSDr, %

RSDR, %

0.037

1.977

2.677

0.077

0.104

No outliers removed
9

Raw

10

20

1.385

0.027

10

Raw

10

10

3.455

0.037

1.068

0.103

11

Raw

10

10

4.470

0.153

3.425

0.429

12

Raw

10

10

4.960

0.151

3.035

0.422

13

PH

10

10

0.965

0.034

3.497

0.094

14

PH

10

10

1.930

15

PH

10

20

3.218

16

PH

10

10

0.042
0.030

4.010

0.030

2.185
0.919

0.052

0.919

0.118
0.083

1.288

0.083
0.145

Statistical outliers removed

9

Raw

18

1.383

10

Raw

10

10

3.455

0.037

1.068

0.103

11

Raw

4.517

0.043

0.959

0.121

12

Raw

4.917

0.066

1.345

0.185

3.568

Summary of raw materials

13c

PH

10

10

0.965

14

PH

10

10

1.930

15

PH

10

20

3.218

PH

10

10

4.010

16

Summary of PH materials

Summary of Gerber by pipet (raw and PH materials)

a
b

0.017

0.017

0.035

0.047

1.205

1.205

0.034

0.030

1.315

0.047

0.047

3.497

0.042
0.030

2.538

0.052

0.919

0.133
0.094

2.185
0.919

0.098

0.118
0.083

1.288

0.083
0.145

2.531

0.030

0.040

0.919

1.593

0.083

0.114

3.049

0.024

0.044

0.788

1.435

0.068

0.124

Raw = raw milk; PH = pasteurized homogenized milk.

Summaries were calculated from the individual material performance statistics with outliers removed. The summary means were calculated
by averaging. All other statistical summaries were derived from variance averaging.
All data for material 13 were included in the final analysis, although 2 single Grubbs outliers were identified. Inclusion of these data was
justified because outliers were identified only because the difference between the remaining data for all laboratories was 0.00% fat.
Furthermore, method statistics for material 13 with the outliers left in were similar to statistics generated for all other materials.

Ref.: J. AOAC Int. 84, 15031505(2001)

Results

Interlaboratory Study Results

Data for Gerber by weight and Gerber by pipet methods are
presented in Tables 1 and 2, respectively. No data were identified as invalid for either method. For Gerber by weight
method, outliers were identified for material 3 (single Grubbs,
laboratory B), material 7 (single Grubbs, laboratory H), and
material 8 (single Grubbs, laboratory G, and double Grubbs,
laboratories H and K). For Gerber by pipet method, a Cochran
outlier was identified for material 9 (laboratory E) and single
Grubbs outliers were identified for materials 11 (laboratory H)
and 12 (laboratory G). Single Grubbs outliers were also identified for laboratories B and E for material 13. However, these
outliers were identified only because test results of the other
9 laboratories for this material were identical. Furthermore,
the statistical method performance of material 13 without

elimination of outliers was similar to that of all other materials. As a result, the decision was made to include all data from
material 13 in the analysis.
Interlaboratory study results, with and without statistical
outliers removed, for Gerber by weight and Gerber by pipet
methods are presented in Tables 3 and 4, respectively. There
were no meaningful differences in interlaboratory study results (statistical outliers removed) between raw and pasteurized materials when parameters were summarized by material
type. When performance parameters for raw and pasteurized
homogenized materials were pooled within method (statistical
outliers removed), interlaboratory study results for Gerber by
weight and Gerber by pipet were similar.
The milk fat content of the 8 materials ranged from 0.959
to 5.477% for Gerber by weight method and from 0.965 to
4.917% for the Gerber by pipet method. There was a statistically significant (p < 0.05, linear regression) increase in RSDR
as fat content decreased for both methods. No significant
trends for sR were observed. Only 2 estimates of repeatability

1506 KLEYN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 5, 2001
Table 5. Comparison of milk fat test results by the ether extraction method with the Gerber by weight and Gerber by
pipet methods
% Fat, Gerber by weight vs ether extraction

% Fat, Gerber by pipet vs ether extraction

Typea

Gerber by
pipet

Ether
extraction

Differenceb

13

PH

0.965

1.038

0.073

Raw

1.383

1.457

0.074

Typea

Gerber by
weight

Ether
extraction

Differenceb

Material

PH

0.959

1.012

0.053

PH

1.950

1.994

0.044

Material

PH

3.210

3.213

0.003

14

PH

1.930

1.987

0.057

Raw

3.659

3.672

0.013

15

PH

3.218

3.273

0.056

Raw

3.789

3.797

0.008

10

Raw

3.455

3.498

0.043

PH

4.269

4.263

0.006

16

PH

4.010

4.054

0.044

Raw

5.200

5.200

0.000

11

Raw

4.517

4.579

0.063

Raw

5.477

5.503

0.025

12

Raw

4.917

4.998

0.081

3.564

3.582

0.017

Mean

3.049

3.111

0.061

Mean
a
b

Raw = raw milk; PH = pasteurized homogenized milk.

Discrepancies in the last decimal place are due to rounding.

performance were available for each method; therefore, statistical tests to determine the relationship between fat level and
repeatability parameters were not conducted.

between Gerber by pipet and ether extraction methods was not

related to fat level (p > 0.05).
Discussion

Comparison with Ether Extraction

Fat test results for Gerber by weight and Gerber by pipet
methods were compared with ether extraction values in Table 5. Results for Gerber by weight method averaged 0.017%
lower in absolute fat than the Mojonnier ether extraction, and
this difference was marginally statistically significant
(0.05 < p < 0.10). Results for Gerber by pipet method averaged 0.061% lower in absolute fat than the ether extraction,
and this difference was highly significant (p <0.01). There
was a slight significant trend (0.05 < p < 0.10) toward closer
agreement between Gerber by weight and ether extraction
methods as fat level increased. The difference in test results

Because Gerber interlaboratory study results were unaffected by material type (raw or pasteurized, homogenized) or
method of test portion addition (weight or pipet), performance
parameters determined for all materials and methods were
pooled to estimate overall Gerber interlaboratory study results
and increase the number of materials used to determine repeatability (Table 6). Repeatability and reproducibility standard deviations, as well as the r and R values, were not correlated with fat level, and, thus, each is summarized by a single
parameter. Because the sR was fairly constant, relative
reproducibility (RSDR) was greater at lower fat levels (12%)

Table 6. Comparison of interlaboratory study statistics for determination of fat in milk (g/100 g) by Gerber, Babcock,
and ether extraction methods
Interlaboratory study results, % fat in milk (g/100 g)
AOAC
Method No.

Method

Material type

Current study

Gerbera

Raw, PH

Mean

sr

sR

4.156

0.026

0.047

1.437

RSDr, %

RSDR, %

0.625 (for whole)

1.131 (for whole)

0.074

0.132

1.809 (for 12% fat) 3.271 (for 12% fat)

989.04

Babcockb

Raw

4.110

0.037

0.047

0.901

1.147

0.105

0.133

989.05

Ether extractionc

Raw, PH

3.886

0.015

0.020

0.396

0.512

0.044

0.056

b
c

sr and sR values for the Gerber method were calculated by variance averaging individual material performance statistics (16 materials) with
outliers removed. RSDs were calculated from grand sr and sR using means from 11 milk materials within 26% fat (14, 7, 8, 1012, 15, and
16) and the 5 milk materials within 12% fat (5, 6, 9, 13, and 14).
Ref. 1.
Ref. 2.

KLEYN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 5, 2001 1507

than fat levels found in whole milk. Trends in RSDr could not
be identified because of the limited number of materials examined, but the nature of the test (readability maximum
0.05% fat) suggests that RSDr would increase proportionally
in lower (12%) fat milk. For this reason, relative repeatability
and relative reproducibility are summarized by fat level.
Table 6 presents the summary method performance statistics and compares them with other AOAC chemical methods
used to determine fat content of milk. The within- and between-laboratory performance of the Gerber method is very
similar to that of the Babcock method (AOAC Method
989.04; 1). Because the Babcock method is limited to raw
milk testing, the Gerber method has the advantage of application to pasteurized homogenized milk. The interlaboratory
study results of ether extraction (AOAC Method 989.05; 7),
the reference test, are clearly superior to either the Babcock or
the Gerber methods, but for routine application where the authority and precision of reference testing is not required, both
the Gerber and Babcock tests have acceptable and similar performance when conducted properly.
Compared with the ether extraction reference method, fat
test results were slightly lower (0.017% absolute fat) for the
Gerber by weight method and significantly lower (0.061%)
for the Gerber by pipet method. Of course, these differences
are crude estimates only, as the amount of data available was
limited. The original justification for selecting a weight addition of 11.125 g (11.13 0.03 g) and a pipet that delivered
10.77 mL was evidence of good agreement with ether extraction in an in-house comparison (4). However, the in-house
Mojonnier ether extraction reference method in use at that
time had not been collaboratively studied and specified 2 extractions (5). The current Mojonnier ether extraction method
(AOAC Method 989.05; 7), collaboratively studied, requires
3 extractions to maximize fat recovery and improve precision
(7, 9). In the Gerber collaborative study, the participation of
many laboratories to arrive at Gerber test values and the use of
Mojonnier Method 989.05 with 3 extractions to optimize fat
recovery and precision increase the ability to detect biases and
define differences between methods.
Compared with ether extraction, a trend towards underestimating fat content at lower fat concentrations was observed
for the Gerber by weight method but not for the Gerber by
pipet method. This difference may be due to the effect of density on the amount of sample actually delivered by the pipet.
The density of milk decreases as fat content increases, and the
weight of the test portion delivered increases with decreasing
milk fat content when a constant volume is used.
A weakness in the collaborative study was that only one
laboratory conducted the ether extraction reference test. This
was somewhat mitigated by the expertise of this laboratory,
which is under the direction of the AOAC Associate Referee
for the Mojonnier ether extraction method (9). Additionally,
this laboratory participates in a formalized interlaboratory
proficiency testing program (7 materials in blind duplicate every 2 months). This program, begun in 1990, documents that
method bias for this laboratory is small, averaging 0.004%
lower in fat than all laboratory means (10). In terms of

within-laboratory precision, the repeatability standard deviation for the ether extraction reference testing for the Gerber
studies (20 laboratory samples, tested in known duplicate, representing 16 materials) was 0.007% fat, which is well within
the sr of 0.015% fat for the method (7).
Recommendations
The Associate Referee recommends that the Gerber by
weight method be adopted Official First Action with applicability limited to whole milk because of limited evidence of a
bias between Gerber by weight and ether extraction results at
fat levels of 1 to 2%. At the request of AOAC INTERNATIONAL, the interlaboratory study results accompanying the
method description are listed separately for the raw whole
milk and pasteurized homogenized whole milk materials, although performance for the 2 types of whole milk materials
was equivalent.
At this time, the Associate Referee does not recommend
adopting the Gerber by pipet method using the 10.77 TD pipet
because of a significant bias between this method and ether
extraction results at all fat levels tested. When a suitable pipet
volume is identified, the Gerber by weight method can be
modified to include the option of either weighing or pipeting
the test portion.
Acknowledgments
The authors thank Dr. Kleyns family, including his brothers Carl and John, his children Frank and Allison, and sister-in-law Mary, for giving us permission to publish his material. The associates of Dr. Kleyn felt it important to bring his
work to completion. Jeffrey Bloom at Weber Scientific organized the effort. Researchers at Cornell University, Joanna M.
Lynch and David M. Barbano, evaluated the data and conducted the statistical analysis. Martin W. Mitchell of Certified
Laboratories, Inc. assumed the role of Associate Referee to
oversee the method now and in the future. This report was prepared as a tribute to Dr. Kleyn for his lifelong contributions to
the dairy industry and the field of dairy chemistry.
The authors thank the following collaborators for their participation in the study:
Lynda S. Cooper, Velda Farms, Miami, FL
Elizabeth Cusak, Certified Laboratories, Corona, NY
Mike Fick, Crowley Foods, Inc., Weeks Division, Concord, NH
Tom Hanks, Wyoming Department of Agriculture, Division of Laboratories, Laramie, WY
Mary K. Hesen, Valley Farms Dairy, Inc., Williamsport,
PA
Julie Johnson, Dairy Quality Control Institute, St. Paul,
MN
Dick H. Kleyn, Rutgers University, Cook College, New
Brunswick, NJ
Fayette Mercer, Florida Department of Agriculture, Central Dairy Laboratory, Winter Haven, FL
Denise Monahan, Welsh Farms, Long Valley, NJ

1508 KLEYN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 5, 2001

Barbara Peat, New York State Department of Agriculture

and Markets, State Food Laboratory, Albany, NY
Marquerite Petit, Crowley Foods, Inc., Albany, NY
References
(1) Official Methods of Analysis (1995) 16th Ed., March 1998
Revision, AOAC INTERNATIONAL, Gaithersburg, MD,
sec. 33.2.27
(2) International Organization for Standardization (1976) ISO International Standard 2446, MilkDetermination of Fat
Content (Routine Method), ISO, Geneva, Switzerland
(3) International Dairy Federation (1991) International Provisional IDF Standard 152, Milk and Milk Products.
Determination of Fat Content. General Guidance on the Use
of Butyrometric Methods, Brussels, Belgium

(4) Kleyn, D.H., Trout, J.R., & Weber, M. (1988) J. Assoc. Off.
Anal. Chem. 71, 851853
(5) Standard Methods for the Examination of Dairy Products
(1985) 15th Ed., G.H. Richardson (Ed.), American Public
Health Association, Washington, DC
(6) Vester, C.F. (1959) Neth. Milk Dairy J. 13, 5972
(7) Official Methods of Analysis (1995) 16th Ed., AOAC INTERNATIONAL, Gaithersburg, MD, sec. 33.2.26
(8) AOAC INTERNATIONAL (1997) AOAC Official Methods
Program Manual on Development, Study, Review, and Approval Process for AOAC Official Methods, AOAC
INTERNATIONAL, Gaithersburg, MD
(9) Barbano, D.M., Clark, J.L., & Dunham, C.E. (1988) J. Assoc.
Off. Anal. Chem. 71, 898914
(10) Lynch, J.M., Barbano, D.M., Healy, P.A., & Fleming, J.R.
(1994) J. AOAC Int. 77, 976981