33.2.27
Proper rate of centrifuge rotation for known
AOAC Official Method 989.04
Fat in Raw Milk
Babcock Method
Final Action 1920
Revised First Action 1989
Revised First Action 1997
Final Action 2000
(Applicable to raw, whole milk.)
Results of the interlaboratory study supporting acceptance of the
method (for sections A–D):
sr = 0.037; sR = 0.047; RSDr = 0.901%; RSDR = 1.147%; r = 0.105;
R = 0.133
A. Principle
Known weight of milk is placed into Babcock bottle and H2SO4 is
added. Generated heat releases fat, which is then isolated by
centrifuging and addition of H2O. Fat is quantitated in graduated
portion of Babcock bottle. Result is expressed as % fat by weight.
B. Reagents
(a) Sulfuric acid.—Specific gravity 1.82–1.83 at 20°C.
(b) Mercury.—Purity ³99.99%. Used for testing accuracy of
Babcock bottles.
C. Apparatus
(a) Standard Babcock milk-test bottle.—8%, 18 g milk-test
bottle, total height 160–170 mm (6.3–6.7 in.). Bottom of bottle is
flat, and axis of neck is vertical when bottle stands on level surface.
(1) Bulb.—Capacity of bulb to junction with neck is ³45 mL.
Bulb may be either cylindrical or conical. If cylindrical, od of base
must be 34–36 mm; if conical, od of base must be 31–33 mm, and
maximum diameter, 35–37 mm.
(2) Neck.—Cylindrical and of uniform diameter from ³5 mm
below lowest graduation mark to ³5 mm above highest. Top of neck
is flared to diameter of ³10 mm. Graduated portion of neck is
³75 mm long and is graduated in whole %, 0.5%, and 0.1% from 0.0
to 8.0%. Graduations may be etched, with black or dark pigment
annealed to graduation, or may be unetched black or dark lines
permanently annealed to glass. Graduation line widths £0.2 mm.
Tenth % graduations are ³3 mm long; 0.5% graduations are ³4 mm
long and project 1 mm to left; and whole % graduations extend at
least half way around neck to right and project ³2 mm to left of 0.1%
graduations. Each whole % is numbered, with numeral placed to left
of scale.
Vertical line must be etched and annealed with black or dark
pigment or must be etched into dark stripe permanently annealed to
glass. Etched vertical line is located 1 mm to right of 0.1%
graduation marks and extends ³1 mm above 8% line and ³1 mm
below 0% line. If vertical line is etched into dark vertical stripe, then
dark vertical stripe must extend ³1 mm above the 8% line and
³1 mm below 0% line. Dark vertical stripe should be £2.0 mm wide
and etched line must be £0.2 mm wide.
Zero line (£0.2 mm wide) must be etched and annealed with black
or dark pigment, or not darkened but etched into dark horizontal
stripe (£2.0 mm wide) permanently annealed to glass. Zero and
vertical etched lines, with or without annealed pigment, should be of
sufficient depth to hold point of dividers.
Capacity of neck for each whole % on scale is 0.200 mL.
Maximum error of total graduation or any part thereof must not
exceed 0.008 mL.
Table 989.04.
diameter of wheel to achieve 164 ´ g
Diameter of wheel, cm Diameter of wheel, in. rpm
35.6 14 909
40.6 16 848
45.7 18 800
50.8 20 759
55.9 22 724
61.0 24 693
(3) Testing.—Accuracy of each bottle shall be determined before
use (usually by manufacturer or certified laboratory). Bottle
calibration accuracy is determined by placing bottle upside down on
Babcock bottle calibration apparatus (Nafis tester or modified Nafis
tester) that is capable of delivering known volumes of Hg into
Babcock bottle neck. Bottle calibration apparatus delivery is
cal ibrated and vol ume of Hg contained be tween 8 and 4%
(0.800 mL), 4 and 0% (0.800 mL), and 8 and 0% (1.600 mL) marks
is de ter mined. Ac cu racy of bot tle neck cal i bra tion may be
determined before or after attachment to bulb. Accuracy should be at
least ±0.04% on scale of 18 g milk-test bottle. [Reference: J. AOAC
Int. 78, 463(1995).]
(b) Pipet.—Stan dard milk pipet con forms to fol low ing
spec i fi ca tions:
Total length . . . . . . . . . . . . . . . . . . . . . . . £350 mm
OD of suction tube . . . . . . . . . . . . . . . . . . . . 6–8 mm
Length of suction tube. . . . . . . . . . . . . . . . . . . 130 mm
OD of delivery tube [must fit into bottle, (a)] . . . . . 4.5–5.0 mm
Length of delivery tube
(slightly longer than bottle neck, must not touch
milk test portion when delivered) . . . . . . . . . 105–125 mm
Distance of graduation mark above bulb . . . . . . . . 15–45 mm
Nozzle parallel with axis of pipet, but slightly
constricted so as to discharge in 5–8 s when filled
with H2O
Graduation, marked to contain 17.6 mL H2O at 20°C
when bottom of meniscus coincides with mark on
suction tube
Maximum error in graduation . . . . . . . . . . . . . . £0.05 mL
To test pipet, place tip of pipet against firm rubber surface, clamp
pipet in vertical position, and use buret (Class A—graduations
£0.05 mL) to fill pipet to graduation mark with H2O (at 20°C).
(c) Acid measure.—Device used to measure H2SO4 should deliver
in range from 10 to 20 mL. Use device that can be set to consistently
deliver appropriate amount of acid to obtain desired milk acid
reaction temperature in D.
(d) Centrifuge or “tester.”—Standard centrifuge, however
driven, must be constructed throughout and so mounted as to be
capable, when filled to capacity, of rotating at necessary speed with
minimum vibration and without liability of causing injury or
accident. Centrifuge must be heated, electric or otherwise, to
46°–51°C during centrifuging. It must be provided with speed
indicator, permanently attached, if possible. Proper rate of rotation
may be determined by reference to Table 989.04. Rotation speed
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with full centrifuge should be checked periodically with tachometer.
“Diameter of wheel” means distance between inside bottoms of
opposite cups measured through center of rotation of centrifuge
wheel while cups are horizontally extended. Relative centrifugal
force is approximately 164 ´ g.
(e) Dividers or calipers.—For measuring fat column.
(f) Water bath for test bottles.—Provided with thermometer and
device to maintain temperature of fat column at 48° ± 1°C.
(g) Wa ter bath for tem per ing milk sam ples prior to
weighing.—Provided with thermometer and device to maintain
temperature of milk at 38° ± 1°C.
(h) Supply of hot water added after 1st and 2nd
centrifugations.—Provided with thermometer, device to maintain
temperature of distilled H2O at 50°–52°C, and device to deliver H2O
into Babcock bottles.
(i) Bottle shaker.—Variable speed and matched to maximum
capacity of centrifuge.
(j) Digital thermometer for measurement of milk-acid reaction
temperature.—Capable of reading temperature to the nearest degree
in range of 100°–120°C. Use acid-resistant probe, with small
diameter (£0.5 mm) to ensure rapid response time. Length of
temperature probe should be such that its tip is ca 1 cm above bottom
of bottle, when fully inserted.
(k) Reading light with magnifying glass.—As background when
measuring fat columns. Light should be diffused (soft white color)
and provide illumination from angles above and below level of fat
column. Magnification device must be used to aid reading.
(l) Tachometer.—For determining rotation speed of centrifuge
(capable of measuring between 600–1000 rpm).
D. Determination
(a) Test sample preparation and temperature adjustment.—Place
test sample in water bath at 38° ± 1°C. Level of H2O should be at or
above milk level. Mix test sample 10´ by inversion. If fat line
remains on inside surface of container, run hot tap water (ca
50°–60°C) over outside surface for 15–20 s. Mix test sample
thoroughly by inversion and pipet immediately. Do not allow test
samples to remain in water bath more than 15 min after reaching
38°C.
With pipet, C(b), transfer 17.6 ± 0.05 mL prepared test portion at
38°C to milk-test bottle. Also, transfer 17.6 ± 0.05 mL milk at 38°C
to another milk test bottle for measurement of milk-acid reaction
temperature. Blow out milk in pipet tip ca 30 s after free outflow
ceases. Adjust temperature of milk in bottle to 20°–22°C. Adjust
temperature of H2SO4 to 20°–22°C.
(b) Mea sure ment of milk-acid re ac tion tem per a ture and
determination of amount of H2SO4 to use.—Before testing group of
test portions, determine correct amount of acid to be used in analysis
by measuring milk-acid reaction temperature. Add 17.5 mL H2SO4
to bottle from (a) containing tempered milk test portion prepared for
measurement of milk-acid reaction temperature. Add acid in one
delivery that washes all traces of milk into bulb and cleanly layers
acid under milk. Fully insert digital thermometer probe down bottle
neck and immediately shake by hand rotation until all traces of curd
disappear. Peak reaction temperature should be 108° ± 2°C. Adjust
amount of H2SO4 added, until reaction temperature is within this
range and fat column is translucent golden-yellow to amber.
Amount of acid required may vary between technicians and between
batches of acid.
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(c) Testing milk test portions.—To test bottle from (a) containing
tempered milk, add appropriate amount of H2SO4 [as determined in
(b)] by delivering acid in one delivery that washes all traces of milk
into bulb and cleanly layers acid under milk. Immediately shake
bottle by hand rotation as in (b), until all traces of curd disappear.
Place bottle in Babcock bottle shaker set at medium speed. Continue
to add acid to all test portions, and then shake full set 1 additional
min. Temperature of milk plus acid in first bottle should not be less
than 50.5°C at time bottles are transferred to centrifuge.
Place bot tles in heated cen tri fuge, coun ter-balance, and
centrifuge 5 min after proper speed is reached. Add distilled water at
50°–52°C until bulb of bottle is filled. Centrifuge 2 min. Add
distilled water at 50°–52°C until top of fat column approaches 7%
mark on bottle neck. Centrifuge 1 min longer at ca 51°C. Transfer
bottle to water bath maintained at 48° ± 1°C, immerse bottle to level
slightly above top of fat column, and leave in water bath until
column is at equilibrium and lower fat surface assumes final convex
form (³5 min).
Remove 1 bottle from bath, wipe dry, and with aid of reading light
and magnification, C(k), use dividers or calipers to quickly measure
fat column (before it begins to cool and contract). Place caliper
points in vertical line on neck of bottle, with 1 point at lowest surface
of lower meniscus and other point at top of upper meniscus. Without
changing distance between 2 points on calipers, move calipers down
bottle neck until lower point rests in etched horizontal graduation
mark at 0%. Place upper point of calipers against bottle graduation
and read test in % to nearest 0.05%. Repeat for each bottle.
Fat column, at time of measurement, should be translucent,
golden-yellow or amber, and free of visible suspended particles.
Reject all tests in which fat column is milky or shows presence of
curd or charred matter, or in which meniscus is indistinct or
distorted; repeat test, adjusting volume of H2SO4 added, to obtain
proper color and milk-acid reaction temperature.
References: JAOAC 8, 4(1924); 8, 471(1925); 56, 1401(1973);
58, 949(1975); 71, 898(1988).
J. AOAC Int. 80, 845(1997).
E. Alternative Apparatus (First Action 1986; Final Action 1991)
(a) Gercock-style milkfat test bottle.—(Available from Forcoven
Products, Inc., PO Box 1556, Humble, TX 77338, USA.) 5%,
162 ± 3 mm high. Bottom of bottle is flat, and axis of flat graduated
tube is vertical when bottle stands on level surface. Quantity of milk
charge is 18 g; 18 g is acid-etched and pigmented in clear space
above 5% graduated line. Specifications:
(1) Bulb.—Capacity of bulb to junction with neck must be
³45 mL. Shape of bulb is cylindrical, 36 ± 0.5 mm od. Wall thickness
1.4 ± 0.2 mm. Word “Sealed”, manufacturer’s number or name, and
matted marking spot must be legibly and permanently marked on
surface.
(2) Side filling tube.—8 mm od, 0.1 mm wall thickness. To enter
bulb below junction of bulb and neck of flat graduated tube along
curved portion of bulb and with minimum angle extend to center of
bulb and maintain vertical axis for 40 mm to height of 3 ± 2 mm
above bottom of bottle.
(3) Neck.—Graduated portion of flat tube is uniform cross
section ³4 mm beyond extremes of graduated scale. Length of
graduated portion of flat neck ³75 mm. Graduated at whole %, 0.5,
0.1, and 0.05%. Graduations are acid-etched, with black pigment
annealed to graduation. Graduation widths ³0.2 mm. Each whole %
and 0.5% line is numbered on left above line and these lines extend Length of nozzle . . . . . . . . . . . . . . . . . . . . 15–20 mm
across flat surface. Smallest graduated line is ³2.5 mm. Intermediate Delivery time . . . . . . . . . . . . . . . . . . . . . . . . 5–8 s
graduation line extends 1 ± 0.2 mm equally on both sides of smallest
graduated line. Graduation:
For raw milk. . . . . . . . . . . . . To deliver 17.6 mL at 20°C
(4) Vertical line.—Vertical line is acid-etched and pigmented on
when bottom of meniscus coincides
right side of graduated flat tube. This line touches intermediate lines
with mark on suction tube
at their extreme right end and extends beyond 0 and 0.5% lines
For processed milk . . . . . . . . . To deliver 17.6 mL at 38°C
³3 mm. Width of vertical lines £0.2 mm. when bottom of meniscus coincides
(5) Error.—Maximum error of total graduation or any part with mark on suction tube
thereof must not exceed volume of smallest unit of graduation (= Maximum error . . . . . . . . . . . . . . . . . . . . £ 0.05 mL
0.01 mL). Permanent markings on bulb:
(b) Gercock-style pipet for raw and processed milk.—To conform For raw milk . . . . . . . . . . . . . . . . . . . . . “Sealed,”
to following specifications: manufacturer’s No. or name,
TD 17.6 mL at 20°C,
Total length: tolerance 0.05 mL, 5–8 s
For raw milk . . . . . . . . . . . . . . . . . . . . . £280 mm For processed milk . . . . . . . . . . . . . . . . . . . “Sealed,”
For processed milk . . . . . . . . . . . . . . . . . . ³280 mm manufacturer’s No. or name,
TD 17.6 mL at 38°C,
Suction tube diameter . . . . . . . . . . . . . . . . 7 ± 0.2 mm od
tolerance 0.05 mL, 5–8 s
Wall thickness . . . . . . . . . . . . . . . . . . . 0.9 ± 0.04 mm
Suction tube length . . . . . . . . . . . . . . . . . . . . 130 mm
Reference: JAOAC 69, 831(1986).
Delivery tube diameter . . . . . . . . . . . . . . . 5 ± 0.2 mm od
Distance of graduation mark above bulb . . . . . . . . 15–45 mm Revised: March 2002

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