- Email: [email protected]
A Spectrophotometric Method for Detecting Blood in White-Shell Eggs
A Spectrophotometric Method for Detecting Blood in White-Shell Eggs A. W. BRANT, 1 K. H. NORMS 2 AND GILBERT CHIN 1
Agricultural Research Administration, U. S. Department of Agriculture (Received for publication August 29, 1952)
T
1
Bureau of Animal Industry, Beltsv He, Maryland. 2 Bureau of Plant Industry, Soils, and Agricultural Engineering, Beltsville, Maryland. 357
are mistakenly candled out. Candling accuracy with respect to blood- and meatspot detection reported in the literature indicates a range of accuracy of 20 to 90 percent (Nalbandov and Card, 1944; Denton, 194.7; Jensen et al., 1952). At least one device has been patented (Dooley, 1943) with the claim that it detects blood in shell eggs by a spectrophotometric method. No published data has come to the attention of the authors concerning the performance of the device. It has been found in the studies reported here that blood in white-shell eggs exhibits selective light-absorbing properties. Through appropriate instrumentation it has been found that the presence of blood in an egg can be detected without the aid of the human eye. PROCEDURE
All light transmittance and reflectance data were obtained using a Beckman Model DU Spectrophotometer.3 In measuring the transmittance of egg albumen, 1 centimeter cuvettes were used with the spectrophotometer unmodified. For the yolk reflectance measurements the intact yolk was placed in a short section of 1inch diameter glass tubing with a Number 7 rubber stopper in the bottom end. The top of this receptacle was covered with a thin cover glass and the rubber stopper was pushed upward until the yolk flattened against the cover glass. Several yolks prepared in this manner were measured in the reflectance attachment of 3
Mention of this and other equipment does not imply endorsement by the U. S. Government.
Downloaded from http://ps.oxfordjournals.org/ by guest on June 12, 2015
HE most common defects found in eggs are blood and meat spots. Published data has shown the incidence of such defects to range from less than 1 percent to nearly 100 percent (Hainan and Day, 1935; Van Wagenen et al., 1937; Quinn and Godfrey, 1940; Nalbandov and Card, 1941; Lerner and Smith, 1942; Jeffery and Pino, 1943; Card and Nalbandov, 1944; Jeffrey, 1945; Lerner, 1946; Nalbandov and Card, 1947; Denton, 1947; Lerner and Taylor, 1947). The extent and degree of consumer dissatisfaction created by the presence of blood and meat spots is not known precisely. It seems safe to assume that spots of almost any size that are the color of fresh blood are offensive to many consumers. Blood spots larger than oneeighth inch in diameter or bloody whites can be considered rather generally unacceptable. In the opinion of the authors, it is not safe to assume that "meat" spots have any well denned effect upon consumer acceptance. Whenever the presence of a spot is detected in a shell egg by candling, the egg is usually given a lowered grade or declared inedible. Candling, however, is an imperfect method of detecting blood and meat spots. The result is a monetary loss to the industry through consumer dissatisfaction with defective eggs that are passed and through loss to producers and handlers of eggs when non-defective eggs
358
A. W. BRANT, K. H. NOREIS AND G. CHIN
^
-——^
Sjf~~
•
CURVES
/
z
\
No. 1 2
CLEAR BLOODY
Reference
600
800
WAVELENGTH IN
AL9UMEN ALBUMEN
Std - Distilled Water
1000 MILLIMICRONS
FIG. 1. Spectral transmittance of clear and bloody egg albumen. Blended samples of total albumen were measured in 1 centimeter cuvettes.
No. I 2 3 4
CURVES DARK YOLK "WHITE" YOLK MEDIUM-COLORED YOLK WITH BLOOD SPOT REFERENCE STANDARD (Opal Glass aver Yellow Paper)
60 3 50 40 30
u 20 10
400
500 WAVELENGTH
600 IN
700
MILLIMICRONS
FIG. 2. Spectral reflectance (45°) of egg yolks. Curves 1, 2 and 3 were obtained using the opal glass over yellow paper as a reference standard. Curve 4 is the spectral reflectance of this reference standard relative to powdered MgCOa.
varied in age from one day to several weeks and had undergone varying degrees of deterioration. RESULTS AND DISCUSSION
Transmittance curves for normal and bloody albumen are shown in Figure 1. These* curves were obtained from blended samples of total albumen from several eggs. The presence of blood in the albumen produced absorption bands peaking at 415, 541 and 575 imt. but appeared to have little effect on light transmittance at other wave lengths. Reflectance curves for yolks are shown in Figure 2. As expected yolk color had a pronounced effect upon the curves in the visible region. Curve 1 was obtained from a yolk produced by birds fed a diet containing fairly large quantities of yellow corn and alfalfa meal. It was somewhat darker than the yolk color usually produced by confined birds fed ordinary commercial rations. Curve 2 was obtained from a yolk produced by birds fed a diet
Downloaded from http://ps.oxfordjournals.org/ by guest on June 12, 2015
the DU Spectrophotometer using a piece of opal glass backed by yellow paper as a reference standard. The transmittance measurements of the shell and shell membrane were made by mounting the samples in specially prepared holders to support the sample between the exit slit and the phototube in the spectrophotometer. In order to obtain enough sensitivity to measure the spectral transmittance of intact eggs with a reasonably small spectral band width the spectrophotometer was modified as follows: The phototube and its current-measuring circuit were replaced by an RCA 5819 photomultiplier tube operating through a sensitivity-switching circuit into a Leeds and Northrup Speedomax millivolt recorder. The tungsten source normally used in the visible region was replaced by a 300-watt zirconium-arc lamp. A special sample holder was constructed to accommodate an intact egg. The measurements were all made in a room with the air conditioned at 75 + 2°F. and less than 50 percent R.H. The eggs and egg components were brought to room temperature before measurements were made. The eggs used in these studies
SPECTROPHOTOMETER: DETECTION OF SPOTS IN EGGS
CURVES
45
• No 1 2
WHITE SHELL REFERENCE STANDARD (Opol Class)
3
SHELL
MEMBRANE
lil o
z
< 1t-
l_^-^^"^
j 30 CO
z < a: H 1Z Ul
•
-*-.'•-
£ 15 UJ 0-
3 z n-
400
500 600 WAVELENGTH IN MILLIMICRONS
700
FIG. 3. Spectral transmittance of a white egg shell (curve 1) and shell membrane (curve 2). The reference standard for curve 1 was a piece of opal glass. The spectral transmittance of this reference standard and the shell membrane were measured relative to air.
containing no yellow pigments. In comparison with normally colored yolks, it was called "white." When a blood spot was present on a yolk in the area viewed by the instrument, there was a reduced reflectance of all wave-lengths of light between 540 and 620 m/x. In the region from 570 to 600 m/x. the effect of the blood spot was considerably greater than the effect of yolk color. The transmittance characteristics of a white egg shell and a shell membrane are shown in Figure 3. The shell membrane transmitted all wave-lengths equally well but at very low levels. The white shell showed a very rapid rise in transmittance between 400 and 480 m/t. It showed a continuous rise in transmittance at a slow rate between 480 and 800 mil. but exhibited no selective absorption bands. It should not be concluded from Figure 3 that the white shell transmitted more light than the shell membrane. The reference standard for the shell was opal
glass which had a fairly flat transmittance curve at about 6.5 percent against air. Shell transmittance would be of the order of 2 percent compared to air at 100 percent. The reduced light transmittance by the shell and albumen and reflectance by the yolks below 500 m/t. is of interest. Since blood in the albumen showed the strongest absorption at 415 m/x. attempts were made to detect the presence of blood in intact eggs by making light transmittance measurements near 415 m/x. However, the low order of transmittance and reflectance of normal components below 500 m/x. made it necessary to use Spectral band widths too wide to show selective absorption at 415 m/j. when blood was present. The necessity for very wide spectral band widths below 500 m/i> became apparent when the spectral transmittance curves for intact eggs were obtained (Figure 4). The curves in Figure 4 show, however, that the presence of blood does produce detectable effects on
60
50
CURVES No. 1 2 3 4
NORMAL EGG EGG CONTAINING BLOOD SPOT EGG CONTAINING BLOODY WHITE REFERENCE STANDARD ( 7 Thicknesses of Opol Gloss)
\, / /
£•
40
30
3
20
10
n 400
=~**^- -J500 600 WAVELENGTH IN MILLIMICRONS
4_
700
FIG. 4. Spectral transmittance of intact whiteshell eggs. Curves 1, 2 and 3 were obtained using the seven thicknesses of opal glass as a reference standard. Curve 4 is the spectral transmittance of this reference standard relative to air.
Downloaded from http://ps.oxfordjournals.org/ by guest on June 12, 2015
"
359
360
A. W. BRANT, K. H. NORRIS AND G. CHIN
A. B. C. D. E. F. G.
Zirconium-arc light source Kolle flask filled with filter solution Monochromator Egg in front of exit slit of monochromator Phototube housing Recording millivoltmeter and sensitivity switching circuit Drive motor for wavelength scale
light transmittance through intact eggs. These effects appear to peak near 575 m/i. which agrees with the light absorption characteristics of blood shown in the albumen and yolk curves (Figures 1 and 2). From the foregoing observations it appeared feasible to detect the presence of blood in an intact egg by measuring relative light transmittance at 575 m/*. In order to interpret light transmittance levels at 575 m/i. however, a reference standard was required. The curve for the normal4 egg in Figure 4 is only a typical
example. Different eggs were found to have widely varying levels of transmittance even though the curve structures were similar. For this reason it did not seem likely that comparison with a fixed reference standard (such as opal glass) would be practical except through the laborious process of point by point comparisons used for obtaining the spectral curves. Recording spectrophotometers 4 "Normal" refers to eggs containing no blood spots larger than 1/32 inch in diameter.
Downloaded from http://ps.oxfordjournals.org/ by guest on June 12, 2015
FIG. 5. Instrumentation used in the detection of blood in intact white-shell eggs.
361
SPECTROPHOTOMETRY DETECTION OF SPOTS IN EGGS
The operating procedure was as follows: an egg was placed in the modified cell holder of the spectrophotometer (see Figure 5) and the wavelength drive was
120
no
04
350
1
i
1
1
.
*
1
400
450
500
550
600
650
700
WAVELENGTH IN M I L L I M I C R O N S
FIG. 6. Spectral characteristics of three elements of the blood-detection apparatus.
set at 585 mji. The slit width6 was adjusted to give a phototube output current of 8 microamperes with the recorder sensitivity set to read 10 microamperes full scale. In this way the energy transmitted at 585 ran. was standardized for each egg to give the same initial chart reading. The chart drive of the recorder and the motor activating the wave length drive of the spectrophotometer were started simultaneously. During the 20 seconds required for the spectrophotometer to scan from 585 to 565 m/t. the recorder chart moved 3f°. Figure 7 is a reproduction of a portion of a recorder chart showing several typical curves. Curves. 1 and 2 show the range usually encountered for normal white-shell eggs. Curve 3 is typical of the curves drawn when an egg contained a blood spot. Frequently, however, curves similar to 4 and 5 were obtained but no blood was found in the egg. It was decided therefore that 5
Slit width was found to be influenced to some extent by the amount of blood present in the egg. However, for the measurements reported here the spectral band widths were less than 5 m/t. except for the very bloody eggs. A spectral band width of not more than 5 ntyi. gave the best detection of small amounts of blood.
Downloaded from http://ps.oxfordjournals.org/ by guest on June 12, 2015
produce spectral curves automatically but none of the instruments presently available have sufficient sensitivity for measurements through an intact egg. The method finally adopted was to arrange for automatic recording of relative light transmittance of intact eggs through the spectral region between 585 and 565 mp. This region covered the "blood sensitive" wave length of 575 mju. By adjusting the transmittance of each egg to a predetermined level at 585 m/x. the comparative transmittance at 575 m/i. consistently indicated whether or not blood was present. The instrumentation developed (see Figure 5) consists of the previously described modifications to the DU Spectrophotometer with the addition of a low-speed motor drive on the wavelength scale. The shape of the curve drawn by the recorder is a function of the spectral output of the light source, the dispersion characteristics of the prism, the transmittance characteristics of the egg and the spectral sensitivity of the phototube. For convenience it was decided to attempt to arrange the system so that the curves for normal eggs would have transmittance curves between 585 and 565 m/*. that would move upward from the starting point at 585 m/u. This was done by inserting an appropriate filter in the light source. The filter was developed by filling a Kolle culture flask with a mixture of aqueous solutions of light green SF and metanil yellow. The concentration of each of these dyes was adjusted empirically until the desired curves for normal eggs were obtained on the recorder. Figure 6 shows the spectral characteristics of certain portions of the system.
362
A. W. BRANT, K. H. NOEEIS AND G. CHIN WAVELENGTH
IN
MILLIMICRONS
CHART ROTATION
in addition to showing reduced transmittance at 575 nut. the curve must show an upward trend between 575 and 565 nut.; curves 6 and 7 are examples. A total of 600 eggs have been scanned by this apparatus and opened for examination of the contents. The results are shown in Table 1. These data show that blood was indicated in only one egg that contained no blood. When blood was present in spots less than f inch in diameter its presence was seldom indicated. All eggs containing blood spots larger than | inch were identified by the machine except in 4 eggs out of 207.
The presence of blood in eggs detracts appreciably from their market value. Because present candling procedures are inaccurate, instruments were developed that will detect blood in intact white-shell eggs without the aid of the human eye. The spectral characteristics of intact eggs and egg components were determined. When blood was present the wave lengths of light near 575 nut. were consistently absorbed. Instrumentation was developed in which intact eggs could be automatically scanned between 585 and 565 nut. and the relative transmittance recorded. From the recorded curves on 600 eggs it was possible to segregate eggs containing no blood with 99.7 percent accuracy. Eggs containing blood spots over § inch in diameter or with bloody whites were identified with 98.1 percent accuracy. Only 28.6 percent of the eggs containing blood spots less than j inch in size were identified. REFERENCES Card, L. E., and A. V. Nalbandov, 1944. Controlling blood and meat spots. Poultry Sci. 23: 551. Denton, C. A., 1947. Observations on the incidence and characteristics of blood and meat spots in hens' eggs. Poultry Sci. 26: 272-276. Dooley, W. D., 1943. Method and apparatus for de-
TABLE 1.—Accuracy of detecting blood in white-shell eggs by spectrophotometry means
Class
Clear eggs Bloody eggs Slightly bloody eggs All bloody eggs All eggs
Description*
Number of eggs
Spectrophotometric indication Correct
Incorrect
Eggs containing no blood or containing no spots larger than 1/32 inch in diameter.
379
378
% 99.7
1
Eggs containing blood spots larger than 5 inch in diameter. Includes bloody whites.
207
203
98.1
4
Eggs containing blood spots between 1/32 and J inch in diameter.
14
4
28.6
10
"Bloody" and "slightly bloody" classes combined.
221
207
93.7
14
600
585
97.5
15
All eggs were broken-out and classified by examination of the contents.
Downloaded from http://ps.oxfordjournals.org/ by guest on June 12, 2015
FIG. 7. Reproduction of a segment of a chart from the recording millivoltmeter. Each curve represents the results from a different egg. Eggs 1, 2, 4, and S contained no blood. Eggs 3, 6, and 7 contained blood.
SUMMARY
NUNC DIMITTIS
Lerner, I. M., and L. W. Taylor, 1947. Seasonal and daily fluctuations in the incidence of blood spots. Poultry Sci. 26: 662-667. Nalbandov, A. V., and L. E. Card, 1941. Further evidence on the cause and occurrence of blood and meat spots. Poultry Sci. 20: 469. Nalbandov, A. V., and L..E. Card, 1944. The problem of blood clots and meat spots in chicken eggs. Poultry Sci. 23: 170-180. Nalbandov, A. V., and L. E. Card, 1947. The problem of blood and meat spots in chicken eggs. II. Its importance in poultry flocks and a study of the nutritional factors involved. Poultry Sci. 26: 400-409. Quinn, J. P., and A. B. Godfrey, 1940. Inheritance and variation of blood spots in chicken eggs. Poultry Sci. 19: 359-360. Van Wagenen, A., G. O. Hall and H. S. Wilgus, 1937. Variation in egg quahty characters of certain breeds, varieties and strains of chickens. J. Agr. Research 54: 767-777.
N u n c Dimittis
jWarp €ngle ^Pennington
D
R. MARY E. PENNINGTON, the poultry industry's first poultry products research worker, died on December 27, 1952, at St. Luke's Hospital, New York. She was Chief of the Food Research Laboratory of the United States Department of Agriculture from 1907 to 1919. During this time, methods were developed to maintain high quality in eggs, poultry and fish when held at refrigerator or atmospheric temperatures. She had been an independent consultant in the food field since 1922, with offices in the Woolworth Building. Dr. Pennington served with the War Food Administration in World War I, and
as special advisor to the U. S. Quartermaster General's Office in World War II. Some 30 bulletins were edited by Dr. Pennington while Head of the U.S.D.A. Food Research Laboratory. During this period, the first egg quality charts were prepared, and the first specific instructions on the proper dressing, packing and shipping of poultry were developed. For her outstanding service during World War I, she received the Notable Service Medal from the United States Government. In 1940 she received the Francis Garvan Gold Medal from the American Chemical Society.
Downloaded from http://ps.oxfordjournals.org/ by guest on June 12, 2015
tecting the presence of blood in an egg. U. S. patent 2,321,899. Hainan, E. T., and H. D. Day, 1935. An analysis of some egg faults. J. Min. Agr. (London) 42: 236 and 326. Jeffrey, F. P., 1945. Blood and meat spots in chicken eggs. Poultry Sci. 24: 363-374. Jeffrey, F. P., and J. Pino, 1943. The effects of heredity and of certain environmental factors on the incidence of blood spots in chicken eggs. Poultry Sci. 22: 230-234. Jensen, L. S., E. A. Sauter and W. J. Stadelman, 1952. The detection and disintegration of blood spots as related to age of eggs. Poultry Sci. 31: 381-387. Lerner, I. M., 1946. The incidence of blood spots in eggs and its relation to first year mortality. Poultry Sci. 25: 392-394. Lerner, I. M., and W. R. Smith, 1942. Effect of season and heredity on the incidence of blood spots. Poultry Sci. 21:473.
363
Agricultural Research Administration, U. S. Department of Agriculture (Received for publication August 29, 1952)
T
1
Bureau of Animal Industry, Beltsv He, Maryland. 2 Bureau of Plant Industry, Soils, and Agricultural Engineering, Beltsville, Maryland. 357
are mistakenly candled out. Candling accuracy with respect to blood- and meatspot detection reported in the literature indicates a range of accuracy of 20 to 90 percent (Nalbandov and Card, 1944; Denton, 194.7; Jensen et al., 1952). At least one device has been patented (Dooley, 1943) with the claim that it detects blood in shell eggs by a spectrophotometric method. No published data has come to the attention of the authors concerning the performance of the device. It has been found in the studies reported here that blood in white-shell eggs exhibits selective light-absorbing properties. Through appropriate instrumentation it has been found that the presence of blood in an egg can be detected without the aid of the human eye. PROCEDURE
All light transmittance and reflectance data were obtained using a Beckman Model DU Spectrophotometer.3 In measuring the transmittance of egg albumen, 1 centimeter cuvettes were used with the spectrophotometer unmodified. For the yolk reflectance measurements the intact yolk was placed in a short section of 1inch diameter glass tubing with a Number 7 rubber stopper in the bottom end. The top of this receptacle was covered with a thin cover glass and the rubber stopper was pushed upward until the yolk flattened against the cover glass. Several yolks prepared in this manner were measured in the reflectance attachment of 3
Mention of this and other equipment does not imply endorsement by the U. S. Government.
Downloaded from http://ps.oxfordjournals.org/ by guest on June 12, 2015
HE most common defects found in eggs are blood and meat spots. Published data has shown the incidence of such defects to range from less than 1 percent to nearly 100 percent (Hainan and Day, 1935; Van Wagenen et al., 1937; Quinn and Godfrey, 1940; Nalbandov and Card, 1941; Lerner and Smith, 1942; Jeffery and Pino, 1943; Card and Nalbandov, 1944; Jeffrey, 1945; Lerner, 1946; Nalbandov and Card, 1947; Denton, 1947; Lerner and Taylor, 1947). The extent and degree of consumer dissatisfaction created by the presence of blood and meat spots is not known precisely. It seems safe to assume that spots of almost any size that are the color of fresh blood are offensive to many consumers. Blood spots larger than oneeighth inch in diameter or bloody whites can be considered rather generally unacceptable. In the opinion of the authors, it is not safe to assume that "meat" spots have any well denned effect upon consumer acceptance. Whenever the presence of a spot is detected in a shell egg by candling, the egg is usually given a lowered grade or declared inedible. Candling, however, is an imperfect method of detecting blood and meat spots. The result is a monetary loss to the industry through consumer dissatisfaction with defective eggs that are passed and through loss to producers and handlers of eggs when non-defective eggs
358
A. W. BRANT, K. H. NOREIS AND G. CHIN
^
-——^
Sjf~~
•
CURVES
/
z
\
No. 1 2
CLEAR BLOODY
Reference
600
800
WAVELENGTH IN
AL9UMEN ALBUMEN
Std - Distilled Water
1000 MILLIMICRONS
FIG. 1. Spectral transmittance of clear and bloody egg albumen. Blended samples of total albumen were measured in 1 centimeter cuvettes.
No. I 2 3 4
CURVES DARK YOLK "WHITE" YOLK MEDIUM-COLORED YOLK WITH BLOOD SPOT REFERENCE STANDARD (Opal Glass aver Yellow Paper)
60 3 50 40 30
u 20 10
400
500 WAVELENGTH
600 IN
700
MILLIMICRONS
FIG. 2. Spectral reflectance (45°) of egg yolks. Curves 1, 2 and 3 were obtained using the opal glass over yellow paper as a reference standard. Curve 4 is the spectral reflectance of this reference standard relative to powdered MgCOa.
varied in age from one day to several weeks and had undergone varying degrees of deterioration. RESULTS AND DISCUSSION
Transmittance curves for normal and bloody albumen are shown in Figure 1. These* curves were obtained from blended samples of total albumen from several eggs. The presence of blood in the albumen produced absorption bands peaking at 415, 541 and 575 imt. but appeared to have little effect on light transmittance at other wave lengths. Reflectance curves for yolks are shown in Figure 2. As expected yolk color had a pronounced effect upon the curves in the visible region. Curve 1 was obtained from a yolk produced by birds fed a diet containing fairly large quantities of yellow corn and alfalfa meal. It was somewhat darker than the yolk color usually produced by confined birds fed ordinary commercial rations. Curve 2 was obtained from a yolk produced by birds fed a diet
Downloaded from http://ps.oxfordjournals.org/ by guest on June 12, 2015
the DU Spectrophotometer using a piece of opal glass backed by yellow paper as a reference standard. The transmittance measurements of the shell and shell membrane were made by mounting the samples in specially prepared holders to support the sample between the exit slit and the phototube in the spectrophotometer. In order to obtain enough sensitivity to measure the spectral transmittance of intact eggs with a reasonably small spectral band width the spectrophotometer was modified as follows: The phototube and its current-measuring circuit were replaced by an RCA 5819 photomultiplier tube operating through a sensitivity-switching circuit into a Leeds and Northrup Speedomax millivolt recorder. The tungsten source normally used in the visible region was replaced by a 300-watt zirconium-arc lamp. A special sample holder was constructed to accommodate an intact egg. The measurements were all made in a room with the air conditioned at 75 + 2°F. and less than 50 percent R.H. The eggs and egg components were brought to room temperature before measurements were made. The eggs used in these studies
SPECTROPHOTOMETER: DETECTION OF SPOTS IN EGGS
CURVES
45
• No 1 2
WHITE SHELL REFERENCE STANDARD (Opol Class)
3
SHELL
MEMBRANE
lil o
z
< 1t-
l_^-^^"^
j 30 CO
z < a: H 1Z Ul
•
-*-.'•-
£ 15 UJ 0-
3 z n-
400
500 600 WAVELENGTH IN MILLIMICRONS
700
FIG. 3. Spectral transmittance of a white egg shell (curve 1) and shell membrane (curve 2). The reference standard for curve 1 was a piece of opal glass. The spectral transmittance of this reference standard and the shell membrane were measured relative to air.
containing no yellow pigments. In comparison with normally colored yolks, it was called "white." When a blood spot was present on a yolk in the area viewed by the instrument, there was a reduced reflectance of all wave-lengths of light between 540 and 620 m/x. In the region from 570 to 600 m/x. the effect of the blood spot was considerably greater than the effect of yolk color. The transmittance characteristics of a white egg shell and a shell membrane are shown in Figure 3. The shell membrane transmitted all wave-lengths equally well but at very low levels. The white shell showed a very rapid rise in transmittance between 400 and 480 m/t. It showed a continuous rise in transmittance at a slow rate between 480 and 800 mil. but exhibited no selective absorption bands. It should not be concluded from Figure 3 that the white shell transmitted more light than the shell membrane. The reference standard for the shell was opal
glass which had a fairly flat transmittance curve at about 6.5 percent against air. Shell transmittance would be of the order of 2 percent compared to air at 100 percent. The reduced light transmittance by the shell and albumen and reflectance by the yolks below 500 m/t. is of interest. Since blood in the albumen showed the strongest absorption at 415 m/x. attempts were made to detect the presence of blood in intact eggs by making light transmittance measurements near 415 m/x. However, the low order of transmittance and reflectance of normal components below 500 m/x. made it necessary to use Spectral band widths too wide to show selective absorption at 415 m/j. when blood was present. The necessity for very wide spectral band widths below 500 m/i> became apparent when the spectral transmittance curves for intact eggs were obtained (Figure 4). The curves in Figure 4 show, however, that the presence of blood does produce detectable effects on
60
50
CURVES No. 1 2 3 4
NORMAL EGG EGG CONTAINING BLOOD SPOT EGG CONTAINING BLOODY WHITE REFERENCE STANDARD ( 7 Thicknesses of Opol Gloss)
\, / /
£•
40
30
3
20
10
n 400
=~**^- -J500 600 WAVELENGTH IN MILLIMICRONS
4_
700
FIG. 4. Spectral transmittance of intact whiteshell eggs. Curves 1, 2 and 3 were obtained using the seven thicknesses of opal glass as a reference standard. Curve 4 is the spectral transmittance of this reference standard relative to air.
Downloaded from http://ps.oxfordjournals.org/ by guest on June 12, 2015
"
359
360
A. W. BRANT, K. H. NORRIS AND G. CHIN
A. B. C. D. E. F. G.
Zirconium-arc light source Kolle flask filled with filter solution Monochromator Egg in front of exit slit of monochromator Phototube housing Recording millivoltmeter and sensitivity switching circuit Drive motor for wavelength scale
light transmittance through intact eggs. These effects appear to peak near 575 m/i. which agrees with the light absorption characteristics of blood shown in the albumen and yolk curves (Figures 1 and 2). From the foregoing observations it appeared feasible to detect the presence of blood in an intact egg by measuring relative light transmittance at 575 m/*. In order to interpret light transmittance levels at 575 m/i. however, a reference standard was required. The curve for the normal4 egg in Figure 4 is only a typical
example. Different eggs were found to have widely varying levels of transmittance even though the curve structures were similar. For this reason it did not seem likely that comparison with a fixed reference standard (such as opal glass) would be practical except through the laborious process of point by point comparisons used for obtaining the spectral curves. Recording spectrophotometers 4 "Normal" refers to eggs containing no blood spots larger than 1/32 inch in diameter.
Downloaded from http://ps.oxfordjournals.org/ by guest on June 12, 2015
FIG. 5. Instrumentation used in the detection of blood in intact white-shell eggs.
361
SPECTROPHOTOMETRY DETECTION OF SPOTS IN EGGS
The operating procedure was as follows: an egg was placed in the modified cell holder of the spectrophotometer (see Figure 5) and the wavelength drive was
120
no
04
350
1
i
1
1
.
*
1
400
450
500
550
600
650
700
WAVELENGTH IN M I L L I M I C R O N S
FIG. 6. Spectral characteristics of three elements of the blood-detection apparatus.
set at 585 mji. The slit width6 was adjusted to give a phototube output current of 8 microamperes with the recorder sensitivity set to read 10 microamperes full scale. In this way the energy transmitted at 585 ran. was standardized for each egg to give the same initial chart reading. The chart drive of the recorder and the motor activating the wave length drive of the spectrophotometer were started simultaneously. During the 20 seconds required for the spectrophotometer to scan from 585 to 565 m/t. the recorder chart moved 3f°. Figure 7 is a reproduction of a portion of a recorder chart showing several typical curves. Curves. 1 and 2 show the range usually encountered for normal white-shell eggs. Curve 3 is typical of the curves drawn when an egg contained a blood spot. Frequently, however, curves similar to 4 and 5 were obtained but no blood was found in the egg. It was decided therefore that 5
Slit width was found to be influenced to some extent by the amount of blood present in the egg. However, for the measurements reported here the spectral band widths were less than 5 m/t. except for the very bloody eggs. A spectral band width of not more than 5 ntyi. gave the best detection of small amounts of blood.
Downloaded from http://ps.oxfordjournals.org/ by guest on June 12, 2015
produce spectral curves automatically but none of the instruments presently available have sufficient sensitivity for measurements through an intact egg. The method finally adopted was to arrange for automatic recording of relative light transmittance of intact eggs through the spectral region between 585 and 565 mp. This region covered the "blood sensitive" wave length of 575 mju. By adjusting the transmittance of each egg to a predetermined level at 585 m/x. the comparative transmittance at 575 m/i. consistently indicated whether or not blood was present. The instrumentation developed (see Figure 5) consists of the previously described modifications to the DU Spectrophotometer with the addition of a low-speed motor drive on the wavelength scale. The shape of the curve drawn by the recorder is a function of the spectral output of the light source, the dispersion characteristics of the prism, the transmittance characteristics of the egg and the spectral sensitivity of the phototube. For convenience it was decided to attempt to arrange the system so that the curves for normal eggs would have transmittance curves between 585 and 565 m/*. that would move upward from the starting point at 585 m/u. This was done by inserting an appropriate filter in the light source. The filter was developed by filling a Kolle culture flask with a mixture of aqueous solutions of light green SF and metanil yellow. The concentration of each of these dyes was adjusted empirically until the desired curves for normal eggs were obtained on the recorder. Figure 6 shows the spectral characteristics of certain portions of the system.
362
A. W. BRANT, K. H. NOEEIS AND G. CHIN WAVELENGTH
IN
MILLIMICRONS
CHART ROTATION
in addition to showing reduced transmittance at 575 nut. the curve must show an upward trend between 575 and 565 nut.; curves 6 and 7 are examples. A total of 600 eggs have been scanned by this apparatus and opened for examination of the contents. The results are shown in Table 1. These data show that blood was indicated in only one egg that contained no blood. When blood was present in spots less than f inch in diameter its presence was seldom indicated. All eggs containing blood spots larger than | inch were identified by the machine except in 4 eggs out of 207.
The presence of blood in eggs detracts appreciably from their market value. Because present candling procedures are inaccurate, instruments were developed that will detect blood in intact white-shell eggs without the aid of the human eye. The spectral characteristics of intact eggs and egg components were determined. When blood was present the wave lengths of light near 575 nut. were consistently absorbed. Instrumentation was developed in which intact eggs could be automatically scanned between 585 and 565 nut. and the relative transmittance recorded. From the recorded curves on 600 eggs it was possible to segregate eggs containing no blood with 99.7 percent accuracy. Eggs containing blood spots over § inch in diameter or with bloody whites were identified with 98.1 percent accuracy. Only 28.6 percent of the eggs containing blood spots less than j inch in size were identified. REFERENCES Card, L. E., and A. V. Nalbandov, 1944. Controlling blood and meat spots. Poultry Sci. 23: 551. Denton, C. A., 1947. Observations on the incidence and characteristics of blood and meat spots in hens' eggs. Poultry Sci. 26: 272-276. Dooley, W. D., 1943. Method and apparatus for de-
TABLE 1.—Accuracy of detecting blood in white-shell eggs by spectrophotometry means
Class
Clear eggs Bloody eggs Slightly bloody eggs All bloody eggs All eggs
Description*
Number of eggs
Spectrophotometric indication Correct
Incorrect
Eggs containing no blood or containing no spots larger than 1/32 inch in diameter.
379
378
% 99.7
1
Eggs containing blood spots larger than 5 inch in diameter. Includes bloody whites.
207
203
98.1
4
Eggs containing blood spots between 1/32 and J inch in diameter.
14
4
28.6
10
"Bloody" and "slightly bloody" classes combined.
221
207
93.7
14
600
585
97.5
15
All eggs were broken-out and classified by examination of the contents.
Downloaded from http://ps.oxfordjournals.org/ by guest on June 12, 2015
FIG. 7. Reproduction of a segment of a chart from the recording millivoltmeter. Each curve represents the results from a different egg. Eggs 1, 2, 4, and S contained no blood. Eggs 3, 6, and 7 contained blood.
SUMMARY
NUNC DIMITTIS
Lerner, I. M., and L. W. Taylor, 1947. Seasonal and daily fluctuations in the incidence of blood spots. Poultry Sci. 26: 662-667. Nalbandov, A. V., and L. E. Card, 1941. Further evidence on the cause and occurrence of blood and meat spots. Poultry Sci. 20: 469. Nalbandov, A. V., and L..E. Card, 1944. The problem of blood clots and meat spots in chicken eggs. Poultry Sci. 23: 170-180. Nalbandov, A. V., and L. E. Card, 1947. The problem of blood and meat spots in chicken eggs. II. Its importance in poultry flocks and a study of the nutritional factors involved. Poultry Sci. 26: 400-409. Quinn, J. P., and A. B. Godfrey, 1940. Inheritance and variation of blood spots in chicken eggs. Poultry Sci. 19: 359-360. Van Wagenen, A., G. O. Hall and H. S. Wilgus, 1937. Variation in egg quahty characters of certain breeds, varieties and strains of chickens. J. Agr. Research 54: 767-777.
N u n c Dimittis
jWarp €ngle ^Pennington
D
R. MARY E. PENNINGTON, the poultry industry's first poultry products research worker, died on December 27, 1952, at St. Luke's Hospital, New York. She was Chief of the Food Research Laboratory of the United States Department of Agriculture from 1907 to 1919. During this time, methods were developed to maintain high quality in eggs, poultry and fish when held at refrigerator or atmospheric temperatures. She had been an independent consultant in the food field since 1922, with offices in the Woolworth Building. Dr. Pennington served with the War Food Administration in World War I, and
as special advisor to the U. S. Quartermaster General's Office in World War II. Some 30 bulletins were edited by Dr. Pennington while Head of the U.S.D.A. Food Research Laboratory. During this period, the first egg quality charts were prepared, and the first specific instructions on the proper dressing, packing and shipping of poultry were developed. For her outstanding service during World War I, she received the Notable Service Medal from the United States Government. In 1940 she received the Francis Garvan Gold Medal from the American Chemical Society.
Downloaded from http://ps.oxfordjournals.org/ by guest on June 12, 2015
tecting the presence of blood in an egg. U. S. patent 2,321,899. Hainan, E. T., and H. D. Day, 1935. An analysis of some egg faults. J. Min. Agr. (London) 42: 236 and 326. Jeffrey, F. P., 1945. Blood and meat spots in chicken eggs. Poultry Sci. 24: 363-374. Jeffrey, F. P., and J. Pino, 1943. The effects of heredity and of certain environmental factors on the incidence of blood spots in chicken eggs. Poultry Sci. 22: 230-234. Jensen, L. S., E. A. Sauter and W. J. Stadelman, 1952. The detection and disintegration of blood spots as related to age of eggs. Poultry Sci. 31: 381-387. Lerner, I. M., 1946. The incidence of blood spots in eggs and its relation to first year mortality. Poultry Sci. 25: 392-394. Lerner, I. M., and W. R. Smith, 1942. Effect of season and heredity on the incidence of blood spots. Poultry Sci. 21:473.
363