A CHEMICAL AND BACTERIOLOGICAL STUDY OF FRESH

EGGS.’

BY M. E. PENNINGTON.

(Received for publication, November 16, 1909.)

A study of the eggs commonly offered for sale in public markets will readily show that variations exist between those forwhich the same price is charged and which are described under the same label. While literature giving the chemical analysis of the eggs of the common domestic fowls, especially the hen, is fairly plentiful, there are lacking certain data, such as the distribution of the protein forms of nitrogen in egg-white, or the usual fat numbers for the yolk, which are very desirable if comparisons of fresh, high grade eggs, and stale or low grade eggs are to be made. On the other hand, there has been much more done on the phos- phorus compounds, both organic and inorganic, which occur in the yolk of eggs and on the isolation and identification of such substances as cholesterol, certain glycerides, nuclein, etc.; in other words, those substances which would tend to show the presence or absence of eggs when mixed with other materials as in noodles, and tanners’ fine kid dressings.

The presence of bacteria and molds in eggs, their number, distribution and species, is a subject of economic importance as well as of scientific interest, because the keeping quality of an egg depends to a very great extent upon its bacterial content. Our knowledge of egg bacteriology is much more fragmentary than is our knowledge of its chemistry.

Hence, it seems desirable that a careful study be made of fresh eggs of known history-from both a chemical and bacteriological point of view, that there may be a basis of comparison for eggs of unknown history or doubtful quality.

1 Presented at the Congress of Applied Chemistry, held in London, June,

1909. 109

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110 Fresh Eggs

CHEMICAL ASALYSIS OF EGGS.

The earliest analysis of hen’s eggs recorded by Konigl is that made by Bostock of egg-white, about 1855. He determined the amount of water, nitrogen, fat and ash. These constituents have been determined a number of times since then for white and yolk separately and for the entire egg. Lang-worthy2 gives the following figures which may be taken as typical.

TABLE I.

Gross Analysis of Eggs.

Shell.

-

I with shell Whole egg. . . .

r.

11.2 1

White.. . . j without shell

Yolk...........................1 Whole egg boiled. ’ without shell Raw, white shelled egg . 10.7 Raw, brown shelled egg. . . 10.9

Mater.1 NitI- Fat. gen. I

--;----

73.71 13.4ilO.5 86.2i 12.31 0.20 49.51 73.3;

15.7133.3 13.3’ 12.0

65.61 64.8~

11.8~ 10.8 11.91 I 12.2

Ash.

p. c.

0.9 1.0 0.6 1.1 0.8 0.6 0.7

Lebbir? states that of the total weight of an egg the shell is 10.89 per cent, the yolk 15.50 per cent, and the white 29.5 per cent. These figures are based on the examination of six eggs.

Cook,4 gives the distribution of the protein nitrogen in two samples of fresh boiled eggs, whites and yolks separately. The mean of the two analyses follotvs:

r Konig : Chenzie der Menschlichen Nahrungs und Genussmittel, i. p. 98. 2 Langworthy: Farmers Bulletin No. rz8-United States Department

of Agriculture. 3 Lebbin: Zeit. offentl. Chew. vi. p. 148, 1900. 4 Cook: U. S. Department of Agriculture, Bureau of Chemistry, Bulle-

tin No. 115.

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M. E. Pennington

TrlBLE II.

III

~ Total.

Wet Basis Yolk of boiled egg. White of boiled egg.

Dry Basis

2.62 2.479 1.76 1.463

Yolk of boiled egg. 4.953t 4.6831 White of boiled egg. 12.694l 10.537;

bie.

..I-

ngulxble. and Amide.

j peptones. I

0.136 0.049* i 0.092 0.299 0.263* 1 0.036

0.2697 0.0949*: 0.174s 2.1525 1.8036”; 0.2599

* Difhence between uncoagulable and amide nitrogen.

The analytical methods used by Cook are those of the Associa- tion of Official Agricultural Chemists.’ His work was done on a sample from the mixed yolks and whites of four eggs, which, according to the statement of a “reliable local dealer,” were laid on the previous day.

The analyses made in this laboratory were usually on from I 2

to 18 eggs, all less than 48 hours old; generally about 24 hours. They were collected from the nests at short intervals, dated, the hour of collection marked on the shell and then kept in a cool, dark place until the time for analysis. The two breeds, Barred Plymouth Rocks and White Leghorns, were kept separate. We have, therefore, an amplification of Langworthy’s study of dark and light shelled eggs. The quality of the eggs, aside from the question of freshness, was of the best. The hens were young, well and regularly fed, clean, and the nests were clean. The Plymouth Rock eggs were all late summer eggs, September and October, except analysis 184, which was made on March eggs. The Leghorn eggs were early spring eggs, February and March. It is of interest to note that for these well-fed hens there is prac- tically no difference in composition between Fall and Spring eggs. All the chemical analyses, and most of the bacteriological, were made on fertilized eggs. Some bacteriological work has been

1 U. S. Department of Agriculture, Bureau of Chemistry, Bulletin No. I 07. Revised.

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112 Fresh Eggs

done on unfertilized eggs to determine their relative content of organisms.

For the analysis of white and yolk individually it is necessary to separate the two completely. To eliminate all yolk from the white of fresh eggs is a simple matter; to remove the last traces of white which adheres to the yolk is not so simple. The folltiwing scheme, however, serves the purpose, and that it is accurate is indicated by the agreement of the analyses of the yolks given in the tables:

Crack the egg at the equator and separate the white and yolk in the usual housewife fashion, being very careful not to rupture the vitaline membrane.

When as much of the white as possible has been drained off, transfer the yolk, by gently sliding it out of the shell to a piece of clean copper gauze such as is used in the laboratory as a protection from free flames.

With a piece of filter paper absorb around the edges of the yolk, but without touching it, as much of the white as possible; and also take off in this way the white which is underneath the egg.

With a very gentle stream from a wash bottle wash the yolk. This can be done without breaking the membrane. Every trace of the white can be so removed and by very gently rollin g the side of the yolk next to the copper gauze can be brought uppermost, and washed. Filter paper will absorb the excess of moisture.

Then with exceeding care the almost dry yolk can be rolled upon a piece of filter paper and the drying completed, when it is ready for analysis.

A small amount of white is lost according to this plan, but no attempt has been made in the work being presented to determine the relative amounts of white and yolk. It is the composition of each which is desired.

Water, ether-soluble material and ash in both yolk and white are determined according to the usual official methods. The apportionment of protein nitrogen in the egg-white is determined as that coagulable by heat in a solution faintly acid to litmus, and as non-coagulable by heat in the filtrate from the above. This filtrate is also studied by precipitating the albumoses with zinc sulphate and determining the amino bodies by the method of Bigelow and Cook.’ All nitrogen determinations are made by the Gunning modification of Kjeldahl’s method.

i Bigelow and Cook: Jowx. Amer. Chem. SK, xxiii, p. 1485, 1906.

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M. E. Pennington 113

For the complete coagulation of the higher proteins of egg- white it has been found advisable to dilute with twenty times its lveight of water, make faintly acid with acetic acid, using litmus paper as an indicator and heat on an electric plate at about 90~ C. The volume used is, in the beginning, about IOO cc. The beaker is loosely covered -cvith a watch glass and heating continued until the volume is about 50 cc. This gentle heating causes an even distribution of the coagulum; and the cover, by keeping a moist atmosphere, prevents that which adheres to the walls of the beaker from sticking tightly. The method has been found very satisfactory.

The filtrate from the coagulum is divided into tTvo portions, one of which serves for the estimation of the albumoses, the other for amino bodies. Neither of these determinations by the methods named can be accepted with satisfaction. The albu- moses tend to separate as a very finely divided substance impos- sible to filter out and the amino duplicates show discrepancies which we have been unable to explain. Of course, both forms of nitrogen are present in minute quantities only. It is possible that with aged, or otherwise deteriorated eggs, such determina- tions would have a broader meaning. As a check on the zinc sulphate precipitate the amount of nitrogen thrown dou-n by tannin-salt was determined on the same sample, and the sum of the non-coagulable nitrogenous constituents compared tvith the

TABLE III.

i

,”

16.

z zs

9 8:

!

$? icjl %

-----

No. 176. 12 Leghorn eggs.~ 1.65 ~ 1.46 0.195 0.188 ~ 0.176 1

T,eghorn eggs.’ 1.65 1.49 0.193 0.177 0.179

Xo. 1Sl. 12 A-1.70 1.59 0.112 0.145 0.132 B-1.71 ’ 1.64 0.109 j 0.052 0.062

Ko. 18-4. 12 Plymouth A-1.68 0.152 0.115 : 0.151 Eock. B-l. 67

i 1.51 1.54 0.156

j

I- i 0.126 0.110 :

0.005

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114 Fresh Eggs

amount of nitrogen found by direct determination in the filtrate from the coagulable matter. The tannin-salt should precipitate the albumose and peptone, if the latter is present. Its nitrogen content should, therefore, be as great, or greater, in quantity than the nitrogen content of the zinc sulphate precipitate. In but two out of five determinations was this the case.

The tannin-salt precipitate was washed with a mixture of tannic acid and salt of the same concentration as was used for the protein precipitation and kept cold during the washing. The filtrate was clear and there was nothing to indicate a resolu- tion of the precipitate.

The variation in the amount of nitrogen coagulable by heat is not great, and this, when added to the non-coagulable nitrogen, closely approximates the total nitrogen as actually determined. Such figures may be accepted as reliable and probably represent the actual composition of the fresh egg white. For the further analysis of the non-coagulable proteins better methods must be devised.

The analyses of the white of the egg, as evidenced by 165 Ply- mouth Rock eggs and 69 Leghorn eggs shows that there is prac- tically no difference in their composition. (See Table IV).

The analysis of the yolks of the eggs included the determination of the water, ash, ether extract and total solids. There were also determined the iodine, saponification and Hehner numbers, the acid value and the index of refraction. Certain of these con- stants have been determined for egg oil by Kitt’ and Paladina and Loss0 ;2 and Vignon and Meunie? worked on the yolks of hard boiled eggs which were dried to constant weight at 100~ C., then extracted with chloroform. Such a source cannot be considered to represent the fat of a fresh, unaltered egg.

The preparation of egg yolk for the determination of the con- stants may be accomplished in various ways. The aim should be to extract and dry as completely as possible the fat of the yolk, without altering its composition. Therefore, the heat used must be kept as low as possible and the solvent must not remove non-fatty materials. After numerous trials it was

1 Kitt: Chemiker Zeitung, p. 303, 1897. 2 F’aladino and Losso: Analyst, clxi, 1896. 3 Vignon and Meunier: Collegium, No. 128, p. 32.5, 1904

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M. E. Pennington I’5

decided to dry the yolk separated from the white as previously described by extracting first with absolute alcohol in a Soxhlet. The alcoholic extract was evaporated to dryness and the very small amount of solid material-which was almost fat free-was added to the main portion. This gave a dry, tough mass which was ground in a meat grinder then returned to another Soxhlet and extracted with petroleum ether having a boiling point below 60’ C. until it was fat free. Ordinarily about two days were required for the alcoholic extraction and two for the petroleum ether. The petroleum ether was distilled off, and the residue dried in a vacuum over calcium chloride. The fat so prepared was almost free from inorganic matter. It did not contain the total amount of egg lecithin and the iodine number was much lower than when ether is used as a solvent, but the objectionable features of the latter were so numerous that it was soon discarded. So prepared, too, the values obtained from egg fat are comparable with the other food fats which are commonly extracted by petroleum ether.

A determination of the phosphorus content of the fat extracted according to various methods gave the following:

PeY%t Of 2 5 Yolk, dried two days with alcohol, then extracted two days with

petroleum ether. . . . . . . . . . . . . . . . . . . . . . . 2.2~ Dried two days with alcohol, extracted two days with ethyl ether,

then two days with alcohol.. . . . . . . . 2.95 Dried two days in steam oven, at rooT., extracted two days with

ethyl ether, then two days with alcohol. . . . . . 2.66

Water, solids and total nitrogen in egg-yolk show but slight variation in quantity, neither do the eggs of the two breeds vary. On the other hand, there is a greater difference in the constants, even in the iodine value which is probably the most accurate of all. This value, as well as the others recorded, was deter- mined according to the methods of the Association of Official Agricultural Chemists. The acidity was determined by titra- tion with standard alkali in a hot, alcoholic solution, using phenolphthalein as an indicator. The potassium chloride method was not accurate, in that the fat formed solid particles

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TABL

E IV

.

AXAL

YSIS

O

F FR

ESH

EGG

S.

LEGH

ORN

AND

PLYM

OUT

H RO

CK

BREE

D.

Plym

outh

Ro

ck

Eggs

.

No.

133.

9

eggs

No.

134.

G

eggs

Fr

ozen

.

No.

137.

12

eggs

.

No.

13’1

. 18

cgg

a..

No.

141.

18

egg

s..

so.

145.

1s

eg

gs.

I

X0.

146.

1X

egg

s. Fr

ozen

.

No.

147.

1s

egg

s..

So.

149.

18

egg

s. Fr

ozen

. . .

Whit

e.

.’ 11

.86,

88

.14;

0.

04

0.80

1.

691

1 .4

9 0.

144

Yolk

50.7

4,

49.2

6’ 32

.14

Il.37

2.62

1 W

hite.

.. 11

.76

88.2

4’ 0.

03

’ 0.

70

1.62

1.

51

0.15

4 Yo

lk..

2.62

Whit

e...

12.8

0 87

.20~

0.

02

0.45

1.

82

1.63

0.

167

Yolk

._..

52.3

3 47

.67

32.1

8 1.

31;

2.69

1 I

I

Whit

e...1

12

.40

87.6

0;

31.4

4 O.

OOG~

~ 1

.35

0.66

, Yo

lk 50

.63

49.3

7 :

~ ~

Whit

e...1

12

.28;

0.

02

i 0.

621

1.75

Yolk

. 52

.05’

47.9

5 87

.72i

33.1

2 ~

2.58

’ 2.

72

~ ~

60.9

! 18

l.C

Whit

e...~

12

.72~

87

.28’

1.68

1.

55

~ 0.

06

Yolk

52.3

5 47

.65‘

0.01

61

0.71

’ 0.

076i

0.00

4i 33

.08

11.3

9 2.

76

64.2

: 19

1.1

Whit

e...

12.0

6;

ST.9

41

0.02

0.

351

1.72

1.

42

I Yo

lk ,_

.. 51

.64;

48

.38’

32.3

8 ’

1.34

2.

65

Whit

e...

88.0

4~

0.03

6G

.8~

173.

4 11

.96

0.05

0.

47

‘1.69

1.

35

0 09

I

Talk

._..’

52

.47~

47

.53’

32.9

6 1.F

2 W

hite.

.. 11

.70~

88

.301

0.

04

IO.6

0’ 2.

72

1.67

0.

049

O.OO

G 03

.8

176.

C 0.

06

33.2

9 1

2.45

i

/

Yolk

51.9

8 48

.02,

2.

69

60.3

; 17

Z.t

1 .-H

i-l:%

1.46

52

1.46

48

1.45

57

71.8

1.

4627

~ S3

.58’

1.46

64

~ 73

.4

I 1

4592

71.7

1

~ 75

.46

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No.

152.

18

egg

s. W

hite.

..1

ll.lO

/ 88

.90

0.01

01

Yolk

. . .

. .

52.9

9;

47.0

1 33

.33

No.

184.

12

eggs

.. ,(W

hite.

..i 11

.86

0.01

;

0.76

“I

Yolk

. .

. /

52.5

2 33

.09

12.2

7 2.

721

1.68

i

Max

imum

,..

..,...

. ‘W

hite

.

. . I

12.8

01

88.9

0 0.

05

’ 0.

80

0.19

7 0.

122

0.00

6 M

axim

um.,

Yolk.

. ./

52.9

91

1.d

1.63

1 49

.37

33.3

3 2.

55

2.76

M

iminu

m

..,,..

,,._

Whit

e . .

. .

11.1

0 87

.20

0.00

6 0.

35

1.62

1.

42

0.15

4 0.

049

0.00

4 M

inim

um.

. Yo

lk.

. 50

.63

45.8

8 31

.44

1.31

2.

59

Aver

age.

. .

Whit

e _.

.. 12

.05

87.9

8 0.

0241

32

.70

j 0.

63

1.70

1.

54

0.17

1 0.

075

0.00

5!

Aver

age.

. Yo

lk.

. .

51.9

7 .I

47.8

4 1.

77

2.68

1 /

I

No.

172.

12

egg

s.

No.

173.

12

egg

s.

No.

174.

9

eggs

..

No.

176.

12

egg

s..

No.

177.

12

egg

s.

No.

181.

12

egg

s.

Max

imum

. M

axim

um.

Mini

mum

. M

inim

um.

Aver

age.

. Av

erag

e. .

T ‘: ! .I .! / .I :I _i j

Whit

e..

\Yolk

.. /W

hite.

. \Y

olk..

( W

hite.

. ‘\Y

olk..

/ W

hite.

\Y

olk..

J W

hite.

. \Y

olk..

/Whit

e..

\Yolk

.. W

hite.

. Yo

lk..

Whit

e.

Yolk.

. W

hite.

.

Yolk.

. . -7-

-

12.d

87

24

I 0.

01,

52.4

5 47

.55

33.1

9 11

.97’

88.0

3 0.

01,

52.5

5 47

.46

33.1

0

52.9

71

11.9

51

88.0

91

0.01

47

.071

31

.63

12.6

2i 87

.391

O.

Ol$

52.6

4 12

.19

47.3

71

32.9

0

52.1

5 87

.82i

0.02

47

.86’

31.9

3 12

.06

87.9

4 0.

04

52.6

6 47

.331

33

.12

12.6

2 SS

.OSj

0.

04

52.9

7 47

.861

33

.19

11.9

5’ 87

.39~

0.

01

52.1

5,

47.0

71

31.6

3 12

.151

87

.86:

0.

01’

52.5

71

47.4

41

32.6

5 9

Legh

orn

Eggs

.

I II

/I I

62.2

i 18

4.2

/ ~

/ 77

.95

61.4

1 17

7.0

5.8

171.

2 2.

92

78.8

1

66.8

19

1.1

83.5

8 1.

4664

60.3

17

3.4

71.7

1 1.

4557

62.8

; 17

9.9

5.8

171.

21

2.92

76

.10

1.46

26

0.06

16

4.21

i64.1

/66.

7

165.

5

171.

95.9

183.

96.0

185.

515.

9 I 19

4.9iB

.E

! / /

166.

0 2.

971

73.6

0 I

177.

9 3.

02

78.2

1/

179.

6 2.

97i

83.6

3,

88.8

63.5

18

1.1,

5.2

175.

9

165.

0 17

7.46

.7

170.

7

66.7

‘64.1

164.

8<

194.

9 6.

7

171.

9 5.

2

182.

5/5.

9(

188.

8

’ 16

6.0

2.62

I

73.4

1i

177.

5 3.

011

78.4

11

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118 Fresh Eggs

NITROGENOUS CONSTITUENTS CALCULATED ON WATER-FREE BASIS.

Plymouth Rock Eggs. - .-.-___ I

NUMBER OF BAMPLE.

-- l-

No. 133. 9 eggs. . . . . . . . .

No. 134. 6 eggs. J White..

Frozen. , Yolk.,

No. 137. 12eggs.. . . . . . 1 J White. \ Yolk..

No. 139. 18 eggs. . . { ~o~~~: 1

No. 141. 18eggs.. . . . . j’ White. \ Yolk.

No. 145. j’ White..

18eggs.. . . . , Yolk,

No. 146. 18 eggs. Frozen. . 1 White..

,\ Yolk..

No. 147. 18eggs.. . . . . ( White. \ Yolk. .

No. 149. 18 eggs. Frozen. . . ( White. ‘\ Yolk..

No. 152. 1 White.

18 eggs . , Yolk,

No. 184. 12 eggs. . . J White.

11 Yolk..

No. 172. 12eggs ..........

No. 173. 12 eggs. ..........

No. 174. 9 eggs. ..........

No. 176. 12 eggs. ..........

No. 177. 12eggs ...........

No. 181. 12 eggs. ..........

14.2E 4.77 13.8C

5.0: 14.21

5.11 13.7(

5.1( 14.2:

5.2: 13.2(

5.2! 14.2(

5.0: 14.2

5.1’ 14.21

5.11 15.7

5.01 15.1

5.4

Leghorn Eggs.

jYolk..../ 1 White.

‘iI Yolk.. .

1 White.. _/ \Yolk.....i [White...

iYo1k....l Ii White. ‘;I, Yolk., .)

5.44 14.931 13.71

5.49 14.96/ 5.50: 14.081 0.93f

12.5:

12.8,

12.7

12.4

13.1

12.1

11.7

12.9

13.3

12.8

13.E

1.55

1.31

1.21

1.59

1.39

1.141 0.114

0.478~

0.9221

1.535! I

-

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M. E. Pennington 119

in the liquid and did not permit the alkali to penetrate, yielding thereby results that were too low. For example:

Calculate~~ to Acid value.

fr~co$lc Method. per cent. per Yak.

By potassium chloride 2.7 1.36

By ethyl alcohol.. .5 8 2.()I

BACTERIOLOGICAL EXAMINATIOS OF FRESH EGGS.

That perfectly fresh eggs from healthy hens may contain bacteria is a generally recognized fact. That they are some- times sterile is also admitted. Whether the organisms enter the egg during its passage down the oviduct or whether they pene- trate the shell either at the time of laying or afterward are questions on which opinions are contrary. The fact that certain pathogenic organisms characteristic of fowls, as vibros of chicken cholera, have been found in the egg argues for infection in the oviduct, as do the presence of foreign bodies, such as small insects; while the trade experience indicates that organisms can enter through the shell. A recent publication from Storrs Agri- cultural Experiment Station’ states that organisms enter the egg in both ways. In the experiments described the yolk was broken by shaking and the mixed white and yolk tested. The number of organisms per gram or cubic centimeter is not given.

A more complete study of bacteria in the egg has been made by Pernot ,2 who has examined the eggs from ova the size of a pea to the perfect egg, finding organisms at every stage. He has also studied the permeability of the shell to microorganisms and finds that it is not bacteria-tight. This author has studied especially an organism which is pathogenic to chicks and which he calls “ No. 9.” In addition to this he finds the most common species inhabiting the hen’s egg, both yolk and white, to be B. subtilis, B. rvtycoides and B. mesentericus fuscus.

It has seemed desirable to study from a bacteriological stand- point the whites and yolks separately, determining the num-

1 Bulletin So. 55 : Storr’s LAgricultural Experiment Station. a Pernot: Investigation of the Mortality of Incubator Chicks. Bulletin

No. 103, Oregon &\gric. College, Experiment Station.

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TABL

E;

V.

BACT

ERIO

LOG

ICAL

ST

UD

Y O

F FR

ESH

EGG

S.

PLYM

OUT

H RO

CK

BREE

D

Ferti

lized

Eg

gs.

BPEC

IES

IN

WHI

TE.

I Fr

om

Wrig

ht’s

anae

robic

plates

. Fr

om

aerob

ic pla

tes.

From

ae

robic

plates

.

No.

133.

Plym

outh

Rock

- B.

punc

tiform

is (C

hes

ter)

B.

cutic

ularis

(C

heste

r M

. cin

naba

reus

(F

liig

uei)

M.

a&ius

Le

ptot

hrix

hyali

na

B.

detru

dens

(W

right

Brow

n m

old,

33

pe

cent

of all

colon

ies

Brow

n m

old,

15

pe

cent

of all

colon

ies

B.

cutic

ularis

M

. cin

naba

reus

M.

airius

M

. Da

nticii

34

. fe

rvid

mdA

dam

etz

M.

&ii

(Che

ster)

M.

tena

catis

M

. te

tmge

nous

M

. ca

ndica

ns

(Fliig

gei

+ B.

Fl

ilgge

i (C

heste

r) M

. au

rant

iacus

(S

chae

ter)

- B.

pu

nctifo

rmis

24

0 3

M.

aura

ntiac

us

Bact

. Ma

nsfie

ldii

M.

aura

ntiac

us

B

No.

13%

Plym

outh

Rock

A

2 0

i

M.

orbicu

laris

(Rav

enelf

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B 37

oc.

2 zo

oc.

’ 0

ooc.

0

No.

141

Plym

outh

Ro

cks

37OC

. A

20°C

. 32

0°

C.

5

n 37

0 c.

20

” c.

00

c.

No.

145

Plym

outh

Ro

ck’

37’C

. A

20”

c.

20

55 0 5

32 5 0 56 0

u

No.

146.

Pl

ymou

th

Rock

00 c.

37

0 c.

20

0 c.

oo

c.

3i”

C.

200

c.

0”

c.

No.

147.

Pl

ymou

th

Rock

1 3i’

C.

0 0

A 20

0 c.

11

0

16 6

49

12

0 0 0

0 0 8 0 0 19

0 0

0

--.~

.~

26

0

1G

0 0

0

5

13 2

97

22 0 5 3 4 6

25 8 58

285

0 0 0 0 0 0 0 0 0 0 0 0

-

0 0 0 0

0°C.

0

0 13

, 0’

0 + 0 4 0

+

0

M.

a&&

ill.

einna

bare

us

(Fliig

gei)

M.

ovali

s (E

sohe

rick

M.

ci~nn

abar

eus

(Fliig

gei)

Lept

othr

ix hy

alina

(M

igula)

M.

viticu

losis

(Fliig

gei

Lept

othr

iz hy

alinn

(M

iyla)

&e

t. M

ansfi

eldii

M.

cinna

bare

us

(Fliig

gei)

M.

orbic

ularis

(R

aved

)

M.

awog

enes

(M

iller)

M.

punc

tiform

is

M.

orbic

ularis

(R

aven

el)

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TABL

E V-

Cont

inued

.

WHI

TE.

8m

%F :$

.s

4 -

19

23

15

20

279

121

- -.-

-

BmdP

LE.

From

W

right

’s an

erob

ic pla

tes.

From

W

right

’s an

a&ob

ic pla

tes.

From

a&

robic

pla

tes.

-

f M,

a&&s

’ i Le

ptot

hrir

hyain

d (M

igula)

.- -

-- 0 0 8 6

556 39

-

B

Cora

l ye

ast

Cren

othr

ix po

lyspo

ra

(Coh

n )

M.

ferv

idosu

s (A

da-

met

s)

No.

149.

Pl

ymou

th

Rock

. FlY

X2Xl

Lepo

thrix

hy

laina

(M

igula)

B.

pu

nctifo

rmis

B.

alcali

gene

s (P

etru

M.

orbic

ularis

A

[!I

2yis

B

Lusti

gi

- -

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E

No.

152.

Pl

ymou

th

Rook

A B

No.

184.

Pl

ymou

th

Rock

A B

No.

201.

Pl

ymou

th

Rock

eg

g fro

m

ovi-

duct

. Sh

ell

form

ed

-

370

c. 20

0 c.

00 c

.

370

c. 20

0 c.

0” c

. 37

0 c.

200

c. 00

c.

370

c. 20

” c.

00 c

. 37

0 c.

200

c.

00 c

.

370

c.

200

c,

OO

C.

If - lask

1

Ike

159 36

15

16

42

0 0 0 0 0 0 77 0

0 0 0 0 8 0 0 0 0 0 0 0 0 0

99

19

316

59

32

0

3 0

0 0

0 0

20

26

14

0 15

0

0 0 2 2 39

118 0

0 c

Brow

n m

old

100

pa

cent

/

c

B. p

u?ut

ifor?

nis

M.

cinn

abar

eus

r Bro

wn

mol

d \b

4. lm

ti.9

B.

sicc

w

100

per

cent

~

B.

siceu

s 50

per

ce

nt

1 Br

own

mol

d,

50 p

er

/ ce

nt

I

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TABL

E VI

.

BA

CTE

RIO

LOG

ICA

L S

TUD

Y

OF

FRE

SH

E

GQ

S.

LEG

HO

RN

B

RE

ED

Ferti

lized

Eg

gs.

8PEC

IE8

IN

YOLK

.

-. -- l 1

j

-I-

From

W

right

’s an

aiko

bic

plat

es

From

W

right

’s an

a&ro

bic

plat

es.

From

n&

obic

pl

ates

. Fr

om

atiro

bic

plat

es.

Sbep

toth

rix

chro

mog

enc

(Gas

perin

ii)

50

pe

oent

N.

172.

Le

ghor

n A

M.

orb&

x&w-

is (R

aven

eI

) 334

per

ce

nt

Whi

te

yeas

t, 33

f pe

r ce

nt

Stre

ptot

hrix

aura

ntia

- cu

a-33

8 pe

r ce

nt

0 0

-

’ M.

cinna

bare

us

50 p

er

cent

St

rept

othr

iz ch

rom

ogen

s .

50 p

er

cent

‘M.

mse

ttace

us

(Zim

mer

m

an)

339

per

ten

Lepo

thrix

hy

alin

a (M

igul

a)

33f

per

ten

M.

ferv

idos

us

(Ada

-

i In

&Z)

334

per

cent

B

Whi

te

yeas

t 20

per

ce

nt

cora

l ye

ast

20 p

er

cent

M

. or

bicu

laris

(R

aven

el

) 20

per

ce

nt

M.

Zact

is 20

pe

r ce

nt

Stre

ptot

hrix

albi

do

ross

i do

ria

20 p

er

cent

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I3

No.

174.

Le

ghor

n A

No.

1 i6.

Le

ghor

n A R

No.

17;.

Legh

orn

A

- T I

3i'C.

0

20°C

. 4

00

c.

0

37oc

. ~

5

20°C

. ~

0

0°C.

0

0 0 0

0 0 0 I

O

0

37”

c. /

01

0 2o

oc.

0 0

0

i I 00

c.

3 0

0

37°C

. 13

0

20”

c.

0 0

0”

c.

’ :;

0

3iOC.

12

0

~

11 5 0 0 0 0

20°C

. oo

c. /

0 0 :

0 I

i 0

~ 0

37°C

. 7

0 0

20°C

. : o’

c.l

,“;

0;

; O

1 O

‘0

” __

--

0 O

~ I i

oi 0

0 0 0’ 0

0 0 :

0 ’

0 ~

0

B.

detru

dens

(W

right

) 10

0 pe

r ce

nt

Wllite

ye

ant

50

pet’

rent

W

hite

mol

d 50

pe

r re

nt

B.

plcnc

tijorn

is 50

pe

r ce

nt,

Stre

ptot

hk

farc

inicn

50

pe

r re

nt

M.

tena

catis

10

0 pe

r ce

nt

i Stre

ptot

hrix

jarcin

icn

50

per

cent

St

rept

othr

ix ch

rom

o-

gelza

50

pe

r ce

nt

M.

cand

icans

(F

liig-

gei)

100

per

cent

Stre

ptot

hrix

aura

ntiac

us’

100

per

cent

B.

siccu

s 33

Q pe

r cm

B.

pu

netifo

rmis

(Wrig

ht)3

3*pe

rcen

Le

ptot

hrix

hyali

na

(Wrig

ht)

33Q

perce

n

Stre

ptot

hrix

albido

m

m

doria

10

0 pe

r ce

nt

B.

siccu

s 33

3 pe

r te

n M

. ca

ndica

ns

(Fliig

gei

336

per

cent

St

rept

othr

ir (Is

rael

Krus

e)

33Q

per

oen

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TABL

E VI

-Con

tinue

d.

YOLK

.

NUMB

ER

OF

ORGA

N-

If346

PE

It GR

AM.

From

ae

robic

plates

.

, _-

SAMP

LE.

21

0

From

W

right’

s ma

&rob

ic pla

tes.

From

W

right’

s an

aerob

ic pla

tes.

From

ae

robic

plates

.

--

i

Stre

ptot

hris

aura

ntiac

u.

100

per

cent

B.

siccu

s 33

$ pe

r ten

St

repto

thrix

farcin

ica

333

per

cent

Stre

ptot

hriz

chro

mog

em

(Gas

perin

ii) 33

4 pe

ce

nt

M.

cand

icans

50

pe

r ce

nt St

rept

othr

iz ch

rom

o-

gena

50

per

cent

No.

177.

Legh

orn

B

No.

181.

Legh

orn

A

B.

punc

tifom

is 33

9 pe

r ce

nt

M.

cand

icans

33

f pe

r- ce

nt St

rept

othr

ix fa

rcini

ca

33$

per

cent

Stre

ptot

hriz

farc

inica

10

0 pe

r ce

nt B

M.

cand

icans

33

4 pe

r ce

nt Br

own

mold

33

4 pe

r ce

nt M

. au

rantia

cus

333

pe

cent

r -

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No.

202.

Le

ghor

n

A B

No.

205.

Le

ghor

n A B

No.

209.

Le

ghor

n

.4

B c

No.

216.

Le

ghor

n A B

370

c.

200

c.

0”

c.

370

c.

200

c.

00 c

.

370

c.

200

c.

00 c

. 37

0 c.

20

” c.

0°

C.

37”

c.

200

c.

00 c

.

370

c.

200

c.

0°C.

370

c.

200

c.

00

c.

370

c.

200

c.

00

c.

370

c.

200

c.

00 c

.

0 5 1 0 2 0 0 0 2 0 0 0 11 0 0 0 0 0 3 0 0 0 0 0 10 0 5

0 ~

0)

0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 a 0 0 0 0

41

0 2:

0

0:

0 29

8 0 6 2 0 2 12 0 12 2 0

10 5 0 12 0 0 1 0 0 6 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

f M

. au

rant

iacus

10

0

1 pe

r ce

nt

Whit

e m

old

100

per

cent

Gree

n m

old

100

per

cent

Lept

othr

is hy

alina

40

pe

r ce

nt

I

B.

punc

tiform

is 60

pe

r ce

nt

M.

VitiCUhiS

100

,,W

per

cent

B.

detru

dens

(W

right

,) 10

0 pe

r ce

nt

Cora

l ye

ast

100

per

cent

B.

sicm

s 10

0 pe

r ce

nt.

Brow

n m

old

100

per-

cent

B.

siccu

s 10

0 pe

r ce

nt

B.

gin&

mm

(R

aven

el)

50

per

cent

B.

sic

cus

50

per

cent

Brow

n m

old

100

per

cent

/

1 M

. cin

naba

rewr

10

0 pe

r,

‘\ ce

nt

Brow

n m

old

100

perc

ent

I B.

siccu

s 50

pe

r ce

nt

M.

cinna

bare

us

50

per

cent

B.

siccu

s 10

0 pe

r ce

nt

i

B.

detru

dens

(W

right

s)!

50

per

cent

I

/ Br

own

mol

d 5O

pero

ent

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0 0 0 0 0 0 0

0 0 0 0 0 0 C

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- - -.

330003006 I

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130 Fresh Eggs

ber and also the kinds of organisms present and the relation which they bear, both numerically and specifically, to the tem- perature at which the cultures are incubated. Such information is of practical importance since the preservation of eggs by cold has become a universal method for the supplying of the market during the non-laying period.

The technique required for the study of the bacteria of white and yolk separately is simple. Because of the inaccuracy of measuring from small pipettes such viscous materials as egg yolk and white a weight basis was adopted and obtained as described below:

Scrub the egg well in clean water. Then soak in bichloride, I to IWO for a few minutes. Wash off the e gg with sterile water and place upright in a suitable holder. With sterile instruments crack the end andwith sterile forceps remove small pieces of shell without rupturing the egg membrane below, until a sufficient space is made to introduce a sterile pipette.

Rupture the shell membrane with sterile forceps and with a sterile pipette withdraw about 2 cc. of the white. Place this in a small tared flask containing broken glass, and reweigh. Add IO cc. of physiological salt solution and shake for ten minutes. The glass cuts the white of the egg and the solution is fairly satisfactory. Plate definite volumes as USUd.

Pipette off, so far as possible, the white of the egg leaving the yolk unbroken. With a sterile wide-mouthed pipette puncture the vitaline membrane and withdraw about 2 cc. of yolk, which is placed in a tared flask and treated exactly as the white.

Equal portions from three eggs were mixed and examined at the same time, the A and B lots in Table V representing, there- fore, 6 eggs of the same sample.

The media used were plain nutrient agar, litmus-lactose-agar, and nutrient gelatin. Plates were grown with and without air access that anaerobes might develop should they be present. They were incubated at three temperatures; namely, 37’, 20’

and o” C., and counts made of the colonies developing after periods of 72 hours, 2 weeks and 6 weeks, respectively. An endeavor has been made, also, to isolate and identify the different species occur- ring in both yolks and whites, and, as will be observed in the table, the relative proportions in which various species occur have been noted in a number of cases.

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M. E. Pennington 131

The tabulated findings show (Table VI) that organisms are usually to be found in both yolk and white. In the 57 experi- ments 18 had a decidedly greater number of bacteria in the yolk; I I had the majority in the white and 21 had an almost even distribution; 7 were sterile. All the Plymouth Rock eggs except sample No. 184 were laid during September and October, and these eggs contained a greater number of bacteria than the early spring eggs, as evidenced by the Leghorn eggs and the one sample of Plymouth Rock eggs above noted. It may be that this differ- ence in bacterial content depends on the breed and the conditions as well as the season.

The tabulated findings of the eggs of the Leghorn chickens show about the same number of organisms in the fertilized and unfertilized specimens. Whether this is a function of breed or of season remains to be determined. It is of interest to note, how- ever, that the fertilized Leghorn eggs were from the same farm, and laid under the same conditions, as the fertilized Plymouth Rock eggs. The unfertilized Leghorn eggs were from a neighbor- ing farm where conditions were similar, yet not exactly the same.

The number of species of bacteria found is noteworthy-36 in the IOO eggs from which the varieties were isolated. A list of them follows :

B. punctiJormis B. cwtic&ris 8. cin?zabareus H Flztggei 8. Ravesccns B. az4ra+&acus

H. alcaligcnes (Petruschky) B. sicc~s H. detrudens (Wright) B. ginglymzu (Ravenel)

M. ciwzabarerw (Fluggei) Al. aeriits Ad. awantiacm M. Da&l-ii M. /erz@dows (.Idametz) M. al&

M. tenacatis M. tetragenzts AT. candicans (Fluggei)

M. orbicularis (Ravenel) Al. o&is (Escherick) M. viticulosis (Fluggei) M. a&rognes (Miller) 114. versicolor (Fluggei) M. punctiformis M. lactis Ad. Lmtigii M. rosettaceus (Zimmermann)

Streptothriz chromogena (Gasperinii) Streptothrix aurantiacsus Streptotlzrix albido roasi doria Streptotlzrix farcinic Streptothrix Israel Kruse Crenothrix polyspora (Cohn) Leptothrix hyalina (Migula)

Molds.-Brown, white and green.

Yeasts.--Coral and white.

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132 Fresh Eggs

A study of eggs which are not fresh is now in progress in this laborotory, the results of which will be published as soon as the work is completed.

The chemical analyses recorded in this paper were made by J. S. Hepburn, J. I. Burrell, and M. 0. Stafford; the bacteriologi- cal work was done by E. Q. St. John and E. Witmer.

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M. E. PenningtonEGGS

FRESHBACTERIOLOGICAL STUDY OF A CHEMICAL AND

1910, 7:109-132.J. Biol. Chem. 

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