-Spoilage of Shell Eggs by Pseudomonads

R. PAUL ELLIOTT

Food and Drutg Administration, Federal Office Building, San Francisco, California

Received for publication January 14, 1954

Previous to the recent introduction of black-light'candlers (Lorenz, 1950), the egg industry has lackedan adequate tool to protect consumers against shelleggs spoiled by pseudomonads. Such eggs cannot bedetected except in the later stages of decompositionwith the ordinary white-light candler. However, withthe new black-light lamps, white eggs spoiled by thepigment-producing species of the genus Pseudomonascan be detected becausc the albumen fluoresces brightlythrough the shell. Spoilage by pseudomonads in browneggs is not so easily detected by this new technique.The pseudomonads are soil and water forms (Breed

et at., 1948) and enter the egg after it has been laid.Most workers agree that the washing of dirty eggstends to increase spoilage of shell eggs from this cause,because the bacteria enter the shell more readily whenit is wet and when the water is colder than the egg(Haines and Moran, 1940; Gillespie et al., 1950;Lorenz et al., 1952; Starr, et al., 1952; McNally, 1952).The presence of more than about 1 per cent fluorescenteggs in a batch indicates the probability either thatthe batch was dirty or that it was washed, or both(Lorenz and Starr, 1952). Development of fluorescencein eggs after exposure to conditions favoring penetrationof pseudomonads is progressive. Lorenz et al. (1952)found that after exposure a few eggs in the lot becamefluorescent within a day or two but 6 or 8 weeks at15 C were required for all or most susceptible eggs tobecome fluorescent. Thus a lot of eggs with a lowincidence of spoilage by pseudomonads may develop amuch higher incidence on further storage. In fact,various workers have pointed out that such spoilage ismost serious in stored eggs.The objects of the present work were 1) to determine

the effect of age of the egg on susceptibility to subse-quent infection by pseudomonads, 2) to compare theirtime of entry into the albumen with the developmentof the fluorescence in the albumen, and 3) to determinethe relation of odor, bacteria count, and fluorescenceduring the process of decomposition.

EXPERIMENTAL METHODS AND RESULTS

Materials and EquipmentClean, large, unwashed white eggs were used through-

out. A single strain identified as Pseudomonas ovalisI Black light is a popular name for filtered ultraviolet

radiation of wave lengths between 3200 A and 4000 A.

was used in all inoculation experiments. This strainwas isolated from a fluorescent egg and identified bythe classification outline for the genus Pseudomonasto be used in the forthcoming seventh edition ofBergey's Manual (Haynes, 1953).

All tests for fluorescence were made under blacklight. The black-light candler used was a Vogelitemodel 205. Its light source was a General ElectricH100 SP4 spot behind a 5-mm Corning 5876 filter.Fluorescence of materials other than eggs in the shellwas detected using a Vogelite model 6W101 handfixture. Its light source was a 6-watt General Electric360 BL lamp encased in a 1-inch tubular Corning5874 filter. Both of these pieces of equipment weremanufactured by the Vogel Luminescence Company,San Francisco, California.

All bacteria counts were made on BBL trypticaseglucose extract agar.

Effect of Storage on Susceptibility to InfectionAlmquist and Holst (1931) found that shell porosity

was low in fresh eggs but it increased with length ofstorage. Haines (1938) found second-quality eggs werepenetrated by bacteria more readily than were first-quality eggs. Romanoff (1943) reported that shellpermeability to various gases increased considerablywith aging, particularly at high humidity, high tem-perature, and high carbon dioxide, and decreasedslightly on storage in low humidity and high carbondioxide.Two experiments were conducted to determine the

effect of age of the egg on its susceptibility to subse-quent infection by Pseudomonas ovalis. In the first, alot of 12 dozen freshly laid eggs was split into twoequal batches. The first batch was warmed to 35 C,then immersed 5 minutes in a cold-water suspension ofPseudomonas ovalis. The second batch was held 58days at 15 C and relative humidity of 87 per cent,then warmed to 35 C and immersed in a similar sus-pension of the same organism. As a control, a lot offreshly laid eggs was immersed at the same time as theaged batch. In the second experiment, 15 dozen oiledcommercially cold-stored eggs were similarly immersedin parallel with 15 dozen freshly laid eggs. All eggswere stored at 15 C and 87 per cent relative humidityand candled with the black-light candler at leastthrice weekly.

Results are shown in figures 1 and 2. It is evident158

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SPOILAGE OF SHELL EGGS BY PSEUDOMONADS

0 10 20 30 40 50 60 70DAYS AT 15°C. AFTER INOC.IILATION

FIG. 1. Effect of aging on susceptibility of shell eggs tosubsequent infection by Pseuldomonas ovalis. Before inoculationby immersion, lot 1, batch 1 was held 58 days at 15 C and 87per cent relative humidity.

that the aged or stored eggs became infected morerapidly than did fresh eggs.

Penetration of the MembranesThe shell and outer membrane of the egg contain

many pores through which bacteria penetrate withmoderate ease, especially when subjected to pressurefrom the outside, as is the case when the egg cools(Moran and Haines, 1938). However, the inner mem-brane has much smaller holes. Romanoff (1943) foundthat the inner shell membrane of the egg delayed thepassage of air 3 to 17 per cent as compared with shellsw^ith this membrane removed. Gillespie and Scott(1950) and Haines (1938) have pointed out variousindirect indications that this membrane is not as easilypenetrated by microorganisms as was once supposed.However, no direct evidence proving this has previouslybeen recorded.Four eggs, rejected by a commercial candler because

they had small fluorescent spots in the albumen, werebroken aseptically into sterile cellophane bags. Thou-sands of bacteria were recovered from the fluorescentspots of three of the eggs, whereas no bacteria wererecovered from the fluorescent area of the fourth egg.These eggs were held 13 days at 15 C. DOuring this time,the first three eggs became completely fluorescentand developed strong odors of decomposition and highbacteria counts. However, the fluorescent spot in thefourth egg had disappeared and the odor was normaland no bacteria could be recovered.

100

Ss U, s

~-so!O 0 20 3 00O 6

zuJ

6 20i40.

I0

30 10 2 30 4 50 6

DAYS AT 15°C. AFTER INOCULATIONFIG. 2. Effect of oiling and commercial cold storage on

susceptibility of shell eggs to subsequent infection by Pseudo-monas ovatis. The freshly-laid eggs were 4 days old; and thecold-stored eggs were held in a commercial cold-storage roomat 30 to 32 F and 85 per cent relative humidity for 108 daysbefore immersion inoculation.

INOCULUM ::: WINDOWIN AIR CELLC7

\ ~~~~OPENED\ , }t~'HERE

\ ,' / ~AFTER STORAGE

FIG. 3. Inoculation experiment to show delay in penetra-tion of bacteria through inner shell membrane.

An experiment was conducted to investigate thecause of this phenomenon. Fifty eggs at 15 C werecandled, then immersed for 1 minute in 1-1000 HgCl2at 25 C. On the side of each egg a window was cut,with a sterile saw, through the shell but not throughthe membrane (figure 3). The window was temporarilycovered with a sterile cover slip. A hole was cut carefullyinto the air cell to avoid rupturing the inner membrane.As indicated in table 1, 0.5-mI aliquots of the inoculawere placed in the air cell. These inocula were preparedfrom a highly fluorescent strain of Pseudomonas ovalisgrown in asparagine broth (Clara, 1934)2. A sterile

2 Clara, in a study of plant pathogens, used a mediumconsisting of asparagine 0.1 per cent, MgSO4*7H20 0.05 percent, and K2HPO4 0.05 per cent. This combination of chemicalswas first suggested by Sullivan (1905) for production of bac-terial pigments.

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R. PAUL ELLIOTT

TABLE 1. Penetration of Pseudomonas ovalis and its water-soluble pigments through the inner air cell membrane of the hen's egg

Sterile filtrateWashed suspensionWashed suspensionWashed suspensionWashed suspensionWashed suspensionWashed suspensionWashed suspensionWashed suspensionWashed suspensionWashed suspensionWashed suspensionWashed suspensionWashed suspensionWashed suspensionWashed suspensionFluorescent cultureFluorescent cultureFluorescent cultureControl

1-311

2-33668881010101010301

1-238

SpotNoneNoneSpotNoneSpotSpotSpotSpotSpotSpotSpotBright, generalBright, generalBright, generalBright, generalSpotSpotModerate, generalNone

0

ot0

0

0

0

0

0

0

1,000,000

48,000

3,700,000

280,000

19,000, 000

31,700,000

300-350 million

390,OO0Ok0

0

0

NoYesYesYesNoYesYesYesYesYesYesYesYesYesYesYesYesYesYesNo

CONDITION AFTER 1 WEEK IN STERILE BOTTLE*

No odor, diffuse fluorescenceNo odor, no fluorescenceSpoiled, bright fluorescenceNo odor, diffuse fluorescenceNo odor, no fluorescenceSpoiled, bright fluorescenceNo odor, diffuse fluorescenceNo odor, diffuse fluorescenceSpoiled, bright fluorescenceSpoiled, bright fluorescenceSpoiled, bright fluorescenceSpoiled, bright fluorescenceSpoiled, bright fluorescenceSpoiled, bright fluorescenceSpoiled, bright fluorescenceSpoiled, bright fluorescenceSpoiled, bright fluorescenceNo odor, diffuse fluorescenceNo odor, diffuse fluorescenceNo odor, no fluorescence

* The term "spoiled' indicates strong odors of decomposition were present.t One of these eggs spoiled from another organism. Pseudomonads were not present in the albumen.$ Membrane not intact.

filtrate was obtained by Seitz filtration of the brothculture, and a washed suspension was obtained byrepeated centrifugation and suspension of the cells insaline.The inoculated eggs were stored at 15 C at the

approximate angle indicated in figure 3. At 1-dayintervals representative eggs were opened asepticallyat the small end. The cover slip was removed and themembrane window was punctured with a sterile scalpelas the egg contents were dropped into a sterile bottle.The hole on the side allowed air to enter so that theinner membrane was protected from rupture at the aircell. The membrane was examined carefully for breaksand was transferred to asparagine broth for recoveryof pseudomonads producing fluorescent pigments. Asample of the fluorescent portion of the albumen or ofthe normal albumen, whichever was the case, was

plated. The remaining egg contents were held in sterilejars 1 week at 15 C to test for the presence of verysmall numbers of pseudomonads. Odors of decomposi-tion and intense fluorescence after the further storagewere taken as indications that bacteria had penetratedthe inner membrane.

Results are presented in table 1. The sterile fluores-cent filtrate passed through the membrane in 1 day.The pigment was then diffused, but was detectableafter the additional week in a bottle. Eggs inoculatedwith a washed suspension or a fluorescent cultureshowed fluorescent spots in the albumen within 1 or 2days. There were two exceptions in which the bacteria

were killed by contact with the membranes. Bacteriawere found to have penetrated the membrane of one

egg at 1 day, of two eggs at 6 days, and of two more

at 8 days. On the eighth day, one membrane had stillnot been penetrated, although a spot of fluorescencehad appeared earlier in the albumen. At 10 days, allhad been penetrated, and at 30 days bacteria countswere over 300 million per gram of albumen.Thus pseudomonads do not necessarily enter the

albumen immediately on exposure but may form a

colony on the inner shell membrane. Here, in the firstfew days, they use the nutrient material in the albu-men; and their waste materials, including water-soluble pigments, diffuse back into the albumen. Laterthe bacteria penetrate in small numbers and multiplyrapidly. The disappearance of the fluorescent spotobserved in the preliminary experiment was probablydue to dispersion. The fluorescent pigment in Pseudo-monas-infected egg albumen does not fade noticeablyeven on storage for several months. It is also sufficientlyheat-stable so that fluorescent eggs retain brightfluorescence even when hard-boiled.

Development of Colony and Fluorescence in the Egg

In all observed instances, definite fluorescent spotsin the albumen developed to full fluorescence when theeggs were held in the shell. In one experiment, 24million bacteria were placed in the air cells of each of18 eggs. Each day the eggs were candled. An average

of 7/1 days after fluorescent spots appeared in the

1-67-1314

15-2223, 2425, 2627-30

3132333435363738

39-42431

44-4647

48-50

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SPOILAGE OF SHELL EGGS BY PSEUDOMONADS

outer albumen was required for the fluorescence todisseminate throughout the albumen. The extremeswere 2 and 11 days.

Further tests were conducted to compare roughlythe degree of fluorescence with the bacteria count pergram. Shell eggs were inoculated in the air cell, thenstored at 15 C. At intervals, eggs in various stages offluorescence were broken aseptically into sterile j ars

TABLE 2. Nutmtber of bacteria present in eggs at variouts degreesof fluorescence

APPROX. DAYS STORED AVERAGE

DAYS FROM PE ETAFTER FULL BACTERIANO. FIRST SPOT OF ALBUMENFLOE-

DEGREE OF BRIGHT- COUNT PEROF OF LUR^ES- FLUJORES- NTESS OF CtN e

EGGS PLATING CENT BEFORE FLUORESCENT AREA EGG*PLATINGTINBEFORE1PLATED PLTN(X1)

5 1-2 5-25 - Moderate 464 1-7 50-75 - Moderate 15,7007 5-9 100 0 Spotty, diffuse 30,600

15 3-13 100 0 MIoderate 65,20010 9-16 100 8-14 Bright 121,000

* All eggs weighed about 50 grams.

uJ.a

-

to

aU

0m

w

In0

z

0

and mixed with Butterfield's phosphate buffer andglass beads. An aliquot of this was taken for the bac-terial count. Since it was difficult to follow the develop-ment of the colony in shell eggs with accuracy, severalfresh candled eggs were broken aseptically into sterilescrew-cap bottles. Parallel bottles containing wholeegg or egg albumen were inoculated with 100 and 106bacteria per gram to study the effect of presence offree yolk material and size of inoculum on the spoilagepicture.

Table 2 demonstrates how the bacteria count inshell eggs climbs rapidly as the fluorescence advancesfrom the site of primary infection through the albumen.Figure 4 shows that in egg pulp inoculated and storedin bottles, large inocula result in most rapid spoilageand the presence of yolk material speeds spoilage.

Odors in Pseudomonas-Infected EggsSeveral experiments were conducted to determine

whether or not a trained panel could detect raw fluores-cent shell eggs at various stages of infection merely

10O

6

3 3

3 3 3

22. .*0 .......

21

0

3

632 3 3........ F..O

3 3.O..*-.

O. 0 ALBUMEN

O--0O ALBUMEN &YOLK

F: FLUORESCENCE BEGINS1: PASSABLE, SOME ODOR2: DECOMPOSED, BORDERLINE3: DECOMPOSED, REJECT

0 2 4 6 8 10 12 14 16DAYS AT 150C.

FIG. 4. Spoilage history of egg material inoculated with Pseudomonas ovalis and stored at 15 C in screw-cap bottles.

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R. PAUL ELLIOTT

TABLE 3. Effect of soft cooking on odor of eggs fluorescentutnder black light

EGGNO.

2345678910111213

APPEARANCE*

NormalNormal, weak yolkNormal, weak yolkNormalNormal, weak yolkNormal, weak yolkNormal, weak yolkDarkened yolkBrown liquidNormalNormal, weak yolkSlightly dark volkNormal, weak yolk

14 1Normal control

ODOR RAW

Panel members

A B C D

0 0 0 0

0

0

0

0

0

0

10

0

0

1

0

10

0

130

113

0

20

0

0

0

0

0

0

0

0

0

3

0

310

1

0

10

0

10

0

3

0

ODOR SOFT-COOKED

Panel members

B C D E

3 2

0

0

0

3

1

2

3

3

0

3

0

32131333330

3

0

3331330

33333

0

20

12111

33

0

11

0

VIABLEBACTERIAPER G(X 106)

650960

1,720660630780650740

2,330234650610

Notdone

Notdone

* All eggs except number 14 had green fluorescent albumenby black light and light green albumen by daylight.

Key: 0: Passable, no odor.1: Passable, some odor.2: Decomposed, borderline.3: Decomposed, reject.

by smelling them. Smelling the raw egg was found tobe an unreliable test for detection of Pseudomonas-infected eggs. Eggs which had come recently to fullfluorescence were almost never rejected. Odors were

not detected in all infected shell eggs even when theywere stored up to 52 days in the fluorescent condition.In white light the appearance of such stored eggs was

often nearly normal, except for the light green wateryappearance of the albumen. On the other hand, some

eggs had degenerated to a dark brown liquid. Manyof these were odorless rots.However, several dozen naturally-infected and

artifically-inoculated eggs were broken asepticallyinto closed or partly closed sterile containers. In a

few days they all developed strong odors of decomposi-tion. The point at which odor appeared strong enoughfor a panel to reject inoculated egg material paralleleda bacteria count of approximately 107 per gram (seefigure 4). When highly odorous Pseudomonas-infectedegg albumen in a bottle was held 36 days, the odordisappeared so that a panel did not reject it. The onlyindication of abnormality was its watery, slightlyyellow-green appearance in daylight and its brightfluorescence under black light. The odorous compoundshad apparently evaporated during the occasionalmoments the bottle was opened for detection of odor.The odor of whole egg treated the same way fadedalso, but not to the point where none was detectableafter 36 days of storage. Fluorescent eggs in the shellheld in airtight jars for 2 days produced odors of de-

composition in the air of the jars. It seems clear thatodors of decomposition are produced by pseudomonadsin shell eggs, but that they evaporate through the shellas they are formed.An experiment was conducted to determine whether

or not soft-cooking would have an effect on the in-tensity of odors. Eggs which had been inoculated byimmersion were held 26 days at 15 C after they hadbecome fluorescent. They were broken into sterilePetri dishes, a sample for bacteria count was taken,and the raw eggs were smelled by the panel. Then theywere soft-cooked double-boiler style and smelled againby the panel. Results, as shown in table 3, show thatsoft-cooking does indeed intensify the odors caused byPseudomonas spoilage.A series of experiments was conducted to determine

whether or not a panel could detect odors in soft-boiled eggs at various stages of infection from beginningspots to complete fluorescence. A normal egg from thesame lot was included in each group of three as a

control. Panel members were asked to pick out thePseudomonas-infected eggs by odor. All eggs were

smelled while still hot.Results of this series are shown in table 4. There was

an increase in the accuracy with which such eggs were

detected by odor as fluorescence progressed throughthe egg. Eggs stored a few days to a few weeks in

TABLE 4. Accuracy of odor method for detecting fluorescentPseudomonas-infected shell eggs soft-boiled at various

stages of fluorescence*

TESTNO.

1

2

3

4

5

6

7

8

9

HISTORY OF LOT OF EGGSt

Inoculated. Fluorescent spot 1 square

inch or less in sizeInoculated. One-fourth to two-thirds

of albumen fluorescentInoculated. Albumen fully fluorescent,stored thus 0 to 2 days

Inoculated. Albumen fully fluorescent,stored thus 5 to 12 days

Inoculated. Albumen fully fluorescent,stored thus 1 to 13 days

Inoculated. Albumen fully fluorescent,stored thus 2 to 22 days

Rejected fluorescent eggs from com-

mercial lot. Exact age not known,but less than 4 weeks

Rejected fluorescent eggs from a com-

mercial lot about 1 week oldRejected fluorescent eggs from com-

mercial lot 1 to 3 weeks old

NO. OFEGGS

18

15

12

15

12

24

18

29

12

CORRECTDECISIONSBY PANEL

per cent

36.5

60

69

69

70

76

92

78.5

67

* The panel consisted of three to five members.t A normal egg from the same lot was included as a control

in each group of three. Therefore, expected correct decisionsfor each lot if all eggs judged odorless, 33.3 per cent; all eggs

judged fluorescent, 66.6 per cent; and all eggs judged correctly,100 per cent.

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SPOILAGE OF SHELL EGGS BY PSEUDOMONADS

fluorescent condition were detected 69 to 92 per centof the time.

DISCUSSIONThe black-light candler can be used to detect eggs

infected with pigment-producing members of thegenus Pseudomonas even in the earliest stages of de-composition. For this reason, it is a valuable tool forconsumer protection and an economic aid for the eggindustry. If undetected, such eggs will reach the con-sumer with brightly pigmented albumen and withhundreds of millions of bacteria per gram. Furthermore,many of them will possess distinct odors of decomposi-tion.

Fluorescence from pseudomonads can be readilydistinguished from that caused by normal aging, andthat caused by washing the egg in detergents contain-ing blancophors (optical bleaches). No other cause offluoresconce in eggs has been experienced.

ACKNOWLEDGMENTSThe author wishes to acknowledge the help of various

members of the staff of the San Francisco District,Food and Drug Administration, in the panel testing.He especially wishes to thank Inspector Almond D.Davison for his constant advice and assistance.

SUMMARY

Eggs previously stored at 85 to 87 per cent relativehumidity were more rapidly infected by Pseudomonasovalis than were freshly laid eggs when exposed toinfection under identical conditions. The organism,when placed in contact with the inner shell membraneof hens' eggs, formed colonies without penetrating tothe albumen in the first days of storage. Black-lightfluorescent pigments diffused into the egg albumen toform bacteria-free fluorescent spots in 1 or 2 days.The time required for the bacteria to penetrate theinner membrane varied from 1 day to more than 8days. In two eggs, the inoculum in contact with themembrane died before penetration occurred.

In all observed instances, fluorescent spots in thealbumen spread through the entire albumen if the eggwere left in the shell. The spread of fluorescent pigmentfrom the site of primary infection throughout the eggtook 2 to 11 days at 15 C in eggs inoculated in the aircell and examined daily. During this process the bac-teria count increased until, when the entire albumenhad become fluorescent, it had reached 107 per gram.Further storage resulted in counts of 108 to 109 pergram.

Shell eggs that were only partly fluorescent gave noperceptible odor of decomposition. Eggs stored infully fluorescent condition for many days or weeksdeveloped odors of decomposition detectable 69 to92 per cent of the time by a panel, when the eggs were

soft-boiled. Fluorescent eggs broken aseptically intoclosed, sterile containers invariably developed distinctodors of decomposition in 3 or 4 days, detectable evenin the raw state. Similar storage of fluorescent eggsin the shell in closed jars resulted in decompositionodors in the air of the jars in 2 days. Storage of Pseu-domonas-inoculated egg albumen in a screw-cap bottleresulted in the appearance of distinct odors of decom-position when the bacteria count per gram reachedabout 107. The bottle was opened each day or two forsmelling tests. The odor first became intense, thengradually dissipated, until after 36 days from itsfirst appearance it was not detectable by the panel.It was concluded that odorous compounds are pro-duced by pseudomonads in shell eggs but these com-pounds will sometimes evaporate through the shell.

REFERENCES

ALMQUIST, H. J., AND HOLST, W. F. 1931 Variability of shellporosity in the hen egg. Hilgardia, 6, 61-72. C. A. 26,1356 (1932).

BREED, R. S., MURRAY, E. G. D., AND HITCHENS, A. P.1948 Bergey's Manual of Determinative Bacteriology.6th ed. The Williams & Wilkins Co., Baltimore.

CLARA, F. M. 1934 A comparative study of the green-fluorescent bacterial plant pathogens. N. Y. State Agr.Exp. Sta., Cornell, Memoir 159, 36 pp.

GILLESPIE, J. M., SCOTT, W. J., AND VICKERY, J. R. 1950Studies in the preservation of shell eggs. III The Storageof machine-washed eggs. Australian J. Appl. Sci., 1,313-329.

GILLESPIE, J. M., AND SCOTT, W. J. 1950 Studies in thepreservation of shell eggs. IV. Experiments in the mode ofinfection by bacteria. Australian J. Appl. Sci., 1, 514-530.

HAINES, R. B. 1938 Observations on the bacterial flora ofthe hen's egg, with a description of new species of Proteusand Pseudomonas causing rots in eggs. J. Hyg., 38, 338-355.

HAINES, R. B., AND MORAN, T. 1940 Porosity of, and bac-terial invasion through the shell of the hen's egg. J. Hyg.,40, 453-461.

HAYNES, W. C. 1953 Key to the species of genus Pseudo-monas. Mimeo. 7 pp. Presented at Symposium on Tax-onomy, Aug. 13, 1953. Fifty-third general meeting of theSociety of American Bacteriologists, San Francisco.

LORENZ, F. W. 1950 Black light. Am. Egg and Poultry Rev.11, October, 16-18.

LORENZ, F. W., AND STARR, P. B. 1952 Spoilage of washedeggs. I. Effect of sprayed versus static water under dif-ferent washing temperatures. Poultry Sci., 31, 204-214.

LORENZ, F. W., STARR, P. B., STARR, M. P., AND OGASAWARA,F. X. 1952 The development of Pseudomonas spoilagein shell eggs. I. Penetration through the shell. FoodResearch, 17, 351-360.

McNALLY, E. H. 1952 Effect of drying 4aer washing on theincidence of eggs infected by spoilage' micro-organisms.Poultry Sci., 31, 1102-1104. ''

MORAN, T., AND HAINES, R. B. 1938 The porosity of theshell of the hen's egg. Dept. Sci. Ind. Research (Brit.)Repts. Food Investigation, 39-41.

ROMANOFF, A. L. 1943 Study of various factors affectingpermeability of bird's eggshell. Food Research, 8, 212-223. *

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DAVID H. HOLLANDER AND E. ELLEN NELL

STARR, P. B., LORENZ, F. W., AND OGASAWARA, F. X. 1952Spoilage of washed eggs. II. Laboratory versus ranchwashing. Poultry Sci., 31, 215-220.

SULLIVAN, M. X. 1905 Synthetic culture media and the bio-chemistry of bacterial pigments. J. Med. Research, 14,109-160.

Improved Preservation of Treponemna pallidum and OtherBacteria by Freezing with Glycerol'

DAVID H. HOLLANDER AND E. ELLEN NELL

The Department of Microbiology, School of Hyqiene and Puiblic Health, 7'he Johns Hopkins University, Baltimore, Maryland

Iteceived for publication January 18, 1954

Preservation of bacteria at the temperature of dryice (-78.5 C) is a particularly valuable method for themaintenance of such organisms as Treponema pallidumwhich otherwise require continuous animal-to-animalpassage. Turner (1938), Turner and Fleming (1939), andTurner and Brayton (1939) demonstrated that at thetemperature of dry ice T. pallidum and relapsing feverspirochetes retain their virulence for long periods.However, in practice it has become apparent that thefreezing method of preservation, especially in the caseof spirochetes, is not completely reliable, and thatmuch activity may be lost in the process, so that onoccasion it has been difficult or impossible to reactivatefrozen strains of trepoinemes (Turner, 1953).The influence of low temperatures on living cells

has been reviewed by Belehra'dek (1935) and by Luyetand Gehenio (1940). Prudden in 1887 distinguishedtwo types of damage which may result from the freezingof bacteria. Although subsequent investigators havenot always separated these two effects, all the availableevidence tends to support this distinction. This isclearly demonstrated in the studies of Weiser andOsterud (1945) who state in summary:"Death by freezing involves a rapidly acting or

"immediate" death, caused by the freezing and thawingper se, and a "storage death" which is a direct func-tion of time and temperature."The present study is primarily concerned with the

damage which occurs during the freezing and thawing.Prudden (1887) was the first of many observers toreport that repeated freezing and thawing was moreinjurious than a single freezing. Weiser and Lief (1940)in unpublished experiments, determined that bacteriawhich are repeatedly frozen and thawed are damagedat a rate which is the same for each cycle of freezingand thawing so that the loss is exponential.

Different species of bacteria vary in their suscepti-b)ility to damage by freezing and thawing. Haines

1 This study was supported by a grant from the NationalInstitutes of Health, U. S. Public Health Service.

(1938) found that a single freezing and thawing killed89 per cent of Saccharomyces cerevisiae, but only 5 percent of Staphylococcus aureus. The susceptibility ofseveral other bacteria was intermediate. Keith (1913)showed that if the einvironmental coniditions arechanged, the resistance of the organism may be greatlyaltered. When certain substances, such as sugar, milk,or glycerol, were added to the suspending medium,the bacteria were partially protected from the damageof freezing at -20 C. Recently, analogous observationshave been made of the effect of glycerol when othertypes of cells are frozen. Polge, Smith and Parkes(1949) and Smith and Polge (1950) have reportedthat in the presence of dilute glycerol solutions sperma-tozoa of various species can regain their motility andtheir physiological activity (Polge, 1951) after beingfrozen at the temperature of dry ice; Smith (1950)has reported that red blood cells in glycerol are nothemolyzed by freezing; and Hartmann and Conley(1952) have found that blood platelets in the presenceof glycerol are protected from the destructive actionof freezing.The amount of damage from freezing and thawing

may be influenced by the rate and by the temperatureof the freezing and of the thawing. Bacteria ordinarilyshow the least amount of damage when rapidly frozenand rapidly thawed (Turner and Brayton, 1939;Weiser and Osterud, 1945). According to Luyet andGehenio (1940), who believed that the damage iscaused by crystal formation, quick freezing and thaw-ing is essential in order to pass rapidly over the tem-perature range in which crystallization occurs. Luyet(1951) found 28 per cent of a red blood cell suspensionwas hemolyzed after quick freezing and thawing,whereas 96 per cent was hemolyzed during slow freezing.More recently Luyet and Keane (1952) have reportedthat the speed of freezing was not important in pre-serving chicken embryo cells with ethylene glycol,and Smith and Polge (1950) have found that very

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