THE DEVELOPMENT OF MOLD ON COLD STORAGE EGOS AND METHODS OF CONTROL Thesis Submitted to the Faculty of the Graduate School of Michigan State College in p a r tia l fulfilm ent of the requirements fo r the degree o f Doctor o f Philosophy hy Catherine Edwards Michael Department of Bacteriology 1939 ProQuest Number: 10008386 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Pro uest ProQuest 10008386 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 TABLE OF CONTENTS Pci§G PART I . ISOLATION AND IDENTIFICATION Introduction .......................................................................... . . 1 Is o la tio n and Id e n tific a tio n of Molds Discussion PART I I . ........................ 4 . « • • ...................... 17 LABORATORY TESTS ON MYCOSTATIC AGENTS..................................SO PART I I I . COLD STORAGE UNDER COMMERCIAL CONDITIONS WITH ....................................................31 HIGH AND LOW HUMIDITIES I. Influence of high, re la tiv e humidity on growth of molds on eggs and egg packages as compared to low re la tiv e humidity • • • • 33 The e ffe c t of w etting eggs, f i l l e r s , f l a t s , and cases on the keeping Q uality of eggs stored a t various h u m i d i t i e s . ........................... 34 III. The in v estig atio n of wood used in containers. 36 IV. The in v estig atio n of f i l l e r s fo r mold growth. 36 In v estig atio n on the treatment of eggs by myco s t a t s ........................... 37 Investigations on mycostatic tre a te d f i l l e r s and f l a t s . ........................... 38 The influence of the mycostatic agent on the q u ality of the eggs................................ 39 II. V. VI. VII. Discussion SUMMARY . ............................................................... 40 42 TABLES: Table I . Mycostatic p ro p erties of eight copper compounds on PeniciIlium -puberulum . . . . . . . ......................44 Table I I . Mycostatic p ro p erties of eight copper com­ pounds on A spergillus niger • • • • • . • • • • 4 5 Table I I I . Table IV. Table V. Mycostatic p ro p erties of eight copper com­ pounds on a species of A lte rn a ria . . . . . . 46 The mycostatic p ro p erties of three compounds commonly used in food preservation . . . . . . 47 The resistan c e of fourteen c u ltu res of Penic i l l i a to sodium b e n z o a te ......................... Table VI* 48 The resistan ce of fourteen c u ltu res of Penie i l l i a to sodium borate. ...........................................49 Table VII. The mycostatic powers of compounds on market sold fo r mold prevention. • • • • . . . . . . 5 0 Table V III. The resistan c e of fourteen c u ltu res of Penic i l l i a to sodium ortho-phenyl phenate . . . . 51 Table IX. The resistan c e of fourteen cu ltu res of Penic i l l i a to sodium 2 chloro-ortho-phenyl phenate. 52 Table X. The mycostatic powers of fiv e phenol derivativ es on four genera o f m o ld s........................... 53 Table XI. The mycostatic powers of egg-case m aterials impregnated with phenol d e r i v a t i v e s .............................55 Table XII. Table X III. Table XIV* Table XV. The r e s u lts of applying phenol d eriv ativ es by d iffe re n t m ethods.................................................... 56 The mycostatic powers of phenol prepara­ tio n s applied to eggs in cold storage . . . 47 Influence of humidity on growth of molds on eggs and egg packages on fresh market eggs not o il- tr e a te d . ........................• • • • • 58 Influence of humidity on growth of mold on eggs and egg packages on o il- tr e a te d eggs • . 59 page Table XVI* E ffect o f w etting eggs and wet f i l l e r s and f l a t s on keeping q u a lity of eggs stored a t 88 per cent humidity • • • • • • • • - • • • • 6 0 Table XVII* E ffect of w etting eggs and wet f i l l e r s and f l a t s on keeping q u a lity of eggs stored a t 98 per cent h u m id ity ........................... BIBLIOGRAPHY 61 62 ACKNOWLEDGMENTS To Dr* E. A. Bessey and Dr* W. L. Mailman are due the w r i t e r 's appreciation and thanks* not only f o r the aid in the laboratory work and in the w ritin g of th is thesis* but fo r th e ir patience and encouragement while the work was in progress* The w rite r also wishes to express appreciation and thanks to Mr. Eugene A. Toop fo r h is assistan ce and in te r e s t in the cold storage experiments* Thanks are also due the following: Dr* Charles Thom of the United S tates Department of A griculture for iden­ tify in g five cu ltu res of Penicillium submitted to him; Dr* A* Koehler of the Forest Products Laboratory a t Madison* Wisconsin* and Dr. A. J . Panshin* Department of Forestry, fo r id e n tif i­ cation of wood used in egg cases; Mrs* Kathryn Niles of the American I n s titu te of Poultry In d u stries and Miss E. Griswold and Miss D. Grantham* Division of Home Economics* fo r perform­ ing the te s ts on q u ality and ta s te of eggs submitted to them* Credit is also due the Central Fiber Products Co., Dow Chemical Co., United States Cold Storage and Ice Co., American I n s titu te of Poultry In d u stries, and Swift & Co., fo r the supplying of m aterials, fin a n c ia l a id and cold storage f a c i l i t i e s ; without which these experiments could not have been c a rrie d out. PART I • ISOLATION AND IDENTIFICATION Introduction The need of producing a high q u ality storage egg is evidenced by the fa c t th a t approximately 10,000,000 cases of eggs were placed in cold storage during the year 1926 (6). This volume represented approximately 13 per cent of the e n tire crop* Unfortunately the preservation of eggs by cold as practiced to­ day i s s t i l l not the id e a l to which the industry a sp ire s, p a r t i ­ c u la rly fo r long periods of storage* a t temperatures of approximately 0° h The process of holding foods i s a means of delayed de­ composition rather than a process of to ta l or complete in h ib i­ tio n of m icrobiological and enzymatic spoilage. I f the humidity of the storage room i s s u ffic ie n tly high to form a microscopic film of moisture on the surfaces of the foods and th e ir containers, then molds slowly but surely develop. I f . on the other hand, the humidity of the storage room i s kept a t a point where the surfaces In the id e n tific a tio n of the various fungi found by the w rite r in eggs and egg m aterials, assistance was received from Dr. E. A. Bessey* Department of Botany, under whose supervision the mycological po rtions of th is in v estig atio n were carried on. The remainder of the work, including the laboratory te s ts on mycostatic agents and the cold storage in v estig atio n s, was c arried out under the supervision of Dr. W. L. Mallmann of the Department of Bacteriology* The main f i e l d te s ts were conducted a t the p lan t of the United S tates Cold Storage and Ice Company, in Chicago, with the able assistan ce of Mr. Eugene A* Toop. of the foods and containers do not carry a microscopic film of m oisture, then molds are prevented from growing hut the food gives up moisture to the air, and shrinking* with ultim ate loss in q u a lity of the food* r e s u lts . In the operation of a cold storage p la n t, the hum idities a re , a t present, kept a t a point Ju st below th a t which allows the development of microorganisms. This means th a t during storage a gradual lo ss of moisture from the foods r e s u lts . In eggs there i s not only lo ss in weight but the appearance of large a i r sacs, i f the egg i s held too long in storage. The losses due to moldiness vary from season to season, and from one p lan t to another. Many fa c to rs en ter to determine the extent and degree of spoilage th a t occurs. Most of the facto rs under present operating conditions are uncontrollable; a t le a s t there i s no p ra c tic a l means of co n tro llin g contaminations of eggs and egg cases. Moisture content of the storage rooms and storage temperatures cannot be varied s u ffic ie n tly to be of any value in c o n tro llin g the growth of the molds without g rea ter in ju ry to the product i t s e l f . Some method of mold control which w ill function regardless of the humidity and the i n i t i a l contamination i s necessary. Mold has been reported as developing on a number of pro­ ducts in cold storage* The development of mold on meat in cold storage has been frequently observed. Monvoisin in 1S22 (9) 3 found Cladosporium herbarum» Thamni di um elegans* Mucor mucedo# Bhizopus n ig ric a n s# and PeniciIlium glaucum. Bidault in 1922 (2) found Chaetostylum fre s e n ii » Thamnidium elegans» Penicillium crustaceum* Hormodendron cladosporioides» Cladosporium herbarum* Stysanus stem onites* B otrytis m icheli* and some v a rie tie s of Eubotrytis# Wright in 1923 (17) found th a t "black spot" of meat in cold storage could be produced by a number of molds# including Cladosporium herbarum* Mucor mucedo» and Penicillium glaucum# In 1923 Yesair (18) found th at Penicillium expansum was one of the most common molds on the walls and equipment of meat packing houses# In 1927 Weston and Holman (15) demonstrated th a t a mold belonging to the genus Cladosporium was responsible fo r black d isco lo ratio n of eggs. This discoloration# "black spot#" was reproduced in eggs by tre a tin g clean eggs with spore suspensions of Cladosporium herbarum# They also found th at a species of Penicillium could produce a sim ilar condition* In 1929 James and Swenson (8)» in a sim ila r study* found two molds# a Penicillium and a Cladosporium# In order to study the methods and procedures fo r mold con­ t r o l in the cold storage room* from a s c ie n tif ic attack* i t i s f i r s t e s s e n tia l to know exactly the species of molds encountered and the extent of th e ir a c tiv ity . Inasmuch as the lite r a tu r e does not reveal any such stud ies i t was deemed necessary to make a care­ f u l and extensive survey on the incidence and id e n tity of the molds occurring on eggs* f ille r s * fla ts* and cases in the cold storage plant# Is o la tio n and Id e n tific a tio n of Molds In order th a t a wide survey of the various species of molds he made rep resentative of the whole industry* eggs were ob­ tained over a period of two seasons from p lan ts in Chicago* Port Wayne* Detroit* Lansing* Omaha* and Topeka. Fresh eggs from the College poultry plant and moldy eggs from cold storage were cul­ tured* In addition to eggs moldy f ille r s * flats* and c rates from the packing plants* and new f ille r s * fla ts* and c ra te s from a manufacturer of the same were cultured fo r molds. The is o la tio n s of molds were made by using scrapings from the surfaces of affected areas on the various m aterials ex­ amined* These scrapings were smeared on the surface of dextrose agar or beanpod agar and incubated a t room temperature u n til f r u i t ­ ing bodies of the molds appeared. The re su ltin g mixed growths of molds were then p u rifie d by the d ilu tio n p la te method. A fter the p u rity of each culture was d e fin ite ly established* the c u ltu res were tran sfe rred to the proper media upon which id e n tific a tio n s are based. Except where in dicated , the P e n ic illia in th is study were id e n tifie d from Thom's “The P e n ic illin " (13). The genus Penicillium consists of a g reat many species which intergrade and which are exceedingly v ariab le under d iffe re n t conditions of tem­ perature* substratum# and manner of cu ltu re. The students of th is genus* of whom Dr. Charles Thom of the United S tates Department of A griculture i s foremost* recognize many s tra in s w ithin each species. A few species were id e n tifie d by Dr. Thom. Where the d e scrip tio n of an organism f e l l widely outside of any standard d escrip tio n in Thom's key* i t i s designated by i t s culture num­ ber and the d escrip tio n is presented in d e ta il. Organisms belonging to the order Mucorales were id e n ti­ fie d from the d escriptions of N. A. Naumov (11). Several forms of Mucor racemosus were iso la te d which f i t th is species most clo sely but are aberrant in some p a rtic u la r. The id e n tific a tio n of the A sp erg illi iso la te d correspond to the d escriptions of Thom and Church (14). A number of c u ltu res were id e n tifie d only as to genus. These belong to the Fungi Imperfect! and correspond to the des­ c rip tio n of the genus as found in standard works. The descriptions of the aberrant species follow. Penicillium No. 30 Colony a f te r four days on Czapek's agar lig h t green with s lig h t s t e r i l e border; reverse tan; drops absent. Colony a f t e r two weeks b rig h t green with white margin about 2000 u broad; colony 4 cm. broad and about 1000 u deep; d is tin c tly zonate; re­ verse cream to rose; agar f a in t rose color. Colony a f te r 24 days 5.5 cm.* b rig h t green* velvety* deeply zonate a t margin; reverse yellow -rose; agar ro se -ta n . Colony a f te r six weeks dark green* deeply zonate a t margin# with wide submerged margin; drops yellow; reverse dark red; agar dark red-brown; conidiophores s lig h tly rough; metul&e not divaricate* 9.44 - (11.0) - 11.8 u. P e n ic illi showing sterigm ata and metulae in a compact brush or broom; sterigm ata 5.9 - (8.0) - 10.6 u; conidia globose* smooth* 2.77 (3.0) - 3.3 u; g e la tin liq u e fie d Penicillium No. 36 Colony a f te r four days on Czapek's agar pale green with white margin; drops absent; reverse lig h t green. weeks 8.5 cm. Colony a f te r six broad* velvety* th in fe lt* brown to gray-green* zonate a t margin with wide submerged margin; white over-growth in center; reverse white to purple-black; conidi ophores rough* metulae not divaricate* but p e n ic illi subtended by one or more d ista n t secondary divergent branches bearing secondary p e n ic illi often monov e r t i c i l l a t e ; metulae 12.5 - (13.0) - 15.0 u; sterigm ata 7.5 (12.0) - 15.0 u; conidia smooth* globose 3.1 - (3.58) - 4.1 u; g e la tin not liq u e fie d . Penicillium No. 65 Colony a f te r four days on Czapek's agar green with s t e r i l e white margin; drops absent; reverse tan-gray. Colony a f te r twelve days 12.5 cm. x 5.5 cm.* velvety* b rig h t green with white margin 1000 u broad, zonate, about 900 u deep near edge; center about 2000 u deep; reverse orange yellow, zonate; agar colored red-brown; conidiophores erect* rough* asymmetrical* over 300 u long; metulae not d iv arica te 9.6 - (9.65) - 10.0 u; sterigm ata 9.6 (9.65) - 10*0 u; conidia globose, smooth 3.0 - (3.7) - 3.84 u; g e la tin liquefied* Penicillium No. 84 Colony a f te r four days on Czapek's agar green, white mar­ gin; reverse in shades of green; drops c o lo rle ss. Colony a f te r 26 days th in f e l t 3.5 cm. broad, floccose, olive-green, azonate; re­ verse in shades of green; agar uncolored; conidiophores rough; metulae not d iv arica te 10.0 - (11.5) - 15 u; sterigm ata 7.5 - (10*5) 12.5 u; conidia smooth* globose 3.86 - (4.0) - 4.16 u; g e la tin lique­ fie d . Penicillium No. 102 Colony a f te r four days on Czapek's agar lig h t blue; drops c o lo rle ss; reverse yellow -tan-rose. Colony a f te r six weeks 8*5 cm*, flo cco se, gray-green, fa in tly zonate a t edge; wide submerged margin; drops c o lo rless; reverse zones of rose red to cream; agar rose colored; conidiophores rough; metulae not d iv arica te 10 - (11.5) -12.5 u; sterigm ata 7.5 - (9*0) - 10.0 u; conidia globose, smooth* 3.3 - (3*48) - 3.6 u; g e la tin liq u e fie d . Penicillium No. 123 Colony a f te r four days on Czapek's agar floccose, green with white margin; drops co lo rless; reverse lig h t yellow. Colony 8 a f t e r six weeks velvety* 4*5 cm* "broad* dark green th in f e l t f wide submerged margin; s lig h tly zonate a t outer edge; drops co lo rless; reverse zones of pink to rose-tan; conidiophores rough; metulae not d iv a ric a te 12.5 - (16*5) - 20 u;sterigm ata 12.5 - (13.0) - 15.0 u; conidia globose* smooth* 3.75 - (3.83) - 4.1 u; g e la tin not lique­ fie d . Penicillium No* 131.1* Colony a f t e r two months on Czapek’s agar 4 cm. long by 1 1.5 cm* broad* lanate* dark green with narrow white margin; much wrinkled; zonate in age; reverse mouse gray-violet* wrinkled* zonate; conidiophores short* smooth* 48 - (60) - 72 u» asymmetrical; metulae d iv a ric a te 8.4 - (9*6) - 10.8 u; sterigm ata 4.8 - (6*8) - 8.4 u; 3-4-5 a t end of metulae; conidia globose* smooth, 2.3 - (3*1)-3.8 u; spore chains 36 u to 60 u. Penicillium Ho* 166 Colony a f te r four days on Czapek's agar blue; margin very fuzzy; drops co lo rless; reverse lig h t green* Colony a fte r two weeks velvety* gray-blue with white margin 2000 u, azonate; colony 500 u deep* center umbonate; reverse cream; agar s lig h tly cream-colored. Colony a f t e r six weeks dark blue-gray; zonate; reverse cream to rose *Dr. Thom examined th is culture and could not s a tis fy himself as to i t s sp e cific id e n tity ; i t seemed to conform to his 131*1 to which he had not assigned a sp e cific name. shades; conidiophores s lig h tly rough; metulae not d iv aricate 10 - (11*5) - 15 u; sterigm ata 10 - (11*0) - 12.5 u; conidia glo­ bose* smooth, 2*77 - (3.0) - 3.5 u; g e la tin liquefied* Penicillium No* 208 Colony a f te r four days on Czapek’s agar pale blue* very fuzzy with white margin; drops co lo rless; reverse white. Colony a f te r twenty-four days floccose* blue-green; zonate a t margin* re­ verse green-white; agar uncolored; metulae not d iv arica te 10 - (10) 10 u; sterigm ata 7.5 - (9.5) - 10 u; conidia globose, smooth, 3*1 (3*3) - 3.57 u. Mucor racemosus Fresenius The three forms of th is species th a t were most frequently iso la te d were the following. They do not seem to f i t other described species but are quite d is tin c t from one another. Form No. 1. Colony on dextrose agar a f te r ten days broadly spread­ ing, mouse-gray; reverse lig h t tan; colony 3*4 mm. high* very l i t t l e branched; slender sporangiophores 1-3 mm. long; no stolons or rh izoids; sporangia 25-40 u, some sporangia encrusted; columella free* mostly oval* few con ical, 19.2 - (25*4) - 33*6 u x 12.0 - (20.7) - 31.2 u; no chlamydospores; spores very s lig h tly punctate, very s lig h tly rough 4.0 - (6.7) - 11*5 x 3*6 - (5*4) - 9.3 u. This is a small form close to Mucor racemosus, but lacking chlamydospores, not cutinized* and not forming such t a l l sporangia as may occur in th a t species. 10. Form Uo. 2. Colony on dextrose agar a f te r ten days broadly spreading# gray; reverse lig h t tan; colony 1 cm. high; sporangiophores about 2-6 mrn.; branched irre g u la rly ; sporangium membrane rough but not h airy , breaks in to p ieces, does not deliquesce; sporangia 21.6 - (34.5) - 55.2 u; columella s lig h tly darkened; small c o lla r around base of columella; c o lla rs vary in size; colum­ e l l a absent in l a t e r a l branches; columella shape pyramidal to ovoid# 20-30 x 14-20 u; chlamydospores numerous; spores e l l i p t i c a l , very s lig h tly rough* v ariab le in size 3*3 - (6.6) - 9.6 x 2.4 (4.9) 7.4 u. Form Ho. 3. Colonies a f te r three weeks tan; sporangia 33.6 (48) - 84 u; sporangium membrane rough a l l over# breaks up# does not deliquesce; columella mainly panduriform# 19.2 - (25.8) - 32.4 x 12 (18.4) - 19*2 u; pale brown; chlamydospores numerous. frequently contains one, ra re ly 2 chlamydospores. The columella The sporangia are in 2 layers near the base of the t a l l sporangiospores. many branches with numerous sporangia of various s iz e s . There are These small sporangia have a columella but i t i s sm aller, almost sp h erical, about 7.2 x 7.2 u. The chlamydospores are quite numerous near the base of the sporangia. The lower mycelium i s much roughened. Spores hyaline# lig h t brown enmass; spores smooth# variable in size and shape# 3*6 — (5.5) — 12*0 x 3.6 - (5*3) - 9.6 u. The occurrence and incidence of molds on the various m aterials examined are presented in the following l i s t s . The in ­ cidence of the molds does not represent the extent of the mold con­ tam ination, except to measure to a degree the predominance of the species on the p a rtic u la r m aterial examined. Various cold storage p lan ts kindly submitted moldy eggs th a t were found a t the time eggs were removed from storage. These eggs showed very marked evidence of mold contamination on the surfaces. Some of the eggs were completely covered with mycelial growth and f r u itin g bodies. surface. Samples were made by using scrapings from the sh e ll The following molds were iso la te d : Penicillium citreo-roseum Dierckx Penicillium No. 102 Penicillium No. 36 Penicillium asperulum B ainier Penicillium chloro-leucon Biourge Penicillium ochro-chloron Biourge Penicillium oxalicum Currie and Thom Penicillium puberulum B ainier Penicillium citrinum Thom Penicillium janthinellum Biourge Penicillium casei Staub Penicillium crus tosum Thom Penicillium No. 131.1 Penicillium flavo-glaucum Biourge Penicillium viridicatum Westling Penicillium verrucosum Dierekx Penicillium roqueforti Thom A spergillus flavus Link A spergillus niger van Tieghem Fusarium sp. Mucor racemosus Fresenius Mucor lausannensis Lendner Chaetomium globosum Kunze Cephalosporium sp. Protabsidia Blakesleana Lendner Actinomyces sp. No. of is o la tio n s 12 2 5 10 16 4 10 36 19 16 14 16 17 10 6 12 9 15 35 5 50 30 24 15 12 Not counted In all# 400 iso la tio n s were made* Of th is to ta l 214 s tra in s were P e n icillia# 50 s tra in s were A spergilli# and 80 s tra in s were Mucors. A fter examining the mold f lo r a of the e x te rio r of the eggs# the eggs were opened a se p tic a lly and cu ltu res were made from the mold growths in the a i r sac. The molds found growing inside of the eggs were in a l l cases found on the surface of the membrane adjacent to the inner surface of the egg shell# in the a i r sac# and on the inner surface of the s h e ll. In no case did the mold seem to penetrate in ­ to the egg contents. The following organisms were obtained from eggs th at showed evidence of having mold growth on the in side of the s h e ll: Penicillium chloro-leucon Biourge Penicillium oxalicum Currie and Thom Penicillium citreo-roseum Dierckx Penicillium No. 30 Penicillium roqueforti Thom Penicillium No. 102 Penicillium No. 36 Penicillium s.sperulum B ainier Penicillium Kapuscinskii Zaleski Penicillium N0* 65 Penicillium No. 84 Penicillium puberulum B ainier Penicillium citrinum Thom Penicillium janthinellum Biourge Penicillium casei Staub Penicillium No. 123 Penicillium ochro-chloron Biourge Penicillium No. 166 Penicillium No, 208 Penicillium viridicatum West lin g Penicillium verrucosum Dierckx Penicillium crustosum Thom Penicillium No. 131.1 Penicillium flavo-glaucum Biourge Hormodendrum sp. No. of iso la tio n s 34 6 12 8 10 6 12 6 3 4 2 10 6 6 2 3 6 1 2 4 3 4 6 8 4 Of the to ta l of 170 iso latio n s* 166 s tra in s were Penic illia . Only one other genus* Hormodendrum, was iso la te d . This is exceedingly in te re s tin g as i t would appear th at the only organ­ isms capable of penetrating the egg s h e ll to a marked degree were the P e n icillia* Moldy cases obtained from several cold storage houses were examined fo r molds. These cases showed v is ib le evidence of mold development. The boxes were badly discolored by black and green mold spots. Cultures were taken from these areas. The follow­ ing molds were iso la te d : No. of iso la tio n s Penicillium No. 102 2 Penicillium citreo-roseum Dierckx 5 Penicillium Kapuscinskii Zaleski 4 Penicillium asperulum Bainier 4 Penicillium puberulum B ainier 5 Penicillium oxalicum Currie and Thom 3 Penicillium citrinum Thom 3 Penicillium janthinellum Biourge 8 Chaetomium globosum Kunze 4 T hirty-eight iso la tio n s were made. th irty -e ig h t cu ltu res were P e n ic illia . T hirty-four of the Only one other organism* Chaetomium globosum* was iso la te d . In a sim ilar manner, iso la tio n s were made from moldy f i l ­ le r s and f l a t s obtained from cold storage houses. These f i l l e r s and f l a t s lik e the cases showed marked evidence of mold formation. They were spotted with mold growth* generally black or green. In some instances the e n tire surface of the f i l l e r or f l a t was com­ p le te ly covered with m ycelial growth and f r u itin g bodies. The fo l- lowing c u ltu res were iso la te d : No. of iso la tio n s Penicillium corylophilum Dierckx 16 Penicillium chloro-leucon Biourge 25 P e n ic illi vim No. 102 10 Penicillium No. 36 13 Penicillium asperulum B ainier 19 Penicillium ochro-chloron Biourge 19 Penicillium roqueforti Thom 9 Penicillium citreo-roseum Dierckx 12 Penicillium No. 208 1 Penicillium puberulum B ainier 18 Penicillium casei Staub 4 Penicillium viridicatum Westling 7 Penicillium verrucosum Dierckx 9 Penicillium crus to sum Thom 11 Penicillium flavo-glaucum Biourge 14 Penicillium oxalicum Currie and Thom 10 Penicillium citrinum Thom 10 Penicillium janthinellum Biourge 16 Mucor racemosus Fresenius 27 Chaetomium globosum Kunze 4 Cephalosporium sp* 7 Fusarium sp* 3 Aspergillus niger van Tieghem 5 Of the to ta l 269 cultures* 233 s tra in s were P en icillia* Assuming th a t the molds occurring on the eggs* both insid e and outside* f ille r s * and f l a t s are the molds th a t are sig n ific a n t in the spoilage of eggs; a compilation of th e molds iso la te d from these sources was made to determine the re la tiv e re la tio n sh ip of the molds from each source* The data follow. A t o ta l of 843 is o la tio n s were made* I t i s in te re s tin g to note th a t 607* or 70*8 per cent* of a l l organisms Iso la te d were P e n ic illia . The most common species of P e n ic illia were Penicillium chloro-leucon and Penicillium puberulum* The data do not show th at any p a rtic u la r Penicillium i s significan t* The data do shov/ th at the P e n ic illia appear to he the most sig n ific a n t as f a r as egg spoilage i s concerned. Most species appear on the eggs* f i ll e r s * and fla ts* To determine the significance of new cases as a source of molds* a package of case boards was obtained d ire c t from the manu­ fa c tu re r. To be sure th at the molds represented i n i t i a l contamina­ tio n a t the source* cu ltu res were taken from the boards in the in te ­ r i o r of the package. ib le growth of molds. a se p tic precautions. in dextrose agar. These boards without exception showed no v is­ S livers of wood were cut from the board using These s liv e rs were then cultured by embedding From fiv e packages examined only two molds were isolated* namely* Mucor raeemosus and Chaetomium globosum. the boards showed a heavy incidence of both of these molds. All of In the lig h t of studies on eggs* the absence of P e n ic illia and A spergillus shows th a t the new case m aterial c e rta in ly i s not a source of object­ ionable molds. In a sim ilar manner new f i l l e r s and f l a t s were examined. These were also packaged by the manufacturer. were taken from the center of each package* Samples fo r cu ltu re The organisms iso la te d are as follows: Chaetomium globosum Eunze Hormodendrum sp. A spergillus nig er van Tieghem Mucor raeemosus Fresenius Fusarium sp. Penicillium asperulum B ainier Penicillium oxalicum Currie and Thom Penicillium Kapuseinskii Zaleski These data show th a t the molds on the new f i l l e r s and fla ts * although the incidence was low* are genera th a t may cause mold spoilage of eggs* The use of new f i l l e r s and fla ts* although the incidence of molds would he low, does not elim inate, as f a r as th is source of mold i s concerned* the contamination of the egg* The occurrence of molds on fresh eggs was also determined* Fresh eggs from the College p oultry plan t were obtained. Attempts to is o la te the molds by p la tin g some water used to wash the eggs was unsuccessful* showing th a t incidence of mold spores on the sur­ face of the egg was low. That molds were on these eggs was demon­ s tra te d by placing the egg a s e p tic a lly in to a s t e r i l e deep culture dish and pouring dextrose agar over the egg and incubating a t room temperature. By th is means the mold spores present were able to germinate and grow* The molds iso la te d were id e n tic a l with those found on moldy storage eggs* Market eggs shipped to the cold storage p lan t fo r storage were also examined. These eggs showed a much higher incidence of molds than the fresh eggs c ited above. These data show th a t the eggs entering the cold storage p lan t carry on th e ir surfaces the molds th at may ultim ately cause spoilage. To show the a b ility of Penicillium to penetrabe the shells* eggs showing no evidence of mold growth were washed with mercuric chloride and s t e r i l e water and inoculated with the spores by placing them in the a i r sac without contamination of the outer s h e ll sur­ faces. This was done by in je c tin g the spores d ire c tly into the a i r sac with a hypodermic needle. A fter the in fe c tio n was ma.de* the hole made by the needle was covered with p a ra ffin . A fter 14 months there was no evidence of mold growth on the outside of the eggshells. "Upon opening the eggs, the mold was found to be a liv e . The mold had grown extensively between and on both the outer sur­ face of the membrane and the inner surface of the s h e ll. had not penetrated e ith e r the sh e ll or the membrane. The molds The mold was also placed on the unbroken outer surface of the sh e lls of eggs s te r iliz e d as above and kept in a moist atmosphere for th is length of time. The molds were found to have penetrated the sh e lls and were also found to be growing in the a i r sac of the eggs* Discussion All the fungi obtained from eggs and egg m aterials in th is study belong to the large group of organisms which liv e prima­ r i l y as saprophytes in the s o il and on decaying vegetative m aterial in the s o il. They are, however, ubiquitous. the nest and on the feath ers of the hen. through the in te s tin a l tr a c t of chickens. They are present in They pass unharmed Therefore, i t is evident th at eggs are la id in an environment heavily seeded with these spores. That the packing ma.terials also contain these organisms i s demonstrated in th is study. They are present not only on used m aterials but on new cases, f ille r s * and f l a t s . These organisms also in h ab it the packing and storage houses* p a rtic u la rly when the humidity i s high enough to support Although the environment and th e ir growth. the sh e lls of both fresh and moldyeggs contains a large v a rie ty of molds* pen etrate tio n s. not a l l of them into the i n te r io r of the egg under cold storage condi­ Only two genera were included in those is o la te d from the i n te r io r of the s h e ll. These consisted of twenty-five species of Penicillium and four s tra in s of Honnodendrum. Although the genus Penicillium was is o la te d most fre ­ quently in these experiments and was probably responsible fo r the contamination* no one species can be incrim inated as a causa­ tiv e agent fo r the mold spoilage. All the evidence of th is study shows d e fin ite ly th at the mold found on moldy eggs comes from contamination from such sources as the handling and packing both before and a fte r reaching the cold storage p la n t. With conditions conducive to the growth of molds in the cold storage rooms* i t would appear impossible to prevent mold formation irre sp e c tiv e of the sa n ita ry precautions exercised in the handling and packing of the eggs. This statement should not be in te rp re te d to the e ffe c t that sa n ita tio n need not be exercised, because the extent and degree of contamination can be m aterially reduced by san itary precautions. PART I I . LABORATORY TESTS ON MYCOSTATIC AGENTS The only attem pts to control the development of mold on eggs in cold storage a t the present time have been by the ex­ posure of the eggs to e ith e r carbon dioxide or ozone during th e ir storage period. Ewell 1936 (4) and 1938 (5) reports th at 1.5 p.p.m. of ozone in the a is le s of cold storage rooms provides ample p ro tec tio n against molds a t a humidity of close to 90 per cent. However* the lit e r a t u r e reveals some controversy as to the value of ozone when used as a mold preventive ( l ) , although one man has sta te d th a t a f te r 48 hours in an atmosphere containing fiv e p a rts per m illio n of ozone* molds on moldy eggs w ill "actu ally disappear as though they had evaporated.11 Another plan t manager says " i t cannot be done." Probably one of the biggest drawbacks against the use of ozone in th is connection is the cost of in s ta llin g and maintainring the ducts. Most p lan ts have trouble with the corrosive actio n of the ozone on th is equipment ( l ) . In many cold storage houses eggs are stored in rooms with other products. Another d if f ic u lty would a ris e here because of the d estru ctiv e action of ozone on many food products th a t might be stored. Moran 1938 (10) found th at carbon dioxide would re ta rd mold growth and permit a higher r e la tiv e humidity. He found th a t a t a r e la tiv e humidity of 85 per cent, a ir containing two and one- 21. h a lf per cent carbon dioxide was necessary. At 32° F, 60 per cent carbon dioxide was necessary to suppress mold growth i f the atmos­ phere was saturated with water vapor. He also rep orts th at a t 60 per cent carbon dioxide storage the white as a whole was very watery. Dr. Mary Pennington* in a discussion on carbon dioxide* s ta te s th a t i t is d i f f i c u l t to maintain d e fin ite concentrations of a gas in rooms not adapted to gas storage (12)» and that the aver­ age cold storage room i s not designed fo r gas storage. Therefore* i t was deemed necessary in the search fo r a mycostatic agent to fin d a compound th a t was in a so lid or liq u id s ta te ra th e r than a gas* inasmuch as gas storage does not seem to be p ra c tic a l under present conditions in cold storage p la n ts. In the stu d ies to find a su ita b le mycostatic agent of th is kind* i t was f i r s t necessary to find a su ita b le laboratory pro­ cedure th a t would produce re s u lts a t le a s t somewhat comparable to f i e l d conditions. A fter a number of procedures had been teste d i t was found th a t the most su ita b le method was as follows: The myco s t a ti c agent was mixed in varying concentrations with a su itab le s t e r i l e nut­ r ie n t medium, such as beanpod or dextrose agar. The media contain­ ing the mycostatic agents were poured into s t e r il e p e tr i dishes and the agar was allowed to harden. Spores of the various molds were placed on the surface of the medium in a circumscribed area. Growth on the p lates was recorded q u a n tita tiv e ly by the extent and degree of development. Two methods of* co n tro llin g mold growth appeared to he worth te s tin g ; f ir s t* an agent with marked mycostatie p ro p erties with low or negative vapor tension which a c ts by contact with the mold spore or mycelium; and* second* an agent with marked mycos t a t i c p ro p erties with a vapor pressure s u ffic ie n t to carry the in ­ h ib itin g actio n to the mold without d ire c t contact with the mold spore or mycelium. The f i r s t type of compound could a t le a s t theo­ r e t i c a l l y be incorporated into the f i l l e r s and f l a t s to hold the mold growth to the in d iv id u al egg in each c e ll or i t might be brought in to d ire c t contact with the eggs by dipping or spraying a t the time they were placed in the case. The second type of compound could be incorporated into the f i l l e r s and f l a t s or sprayed on the eggs in lik e manner* but not only could these agents in h ib it the growth of molds on the f i l l e r s and fla ts* but they might also maintain s u ffic ie n t vapor tension around the egg to prevent mold formation on the ind ivid ual egg. The f i r s t step in conducting these laboratory studies was to fin d su ita b le members of each type of compound. In order to lim it the amount of work* only three organisms were used in the e a rly stu d ies. These were picked largely a t random except th a t in the case of the PeniciIlium one of the most frequently occurring species was selected. The three organisms were P e n icil- lium puberulum* A spergillus n ig e r* and a species of A ltern aria. L ater stud ies were conducted with additio nal stra in s and the fin a l te s ts were made with mixtures containing 14 d iffe re n t commonly occurring species of molds. Although a number of contact agents was tested* only a few w ill be presented* inasmuch as most of the compounds had l i t t l e or no m ycostatie actio n even in strong concentrations. were several copper s a l t s . Among these Although copper is not perm issible in foods* s t i l l i t was hoped th at d ilu te solutions might show s u ffi­ c ie n t m ycostatie actio n to allow th e ir use in f i l l e r s and f l a t s . This would in no way a ffe c t the eggs. Copper s a lts were selected because they are widely used in the co n tro l of fungus diseases of p la n ts. The la rg e st group of fungicides in present use contains as i t s active agent some form of copper. The following copper s a lts were te ste d : cupric n itra te * cupric oxide* cupric sulphate* cupric carbonate* cupric ammonium sulphate* cupric ammonium chloride* cupric a c e ta te , and cupric c h lo rid e. These compounds were te ste d against Penicillium puberulum, A spergillus n ig e r* and a species of A ltern aria. The data are pre­ sented in Tables I* II* and I I I . The to x ic ity of the copper s a lts varies both with the sp e cific nature of the s a l t as well as with the resistan ce of the te s t organ­ ism. Cupric oxide and cupric carbonate had no mycostatie actio n on a l l th ree organisms in concentrations up to 1.0 per cent, the strong­ e s t concentration te ste d . The remaining s a lts a l l in h ib ited the development of Penicillium puberulum and A ltern aria sp. in concert- tr a tio n s of 0.5 per cent* Only cupric s u lfa te and cupric acetate in h ib ite d A spergillus niger in concentrations of 0.6 and 0.5 per cent* resp ectiv ely . Doran 1923 (3) sta te s that Clark found Asper­ g illu s to be more r e s is ta n t to copper than Penicillium , which agrees with the above data. I t i s commonly believed th at many fa c to rs enter into the amount of resistan ce of spores to toxic elements. Whetzel and Mc- Callan 1930 (16) found th at older spores were more se n sitiv e to copper than young spores. Doran 1923 (3) found th at d ilu tio n s of fungicides th at were toxic near the maximum or minimum temperature fo r germination of spores were not toxic a t the optimum temperature f o r germination. He also found th at KMzopus nigricans was eight times as susceptible to cupric sulphate as representatives of the Uredi n ales. The data show th a t f i r s t the concentration necessary to in­ h i b it mold growth i s f a r too strong fo r p ra c tic a l use* and, second* th a t the marked v a r ia b ility in resistan ce of the three molds would make the use of such preparations vary u n reliab le in p ra c tic a l use. The second se rie s of compounds consisted of compounds hav­ ing low to x ic ity which would not be objectionable in the presence of foods. Of th is group, three are presented, namely, sodium borate, boric acid, and sodium benzoate. Tables IV, V* and VI. The re s u lts are presented in The same te s t organisms were used as those used in the te s ts on the copper s a l t s . In Table IV are presented, the data fo r these three pre­ paratio n s using concentrations varying from one to 0.1 per cent. Sodium benzoate had no in h ib itiv e e ffe c t even in concentrations of one per cent and boric acid showed only s lig h t in h ib itin g action on A lte rn a ria sp. and A spergillus n ig e r. Sodium b o rate, however, showed marked in h ib itin g action even in the weakest concentration on both A spergillus niger and Penicillium puberulum. A ltern aria grew in a l l d ilu tio n s except one per cent# To determine whether or not there might be a difference in species re sista n c e , 14 s tra in s of P e n ic illia were teste d in var­ ious d ilu tio n s of these three compounds. The te s t organisms were seven-day old c u ltu res of Penicillium puberulum (4 s tr a in s ) , Peni­ cilliu m ro g u e fo rti, Penicillium verrucosum, Penicillium c a s e i, Peni­ cillium citrinum , Penicillium crustosum, Penicillium oxalicum, Peni­ cilliu m 131.1, Penicillium flavo-glaucum, Penicillium janthinellum , and Penicillium viridicatum . All 14 c u ltu res grew in a l l d ilu tio n s of sodium benzoate, showing that a l l of the stra in s tested were equally re s is ta n t to th is compound. The re s u lts with sodium borate are presented in Table VT. I t w ill be observed th a t not only does the resistan ce of d iffe re n t species vary a t the same concentration of sodium borate, but th at 4 d iffe re n t stra in s of PeniciIlium puberulum show a varied resistan c e. S tra in 1 is in h ib ited by a concentration of 0.2 per cent, s tr a in 2 a t 0.6 per cent, s tr a in 3 by a concentration of 0.5 per cent, and s tr a in 4 by a concentration of 0.7 per cent. Of the three compounds reported, only sodium borate showed any promise of value and as indicated in Table IX, such marked v a ri­ a b i l i t y occurred in the various s tra in s of P e n ic illia coupled with the lack of in h ib itio n of A ltern aria sp. demonstrated th at the use of th is compound appeared to have l i t t l e value. Two commercial contact agents, "Moldex11 and ‘'Prevento,'1 were submitted fo r examination. The “Moldex11 was recommended as a mold preventative on eggs and "Prevento11 for mold control on fru its . Both were recommended as contact agents. These prepara­ tio n s were teste d in the same manner as the previous experiments and with the same three genera as before. Prom the data presented in Table VII, i t i s c lear th a t the preparation '•Moldex" has slig h t m ycostatie powers, but the preparation "Prevento" has none in the stro n g est concentration tested. As rep resen tativ es of the second type of compounds with high vapor tension a group of phenol preparations were teste d by the agar p la te method. These included sodium-ortho-phenol phenate, sodium 2, 4, 5 trichlorophenate, sodium 2 chloro-ortho-phenyl phenra te , sodium 2, 4, 5, 6 tetrachlorophenate, and sodium pentachlorophenate. When sodium-ortho-phenyl phenate and sodium 2 chloroortho-phenyl phenate were te ste d against the 14 above s tra in s of P e n ic iI lia th e ir mycostatie a b il i ty was found to be considerable and th e ir e ffe c t against the d iffe re n t species quite constant. These data are shown in Tables VIII and IX. The mycostatie value of these compounds, i . e . , sodium— ortho-phenol phenate, sodium 2, 4, 5 trichlorophenate, sodium 2 chloro— ortho-phenyl phenate, sodium 2, 4, 5, 6 tetrachlorophenate, and sodium pentachlorophenate^ was teste d on 4 genera of molds, 3 of which have not been presented above. in Table X. These data are presented These three organisms, namely, A spergillus n ig e r, Chaetomium globosum, and Mucor racemosus were commonly is o la te d from eggs and egg-case m aterial. The mycostatie e ffe c t on these four molds showed Mucor racemosus the most re s is ta n t with Penici H i urn puberulum next. Ina.s- much as the l a t t e r organism represents the most common egg contam­ in a tio n , the in h ib itio n of Mucor racemosus represents an effectiv e d e stru ctio n or in h ib itio n of a l l other molds. Although the mycostatie e ffe c t of these phenol prepara­ tio n s was not constant against the four molds, they showed marked m ycostatie a b il i ty against a l l of these organisms. Compounds sodium 2, 4, 5 trichlorophenate, sodium 2, 4, 5, 6 tetrachlorophenate, and sodium pentachlorophenate appear to be the most e ffic ie n t mycos t a t i c agents in th is group of compounds. To t e s t the value of the phenol d eriv ativ es as mycostatie agents when applied to cases, f i l l e r s , and f l a t s , two-inch squares of cases, f ille r s * and f l a t s were prepared. These were soaked fo r fiv e minutes in the various d ilu tio n s of the phenol preparations and then placed in d iv id u a lly in deep cu ltu re dishes. Subsequently they were seeded w ith a spore mixture of four d iffe re n t genera of molds, namely, A spergillus n ig e r, Penicillium puberulum, Chaetomium globosum, and Mucor racemosus. A piece of moist cotton was placed beneath each square in the deep cultu re dish during the incubation period. This cotton was kept satu rated with water to insure proper moisture conditions fo r the growth of the molds. This method of ap p licatio n of the mycostatie agents did not in­ sure th a t the egg-packing m aterials would soak up the same concen­ tr a tio n of the compound as was present in the solution; b u t, as can be observed in Table XI, the egg-packing m aterials can be made to r e s i s t mold growth by being impregnated with e ffectiv e concentra­ tio n s of these phenol p rep aratio ns. A prelim inary experiment was performed in which various methods of applying the chemical agents were used in an e ffo rt to determine which was most e ffe c tiv e . There were several methods th a t might be effe ctiv e fo r th is purpose, such as introducing the compound into the humidi­ f i e r s , placing a so lu tio n of the compound in the storage room, using the compound in the dry form in the room, or simply impreg­ n atin g the f i l l e r s and f l a t s with the compound. mouth Mason ja r s were used as containers. Two-quart, wide- Four eggs were placed on a wire screen ra ise d above the bottom of the f r u i t j a r by a 2 1/2 inch block of wood* The eggs were enclosed w ithin a minia­ tu re “egg case11 made from egg-case f i l l e r and f l a t m aterial cut to f i t the mar* Water was placed in the bottom of the j a r to keep the atmosphere sa tu rated with moisture* All cases* f i l l e r s , flats* and eggs were sprayed with a heavy suspension of molds before be­ ing packed* Sodium 2 chloro-ortho-phenyl phenate and sodium 2* 4, 5 trichlorophenate were used in three ja r s each* being applied as a dry powder, as a solution* and by impregnating the egg-case m aterials* The data obtained a f te r two months a t room temperature show (Table XII) th a t the impregnation of the packing m aterials i s the most e ffe c tiv e method of applying mycostatie agents to the eggs. In order to determine which of the phenol preparations was the most e ffe c tiv e , paper prepared by the Dow Chemical Co. and containing various amounts of the phenol deriv atives was te ste d fo r i t s mycostatie e ffic ac y . Three types of impregnated paper were used, containing 0.81 per cent sodium 2, 4, 5 trichlorophenate, 0.92 per cent sodium 2, 4, 5* 6 tetrachlorophenate, and sodium pentachlorophenate 0.86 per cent dry weight, respectively. A second set of experimental ja r s was prepared using th is impregnated paper fo r f i l l e r s and flats* The ja rs were se t up as in the previous experiment and the humidity maintained in the same manner as before. A fter three months the eggs in the four control ja r s showed a so lid mass of white and green mold and the f i l l e r s were covered with mold# In the ja r s containing paper impregnated w ith sodium 2* 4* 5* 6 tetrachlorophenate a l l of the eggs were s lig h tly moldy# The f l a t s and f i l l e r s were free from mold# The paper containing 0*86 per cent sodium pentachlorophenate in h ib ited mold growth on the f l a t s and f i l l e r s completely* but the eggs showed a s lig h t mold development a t the points of contact with the fille rs . The paper impregnated with sodium 2* 4* 5 trichlorophenate completely in h ib ite d a l l mold growth both on the eggs and on the f l a t s and f ille r s # Of the phenol preparations te ste d , the most s a tis fa c to ry from the standpoint of in h ib itin g mold growth were sodium 2, 4, 5 trichlorophenate, sodium 2, 4, 5* 6 tetrachlorophenate, and sodium pentachlorophenate# As judged from the laboratory te s ts , these phenol pre­ p aratio n s are b e tte r mycostatie agents fo r use in preventing mold growth on eggs and egg-packing m aterials, not only because they are more e ffe c tiv e mycostatie agents than the copper compounds or any of the other chemical agents te ste d , but also because they have a vapor pressure s u ffic ie n t to exert a mycostatie e ffe c t on the sh e ll when the compound is impregnated in the packing m aterial. PART I I I . COLD STORAGE UNDER COMMERCIAL COI3DITIONS WITH HIGH AND LOW HUMIDITIES With these laboratory te s ts completed, a f ie ld te s t was made using five half-cases of eggs. Four of these half-cases o f eggs were sprayed heavily with a heavy suspension of the 18 species of molds previously described. In each instance the molds were applied as each la y e r of eggs was placed in the cases. f i f t h h a lf-c ase was kept as a c o n tro l. The Three of the wooden cases were spenged with a 0.1 per cent so lution of a mycostat* three phenol preparations being used. As each layer of eggs was placed in the case, 0*1 per cent solution of the mycostat was sprayed lig h t­ ly over the eggs. One of the cases seeded w ith mold spores was tre a te d in the same manner with water without the addition of the mycostat. The f i f t h h a lf-c ase , the control, was tre a te d with water but no molds were applied. The cases of eggs were placed in cold storage fo r fiv e months when the eggs were removed and exam­ ined c a re fu lly fo r mold formation. Table X III. The data are presented in The data show th a t when sodium 2 chloro-ortho-phenyl phenate was applied, only three eggs showed signs of mold forma­ tio n and a l l f l a t s and f i l l e r s were mold fre e . Where sodium 2, 4, 5 trichlorophenate was used only 29.1 per cent of the eggs showed mold as compared to 94.1 per cent in the untreated case. Although the m ycostatie agents were applied to the egg and egg-packing 32. m aterials as a lig h t spray* and the spore load of the eggs was heavy* the phenol preparations used were able to decrease the development of mold on the eggs m aterially# These eggs were stored under the conditions of a low r e la tiv e humidity, about 85 per cent, but the cases* f ille r s * fla ts* and eggs were wet a t the time of storage. Because the humidity was not accurately controlled* and the amount of moisture th at the eggs were exposed to was uncertain* b e tte r storage conditions were sought fo r the succeeding experiments# Humidity i s such a primary fa c to r in the development of mold in cold storage houses th a t i t was thought necessary to t e s t a number of mycostatie compounds under conditions of controlled humidity much higher than i s ever used in cold storage rooms where eggs are stored# Commercially packed eggs were stored in an experimental room* a t re la tiv e hum idities from 92 to 100 per cent and a tem­ p eratu re of 32° F# For comparative purposes* eggs were also stored a t r e la tiv e hum idities of 88 per cent# Solutions of various myco- s t a t i c agents were sprayed over the eggs or containers or both im­ mediately p rio r to storage# At the end of three and six months sto rage, the eggs were examined fo r evidence of mold growth. At ♦These te s ts were made possible through the cooperation of the Central Fiber Products Co., Dow Chemical Co., United States Cold Storage and Ice Co., American I n s titu te of Poultry Industries* and Swift and Company# The main f ie ld te s ts were conducted a t the United S tates Cold Storage and Ice Company plant a t Chicago. the end of six months the eggs were removed from storage, ucandled f o r appearance, and examined fo r o f f - ta s te . Observations from these experiments follow. I. Influence of high re la tiv e humidity on growth of molds on eggs and egg packages as compared to low re la tiv e humidity. Both o il- tr e a te d and regular-pe.ck eggs were included in the experiments. In Table XIV i s presented a comparison of r e la tiv e hum idities of 88 and 98 per cent on regular pack eggs. In th is ta b le are presented data obtained on eggs tre a te d with water or d ilu tio n s of the mycostats, which were so d ilu te as to have l i t ­ t l e or no mycostatie p ro p e rtie s. I t w ill be observed th at a t a r e la tiv e humidity of 88 per cent, with the exception of one batch of eggs, no mold was evident a t the end of six months storage# With a r e la tiv e humidity of 98 per cent, on the other hand, the inr* cidence of mold was extremely high on eggs, f i l l e r s , f l a t s , and cases. The d ra s tic conditions of the experiments a t 98 per cent r e la tiv e humidity were c le a rly demonstrated by the e x te rio r appear­ ance of untreated cases, which presented a so lid mass of mold myce­ l i a l growth and spores. In the case of eggs tre a te d with water and d ilu te solutions of mycostatie agents, the cover of the case some­ times adhered to the top f i l l e r s by the attachment of the mycelial filam ents to both surfaces# In many cases, the egg in each f i l l e r ­ c e ll was completely covered with white filam ents of Mucors* The e ffe c t of humidity on mold development on o il- tr e a te d eggs i s presented in Table XI* was present on eggs, f ille r s * I t w ill be observed th at no mold f l a t s , and cases a t a re la tiv e humid­ i t y of 88; whereas a t a re la tiv e humidity of 98 per cent, f i l l e r s , f l a t s , eggs, and cases showed the presence of mold. The extent of contamination appeared to be le ss but the type of mold was m a te ria lly d iffe re n t. The o il- tr e a te d eggs were covered with spots due to the growth of Penici I l i a on the sh e lls; whereas the regularpack eggs showed very l i t t l e development of P e n ic illia on the sh e lls but showed an abundant development of Mucors. This seemingly s p e c ific development of mold was probably due to d ifferen t i n i t i a l contamination of the eggs# The above data show quite conclusively th at high r e l­ a tiv e hum idities (98 per cent) are more conducive to abundant mold growth than low hum idities (88 per cent)# II. The e ffe c t of wetting eggs, f i l l e r s , f l a t s , and cases on the keeping q u ality of eggs stored a t various humidities# In Table XVI are presented data showing the keeping q u a lity of eggs which were wet or stored in wet f i l l e r s and f l a t s and then stored a t a re la tiv e humidity of 88 per cent. The eggs which were stored in dry f i l l e r s and f l a t s at the end of six months f a ile d to show any spoiled eggs, while of the batches of wet eggs or eggs stored in wet f i l l e r s and f l a t s , four batches showed no spoilage and five batches showed 3, 6, 11, 31, and 95 per cent, re sp ec tiv e ly , of spoiled eggs# The d ata fo r the eggs stored a t a re la tiv e humidity of 98 per cent are presented in Table XVTI. In con trast with the dry eggs stored in dry containers, which showed no spoilage a t a r e la tiv e humidity of 88 per cent, the eggs prepared sim ilarly and stored a t a r e la tiv e humidity of 98 per cent showed 0.5, 6, and 9 per cent spoilage a t the end of three months storage in the three batches of eggs te s te d . The wet eggs or eggs stored in wet f i l l e r s and f l a t s a t a r e la tiv e humidity of 98 per cent showed, a f te r three months storage, spoilage from 3 to 77 per cent fo r the 9 cases l i s t e d in the tab le with the general average well over 50 per cent. The data show th a t w etting the eggs or containers has marked e ffe c t on the ultim ate spoilage of the egg, p a rtic u la rly i f the humidity of the storage room i s high. t e r i a ra th e r than fungi* This spoilage was larg ely due to bac­ I t would appear th a t the presence of a water film on the surface of the egg destroys the pro tectiv e film on the egg surface or th a t i t o ffe rs a favorable medium fo r the growth of b a c te ria th a t u ltim ately penetrate the egg sh e ll and thus invade to the egg contents. High hum idities seem to be predispos­ ing fa c to rs toward egg spoilage. I t i s in te re s tin g to note, how­ ever, th a t the presence of a mycostatie agent on the egg sh e ll or on the f i l l e r s and f l a t s seems to lessen th is b a c te ria l spoilage m a te ria lly . Ill* The in v e stig a tio n of wood used in containers* The cases used in these experiments were manufac­ tured from several kinds of wood* At the completion of three months storage, i t was observed th a t c e rta in containers, which were untreated by m ycostatie agents, fa ile d to show any mold form­ a tio n . whereas other containers also untreated were completely covered with a heavy growth of mold* The cases which were r e s i s t ­ ant to mold growth were made of S itka spruce heartwood.* The cases showing marked mold growth were made of Tupelo gum, swamp black gum, or cottonwood* Gum and cottonwood cases trea te d with sodium 2, 4, 5 trichlorophenate and sodium pentachlorophenate were r e s is ta n t to mold development. These data are in agreement with the findings of Hubert 1938 (7) who demonstrated th a t pentachlorophenol* O-phenyl— phenol, 2 chloro-o-phenylphenol, and te tr a chlorophenol were e ffe c tiv e in the control of fungi in millwork products th at were to be exposed to excessive moisture. IV* The in v e stig a tio n of f i l l e r s for mold growth. Three types of f i l l e r s were used in these experi­ mental stu d ies, namely, brown, gray* and white* In each case of eggs, brown and white or brown and gray f i l l e r s were used. It ♦Samples of each type of case were submitted to Dr. A. Koehler of the Forest Products Laboratory a t Madison, Wisconsin, and Dr. A. J . Panshin of Michigan State College fo r id e n tific a ­ tion* was found th a t the amount of mold growth was much g rea ter with hrown f i l l e r s than with e ith e r gray or white f i l l e r s . In the case of the gray or white f i l l e r s , mold was generally present only a t the point of contact with the egg; whereas, with the brown f i l ­ l e r s , mold growth occurred over the e n tire surface of the f i l l e r . The development of Mucors was much more pronounced on the brown f i l ­ le rs . A ll f i l l e r s tre a te d with 0.5 per cent sodium 2, 4, 5 t r i ­ chlorophenate or sodium pentachlorophenate were r e s is ta n t to mold growth. V. In v estig atio n on the treatment of eggs by myco s ta ts . In the studies c ite d , experiments were set up in two ways, (1) f i l l e r s , f l a t s , and cases treated but eggs untreated, and (2) f i l l e r s , fla ts* cases, and eggs tre a te d . An examination of the d ats shows th a t the condition of the eggs was sim ilar to that of the f i l l e r s and f l a t s used in the same case. The trea te d eggs and the untreated eggs appeared about the same where the f i l l e r s and f l a t s were tre a te d in a sim ilar manner. There appeared to be l i t t l e value in the treatment of the eggs themselves. This i s , of course, exceedingly fortunate because the treatment of eggs would be d i f f ic u lt in commercial p ra c tic e . VI. In v estig atio n s on mycostat treated f i l l e r s and f l a t s . A p ra c tic a l solutio n to the problem of applying the m ycostatie agents to the f i l l e r s and f l a t s without wetting the eggs was thought to be by the chemical impregnation of these packing m aterials a t the time of manufacture. This would elim inate the b a c te r ia l decomposition and lowered eating q u ality of the eggs due to excess wetting before storage. Therefore, the next cold sto r­ age experiment consisted of sto rin g eggs, without any previous tr e a t­ ment, in the experimental cold storage room a t the United States Cold Storage Plant a t Chicago. The f i l l e r s and f l a t s used in packing these eggs had been impregnated with various concentrations of the three phenol p reparations found to be the most e ffe c tiv e in preventing mold devel♦ opment in the previous experiments, namely, 0.81$ sodium 2, 4, 5 trich lorop henate, 0.92$ sodium 2, 4, 5, 6 tetrachlorophenate, and 0.86$ dry weight sodium pentachlorophenate. A fter 79 days in storage the control eggs stored in reg­ u la r f i l l e r s and f l a t s showed mold formation a t the points of contact with the f i l l e r s , whereas the eggs in the treated f i l l e r s and f l a t s showed none. A fter fiv e months these eggs were s t i l l mold-free. In these experiments in which compounds with high vapor tension were used, Azochloramide 100 p.p.m. available chlorine was included as an example of the contact type of compound to deter- ♦This paper was prepared by the Dow Chemical Co., Midland, Michigan. mine whether or not the use of s t e r i l e f i l l e r s and f l a t s would prevent the molding of the eggs stored under such conditions* This compound had been suggested as a possible solution fo r the control of mold on cold storage eggs. This did not prove to be the case as mold development did occur on the eggs stored in f i l l e r s and f l a t s rendered s t e r i l e by the use of Azochloramide. VII* The influence of the mycostatie agent on the quality of the eggs. The phenol d eriv ativ es th at are presented in th is * th e s is are characterized, as sta te d previously, by having vapor p ressures such th a t the vapor given o ff by the chemical i s in suf­ f i c i e n t strength to in h ib it the growth of molds on th e egg surfaces, although the compound is impregnated in the f i l l e r s and fla ts* These vapors are gradually but continuously given off by the com­ pounds. Further, these vapors having ta s te and odor might a ffe c t the flav o r and odor of the egg. I t was deemed advisable to place eggs in storage in f i l l e r s and f l a t s impregnated with a high content of the chemicals to see whether or not the q u a lity would be affected . Eggs were placed in storage using these trea te d f i l l e r s and f l a t s a t the United S tates Cold Storage plant a t Chicago, Octo­ b er 28, 1938. were used: The following concentrations of the mycostatie agents 0.81$ sodium 2, 4, 5 trichlorophenate; 0.92$ sodium 2, 4, 5, 6 tetrachlorophenate; and 0.86$ dry weight sodium penta­ chlorophenate. These concentrations were purposely made much higher than w ill he used to in h ib it mold growth under commercial condi­ tions* At the same time eggs were stored under id e n tic a l condi­ tio n s in ordinary f i l l e r s and f l a t s . Eggs were removed and examined fo r odor and ta s te on Jan­ uary 12, 1939 , and February 10, 1939* These examinations were made by Mrs* Kathryn Niles of the American I n s titu te of Poultry In d u strie s and Miss R. Griswold and Miss D. Grantham, of the Divi­ sion of Home Economics, Michigan State College* The re s u lts of these examinations showed th at the eggs stored in f l a t s and f i l l e r s trea te d with these phenol preparations were no d iffe re n t in odor and ta s te than the eggs stored under id e n tic a l conditions in ordinary f l a t s and f i l l e r s . Discussion The above re s u lts prove quite conclusively th at the most e ffe c tiv e method of co n tro llin g the development of mold on cold storage eggs is by the impregnating of the packing m aterials a t the time of manufacture* by some chemical substance. The id eal substance indicated would be one (a) with maximum to x ic ity toward the fungi p e cu lia r to eggs, (b) available a t low co st, (c) having no odor, and (d) showing some but re la tiv e ly low vapor pressure. The compound should possess th is l a t t e r c h a ra c te ristic because eggpacking m aterials are used for more than one season, and also be­ cause a compound with a re la tiv e ly low vapor pressure would insure m ycostatie p ro tec tio n during the l i f e of the packing m aterials so treated* None of the phenol compounds te ste d possessed a l l of the above c h a ra c te ris tic s , a t the concentration of maximum e f f i ­ ciency. Sodium 2* 3* 4 t r i chloro phenol hs.s the advantage of be­ ing a very e ffe c tiv e fungicidal agent but i t s cost i s re la tiv e ly high for commercial use and i t has a high vapor pressure. Sodium pentachlorophenol does not have as high a to x ic ity toward the fungi te ste d as sodium 2» 4, 5 trichlorophenol under the conditions of these experiments. But i t w ill be remembered th at the condi­ tio n s se t up were f a r more severe than would ever be experienced commercially* The experiments were purposely made more severe to t e s t the mycostatie p ro p erties of the compounds to the f u l le s t ex­ t e n t. Under conditions of ordinary commercial storage sodium pen­ tachlorophenate was found to be the most s a tis fa c to ry mycostatie agent. Sodium pentachlorophenate is also more suited to commercial use because of i t s mild odor, lower vapor pressure, and low cost. SUMMARY 1* Representatives of ten genera were repeatedly iso­ la te d from eggs and egg packing m aterial. The m ajority iso lated “belong to the genus Penicillium , “but no one species predominated. 2. Not a l l of the molds penetrated into the in te r io r of the egg under cold storage conditions. Only two genera were iso­ la te d from the in te r io r of the s h e ll. These consisted of twenty- fiv e species of Penicillium and four s tra in s of Hormodendrum. 3. Two groups of compounds were te ste d for th e ir value as mycostatie agents to “be used to control mold development on egg in cold storage. The f i r s t group were compounds having l i t ­ t l e or no vapor tension which act by contact with the mold spore or mycelium* The second group were compounds with a vapor pres­ sure s u ffic ie n t to carry the in h ib itin g a ctio n to the mold with­ out d ire c t contact with the mold spore or mycelium. The compounds with r e la tiv e ly high vapor pressure proved to be the b est mycostatie agents both in the laboratory t e s ts and under commercial cold storage conditions. 4. Of a l l the compounds te ste d , the following proved to be the most e ffe c tiv e in con tro llin g the development of mold on eggs in cold storage; 2, 4* sodium 2* 4* 5 trichlorophenate, sodium 6 tetrachlorophenate, and sodium pentachlorophenate. Although sodium 2# 4, 5 trichlorophenate was the best mycostatie agent under the d ra s tic experimental conditions used, sodium pentachlorophenate was found to he the most s a tis fa c to ry m ycostatie agent under ordinary commercial conditions. I t was also b e tte r from the standpoint of low cost* s lig h t odor, and a r e la tiv e ly low vapor pressure* 5. Eggs stored in f l a t s and f i l l e r s trea te d with these phenol preparations were no d iffe re n t in odor and ta s te than the eggs stored under id e n tic a l conditions in ordinary f l a t s and f i l l e r s . 6. The most e ffe ctiv e method of applying the fungi- — ::;ei4e was by impregnating the f i l l e r s , f l a t s , and cases a t the time of manufacture. be of l i t t l e value. Treatment of the eggs themselves seems to T a b le I . M y c o s t a t ie p r o p e r t i e s o f e i g h t c o p p e r com pounds on P e n i c i l l i u m puberulum Per cent concentration of copper compounds .8 .9 .7 1,0 • 6 .5 .1 Control Mycostat - - - - - 2+ 3+ 2+ 2+ 2+ 2+ 2+ 2+ 3+ - - - - - 2+ 3+ 2+ 2+ 2+ 2+ 2+ - - - - - - 2+ 3+ Cupric ammonium chloride - - - - - - 2+ 3+ Cupric a c e ta te - - - - - - 2+ 3+ Cupri c chlo r i de - - - - - - - 3+ Cupric n itr a te - Cupric oxide 2+ Cupric s u lfa te - Cupric carbonate 2+ Cupric ammonium su lfa te 2+ 3+ e x c e l l e n t g ro w th ; 2+ g o o d g ro w th ; - no g ro w th 3+ T a b le I I . M y c o s t a t ie p r o p e r t i e s o f e i g h t c o p p e r compounds on A s p e r g i l l u s n i g e r . Per cent concentration of copper1 compounds .7 .9 .8 .5 1.0 .6 .1 Control Mycostat Cupric n itr a te 2+ 2+ 2+ 2+ 2+ 2+ 2+ 3+ Cupric oxide 2+ 2+ 2+ 2+ 2+ 2+ 2+ 3+ Cupric su lfa te - ? ? ? ? 2+ 2+ 3+ Cupric carbonate 2+ 2+ 2+ 2+ 2+ 2+ 2+ 3+ Cupric ammonium su lfa te 2+ 2+ 2+ 2+ 2+ 2+ 2+ 3+ Cupric ammonium chloride 2+ 2+ 2+ 2+ 2+ 2+ 2+ 3+ Cupric ace ta te - - - - - - 2+ 3+ Cupric chloride 2+ 2+ 2+ 2+ 2+ 2+ 2+ 3+ 3+ e x c e l l e n t g ro w th ; 2+ g o o d g ro w th ; - no g r o w th Table I I I , Mycostatic p ro p erties of eight copper compounds on a species of A lternaria. Mycostat Per cent concentration of copper compounds .7 •8 .9 • 6 .5 1.0 • 1 Conti Cupric n i tr a te - - - - - - 2+ 3+ Cupric oxide 2+ 2+ 2+ 2+ 2+ 2+ 2* 3+ Cupric su lfa te - - - + + + 2+ 3+ Cupric carbonate 2+ 2+ 2+ 2+ 2+ 2+ 2+ 3+ Cupric ammonium su lfa te - - - - - - 2+ 3+ - - - - - 2+ 3+ Cupric ammonium chloride Cupric a c e ta te - - - - - - 2+ 3+ Cupric chloride - - - - - - + 3+ 3+ e x c e l l e n t g ro w th ; 2+ g o o d g ro w th ; + f a i r g ro w th ; - no grow th T a b le I V . The m y c o s t a t i c p r o p e r t i e s o f t h r e e com pounds commonly u se d i n fo o d p r e s e r v a t io n . Mycostat and t e s t organism 1.0 .9 Per cent concent r a t i on of mycostat .7 .8 .6 .5 .4 •2 .1 Control .3 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 3+ 2+ 2+ 2+ 2+ 2*- 2+ 2+ 2+ 3+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 3+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 3+ Boric acid: A lte rn a ria sp. A spergillus niger PeniciIlium puberulum 2+ Sodium b o rate: A lte rn a ria sp. A spergillus niger Penicillium puberulum 3+ - 2+ 3+ Sodium benzoate: A lte rn a ria sp. A spergillus n ig er PeniciIlium puberulum 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 3+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 3+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 3+ 3+ e x c e l l e n t g ro w th ; 2+ g ood g ro w th ; - no grow th T a b le V* The r e s i s t a n c e o f f o u r t e e n c u l t u r e s o f P e n i c i l l i a to sodium b e n z o a t e Per cent concentration of sodium benzoate .1 .02 002 .0002 Control Organi sm .2 P. puberulum s tr a in 1 3+ 3+ 3+ 3+ 3+ 3+ P. puberulum s tr a in 2 3+ 3+ 3+ 3+ 3+ 3+ P. puberulum s tr a in 3 3+ 3+ 3+ 3+ 3+ 3+ P. puberulum s tr a in 4 3+ 3+ 3+ 3+ 3+ 3+ P. roqueforti 3+ 3+ 3+ 3+ 3+ 3+ P. verrucosum 3+ 3+ 3+ 3+ 3+ 3+ P. easel P. citrinum 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ P. crustosum 3+ 3+ 3+ 3+ 3+ 3+ P. oxalicum 3+ 3+ 3+ 3+ 3+ 3+ P. 131.1 P. flavo-glaucum 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ P. janthinellum 3+ 3+ 3+ 3+ 3+ 3+ P. viridicatum 3+ 3+ 3+ 3+ 3+ 3+ ■/ 3+ e x c e l l e n t g r o w th . T a b le VI* The r e s i s t a n c e o f f o u r t e e n c u l t u r e s o f F e n i c i l l i a t o sodium b o r a t e . Organi sm .8 P* puberulum s tr a in 1 P* puberulum s tr a in 2 P* puberulum s tr a in 3 P. puberulum s tr a in 4 P* P* P. P. P. roqu efo rti verrucosum casei citrinum crustosum P. P. P. P. P. oxali cum 131*1 flavo-glaucum janthinellum vi r i di catum Per cent concentration of sodium borate ■4 .7 .6 .5 .3 .2 .1 .02 Control . + + + + + + — .. — _ — — — — + + + + + + - - - + + + + + + + + + + + + + + + + + + + + + + + + - - - - + + + + + + + + + + + + 3+ 3+ + 3+ 3+ + + + •f + + + + + + 3+ e x c e l l e n t g ro w th ; + f a i r g ro w th ; - no g ro w th 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ T a b le V I I . The m y c o s t a t ic p ow ers o f com pounds on m ark et s o l d f o r m old p r e v e n t io n . Per cent concentration of mycostat .3 .2 .04 .03 .02 .009 Control Mycostat Test organism Moldex A spergillus niger 3+ 3+ 3+ 3+ 3+ PeniciIlium puberulum 3+ 3+ 3+ 3+ 3+ Chaetomium globosum 3+ 3+ 3+ 3+ 3+ Mucor racemosus 3+ 3+ 3+ 3+ 3+ Prevento .4 A spergillus niger 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ PeniciIlium puberulum 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ Chaetomium globosum 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ Mucor racemosus 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ ex cellen t growth; - no growth. T a b le V III* The r e s i s t a n c e o f f o u r t e e n c u l t u r e s o f P e n i c i I l i a to sod iu m o r t h o - p h e n y l p h e n a t e . Per cent concentration of sodium ortho-phenyl phenate .1 •2 .02 .002 .0002 Control Organi sm P* puberulum s tr a in 1 P. puberulum s tr a in 2 -f + 3+ — * + + 3+ — - T- + + 3+ — — + + P. roqueforti - - ■* + + 3+ 3+ p. verrucosum - - T + + 3+ Pm casei - - * + + 3+ P. citrinum - - T + + 3+ P, crustosum - - * + ■ I* 3+ P. oxalicum - - + + + 3+ P. 131.1 - - 7- + +■ 3+ P* flavo-glaucum - - + + + 3+ P. janthinellum - - * + + 3+ P* viridicatum — + + + 3+ P. puberulum s tr a in 3 P. puberulum s tr a in 4 + f a i r growth; + scanty growth; - no growth. T a b le IX . The r e s i s t a n c e o f f o u r t e e n c u l t u r e s o f P e n i c i l l i a t o sod iu m 2 c h lo r o - o r t h o - p h e n y l p h e n a t e . Organism P. puberulum s tr a in 1 P. puberulum s tr a in 2 Per cent concentration of sodium 2 chioro-ortho-phenyl phenate .1 .02 .002 .0002 Control + + 3+ + + 3+ P. puberulum s tr a in 3 3+ P. puberulum s tr a in 4 + + 3+ P. roqueforti + + 3+ P. verrucosum + + P. casei P. citrinum + + + + 3+ 3+ 3+ P* crustosum + + 3+ P. oxalicum + + 3+ P. 131.1 P. flavo-glaueum P. janthinellum + + 3+ + + + + 3+ 3+ P. viridicatum + + 3+ 3+ e x c e l l e n t g ro w th ; 2+ g o o d g ro w th ; + f a i r grow th ; - no gro w th 53. + to + of molds. to + + 03 CO CO CO CO CO CO + CO + + + + CO + CO + + + CO + CO + CO CO + CO + + + CO + CO + genera on four d erivatives phenol powers of five & + CO co CO + + + + + CO + + CQ + CO CO + + CO CO fe + + CO CO + + CO + + CO CO CO CO CO CO + CO CO CO o o CO ir CO + CO CO tt-i o P o •rl 43 © +p». P © o ao o + + CO CO + + + + + + + CO CO I I I I + CO + CO CO CO a o u© P4 © The mycostatic Table X* CO + © + CO + 43 + CO CO CO 03 43 u u _ C fl £iD & & CO - H P* P EH O 0) 43 cd 43 43 43 o - © I »>» H ■© *» O .q P Pf 43 © 4) < 0 o o p CO om p© Pi o •So 3 3 o P . U !h O Pi t* e © © •H © © rQ 0 -rl 0) oo © © ,0 ©o o CD P X> © o PM-f© a ,g © p H © ci •O*=* r* P cd p H q fl su *4 P P4 Ph p , 1 O t© S P o co O U ,p x i CO O P h Ph a p •t-t *© 0 CO • pq I 0 Pi LO O © rH 4 3 X i © O P T4 © ** U . P co 43 ft o »H £j0 P i Pi © © rH rH *h P O P . *rH © P © P©rQ mM*.H 0 p g p •H CO • O CO 0 JX. 1 1 Pi 0 O x i r—I X Pi 0 O p t, 43 »H a 6 43 o .© O >—I f© o p© © © x> o >* © P P P< O OO P *==!*« rH©. «*■ Control 54. + + to + to + to + to + to + to + >001 + to to + + + to + to + + .002 to + + + + + to + to + ,003 to + to + + + + f+> t + o to f+» t to to to to to to to to to to to DO f£ o h Per cent concentration of mycostat .009 .008 .007 .006 .005 ,004 EU) + + to to to + to S3 £ o u E lO + +» to •ft •ri + to H 03 + + ft +> + Si E lO Si to to & o + .02 CO ,03 f+9 t & o Si ElO >04 +9 S3 .2 CD cd o yt a> to Mycostat Test organism 0 to •r-t £ 0 o to o O £ +9 * <1) •rH CD etoj{ (D f t 0 ft 0 p IQ cti to o Si S o CD o O ft o «aH n ft f t * | a cd o 0 I cd Si «—i a CO 3 «—1 Si •ri CD tD .H <*J S3 ft ft *d) Si Si CD f t E*0 CD ft 0 -S i O ft rH f—1 * +> Si «j •h ^ cd o s3 ■d ■PH© O • • f t ft *f— f H • I «> o+3 3 cd u at •H -P O f | *d 0 H (D O 0) f t f t & u i ft o ft s O CO +9 o 0) f t cd o f t rH o 0 m a p ElO Si o o o S CD O