microbiological examination of fresh

Microbiological Examination of Freshand Frozen Fruits and Vegetables*

FRED W. TANNER, F.A.P.H.A.Department of Bacteriology, University of Illinois, Urbana, Ill.

BACTERIOLOGICAL examinationof foods has a several-fold pur-

pose: (1) to protect the consumingpublic from bacteria causing foodpoisoning and food-borne infections;(2) to determine the relation of theindigenous microbial flora to problemsof food preservation; (3) to secure in-formation concerning the conditionsunder which foods have been producedand handled. These purposes areespecially significant for fresh andfrozen fruits and vegetables.

Despite the fact that bacteriologyhas been of great service in the controlof certain foods, it is almost withoutvalue for routine control of others.These limitations are frequently notrecognized by those not familiar withbacteriological technic. Attempts todetermine the value of various methodsof examination and to arrive at standardprocedures are commendable. Methodsquite suitable for research may not besuitable for routine control work. Timeis an important factor since in controlwork it is essential to have results asquickly as possible.As indicated by the title, only fresh

and frozen fruits and vegetables areconsidered in this paper. It is proper toascertain the actual bacteriological con-dition of such foods as they appear on

*Read at a Joint Session of the Laboratory andFood and Nutrition Sections of the American PublicHealth Association at the Sixty-second Annual Meet-ing in Indianapolis, Ind., October 10, 1933.

the market, in order to determinewhether there is a need for control work.

FRESH FOODSFresh foods may be heavily seeded

with various types of micro6rganismswhich are picked up from the soil, dust,and agents with which they come incontact. A complete review of litera-ture need not be attempted here. Thatpathogenic bacteria might be dis-seminated by fresh fruits, has beenshown by Ehrlich,1 Abbott,2 Rommel,8Ressel,4 Neumann,5 Clauditz,6 Sartoryand Fillassier,7 and others. Sartory andFillassier examined fruits exposed forsale on Paris fruit stands. Numeroussaprophytic species were found to bepresent, most of which could be removedby careful washing. Garcia 8 foundEsckerichia coli to be always present oncommon fruits (apples, lemons, oranges,and bananas). Such data indicate thatundesirable bacteria may be present andjustify health officers and state andfederal inspectors in exercising vigilanceto insure sanitary fresh foods. Smeall 9has quite recently suggested that someof the minor gastrointestinal disturb-ances common during the fruit eatingseason may be due to bacteria on fruitand not to the fruit itself.

Attempts have also been made tostudy the problem by determining thelongevity of certain pathogenic bacteriaon the surface of fruits. Ko 10 was ableto isolate Eberthella typsi for a con-siderable time from fruit which had

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been sprayed with pure cultures.WThether the natural fruit juices arebactericidal, seems to have had littlestudy. Ko found that, in general, theacid juices were destructive; juices frompartially ripened fruit were moregermicidal than those from fully ripenedfruit. Smeall 9 reported that Eberthellatyphi remained viable for 68 days onthe surface of dates.

In general, fresh vegetables presentgreater sanitary problems than freshfruits. Vegetables are raised in soilwhere they are subjected to greaterpossibilities of contamination. Wurzand Bourges 11 reported disseminationof pathogenic bacteria by vegetablesgrown in polluted soil or watered withinfected water. Typhoid fever is saidto increase every year in Paris 12 dur-ing the late summer months, most ofit being attributed to fresh vegetablesgrown on sewage farms. While theoriginal purpose of such lands was forraising hay, or for orchards and pas-tures, excellent crops soon stimulatedtheir use for garden truck, much ofwhich is eaten raw. Every precautionshould be taken to insure that freshvegetables are harvested from un-polluted soil for many of them are eatenwithout cooking. Creel 13 observed norelation between the appearance of thevegetables and the presence of Eber-thella typhi. Vegetables which seemedto be clean to the naked eye, were foundto harbor the organism.

Epidemics of communicable diseaseshave been traced frequently to infectedfresh vegetables. It is necessary,therefore, to know something of theconditions under which such foods areraised. Kurk 14 examined the microbialcondition of vegetables bought on theopen market in Chicago. He particu-larly tried to find members of thecolon-typhoid group, streptococci, andanaerobic bacteria. Escherichia coliwas found on 22 of 29 samples of water-cress, celery, and lettuce. James 15

more recently studied fresh head lettuceand reported many more bacteria onthe exterior of the head than in theinterior. His work again showed thevalue of controlled conditions of storageand handling.

Just how much significance should begiven to the presence of lactose-fermenting bacteria on the foods underdiscussion is open to argument. Noinformation seems to be available onwhich to base an opinion. It is verydoubtful whether they may serve asindicators of pollution of fresh foods inthe same manner as they do of water.

Murillo 16 incorporated pathogenicbacteria in garden soil by spraying thesoil and food products grown thereon.Virulent organisms were found after 36days in soil and after 55 days in sterilesand. Such data indicate appreciablelongevity in soil. Melick 17 foundthat longevity of Eberthella typhi insoil depended on the source and strain,the time varying between 29 and 58days. Under natural conditions radisheswere infected after periods of 28, 35,and 37 days. Garden soil inoculatedwith typhoid excreta yielded viableEberthella typhi for 41, 34, and 35 daysin three experiments. Melick wasforced to the conclusion that fresh vege-tables raised on polluted soil may beunsafe. Furthermore, the ordinarymethods of preparation for the tablemight not render them safe.

These conclusions of actual experi-ments seem to be borne out by epi-demiological experience. Wary18 re-ported an epidemic of typhoid fever ina London suburb attributed to water-cress grown in beds fertilized withsewage. Another outbreak of 18 casesof typhoid fever traced to watercresssandwiches was reported from Phila-delphia.19 Pixley o attributed 2 casesof typhoid fever to uncooked rhubarb.Mores 21 believed that 49 cases oftyphoid fever in an insane hospital hadbeen caused by eating celery which had

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EXAMINATION OF FRESH AND FROZEN PRODUCE

been irrigated with sewage. Since it isknown that some of our pathogenicmicroorganisms are quite hardy, theymay be expected to survive during thecommercial age of fresh foods.

Bacteria apparently do not pene-trate the interior of sound fruits andvegetables. Mills, Bartlett andKessel 22 made such observations withvegetables using bacteria and particlesof carbon. They adhere to the surfaceand may be removed by the proceduresused in preparing them for the table.That these procedures may not alwaysbe reliable has been suggested byMelick.17 It is safer to prevent con-tamination than to rely on proceduresin the kitchen to avoid infection. Thesuperimposition of careful washing onfresh foods raised under satisfactoryconditions, gives foods which harborminimum amounts of foreign matter,including microorganisms. It has beenpointed out above that vegetables whichappear to be clean, may harbor patho-genic bacteria.

FROZEN FOODSFrozen or frosted foods have assumed

an important place on the Americanmarket., While this method of preserva-tion has been used for decades, onlyduring the last one, has it been extendedto a wide variety of foods. Publichealth aspects of frozen foods were dis-cussed by Fellers in 1931. At that time,there were few reports of experimentalwork in the literature and not muchwas known about the microbiologicalcondition of such foods, and some ofthe foods now being frozen were not sopreserved when Fellers prepared hispaper. Since then, results of severalextensive investigations have been re-ported and some of the conclusionswhich Fellers had to reach by analogyare now supported by experimentalwork. The discussion by Fellers dealtadequately with some of the generalhealth problems, such as raw materials,

effect on nutritive values, etc. It is myintention to consider some of the newerfindings. It should be stated that thisis done with no prejudice against suchfoods. They are probably as safe asfresh foods, if properly handled andkept frozen until used. Frozen foodsmust endure the same analysis thatfoods preserved by other methods havein the past. If they are considered tobe perishable and perhaps more sus-ceptible to attack by microorganismsthan are fresh foods, and are handledaccordingly, they will be satisfactoryfoods.

Several different positions have beenassumed by those engaged or interestedin the development of the frozen foodindustry. One of them is that little at-tention need be given to the sanitaryfeatures because to date they have notcaused infections or intoxications.Those who assume this position believethat harm is done the industry by suchdiscussions. Such a position is un-tenable and violates the simplest andmost fundamental laws of epidemiology.Application of such logic to the entirefield of public health would lead tochaos, and discredit the public healthofficer. Dr. Hurty's homely poem onpage 796 of the August, 1933, JOURNAL,"The Fence or the Ambulance," aptlyexpresses the situation. In this case itis better to erect fences and keep themin repair than to neglect them and haveto call the ambulance. It is better toprevent trouble than to cope with itafter it has appeared. The fence inthis case is so easy to construct-simplysound raw materials, properly and com-pletely frozen, and kept frozen untilused by the consumer. The frozen foodindustry must decide whether it iswilling to assume the losses which mightresult from improper handling of frozenfood. The ripe olive industry wasruined for a number of years after theoutbreaks of botulism which were tracedto it. Even today, about 15 years after

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the spectacular Canton, Ohio, outbreak,the ripe olive is viewed with suspicionby some laymen. Nothing is to begained by refusing to face these hazards.On the other hand, a finer and saferfood will be available if they are facedand everything done to prevent spoilage.

There has been a tendency in somequarters to compare frozen or frostedfoods to other preserved foods withoutconsidering the differences which ob-tain. Other preserved foods may havereceived special treatment to destroybacteria or repress their development.The situation with respect to frozenfoods is much different. Even afterstorage for some time in the frozenstate, they contain innumerable micro-organisms in some cases, and are sub-ject to fairly rapid spoilage after thaw-ing. Frozen foods, like fresh foods, areperishable and must be stored underproper conditions. Here defects inquality of frozen foods may occur. De-terioration and possibility of causingfood poisoning, remote as they mayseem, are possible in any food in whichthere is a varied flora.

Freezing does not destroy micro-organisms. A review of the literatureof the effect of freezing on micro-organisms prepared by Wallace andTanner appears in the October andDecember, 1933, issues of The FruitProducts Journal and American VinegarIndustry. This survey shows beyondreasonable doubt that, while freezingdoes materially reduce the number ofviable bacteria in some cases, it by nomeans destroys them. Among thosewhich remain, may be microorganismsof considerable significance. Prescott,Bates, and Highlands,23 and Fellers 24reported appreciable numbers of viablebacteria in various frozen foods. Geer,Murray, and Smith25 have recentlyreported satisfactory reductions in thebacterial content of Hamburg steakafter freezing and storage. In theauthor's laboratory extensive investiga-

tions have been under way for severalyears on the microbiology of frozenfoods, including the analysis of over2,000 cans and cartons of frozen fruitsand vegetables packed under com-mercial conditions. Reports on thiswork will shortly be published.37 Itseems sufficient here to report only someof the general conclusions reached. Theoriginal microbial content of the frozenfoods seemed to depend largely on thecondition of the raw materials. Whenover-ripe, blemished, raw materialswere used, the content of microorgan-isms was high; when sound raw ma-terials were used, the content was low.There were some instances where a con-tainer from a low count pack, con-tained many more micro6rganisms thanthe others. This was probably due tothe presence of small amounts of de-composed food. One bad berry, forinstance, might seed an entire pack orcontainer. A fairly high content ofmicroorganisms apparently has littleinfluence on quality as long as the foodis frozen solid, for there is no satis-factory evidence that bacteria growunder such conditions.

Freezing caused a steady decrease inthe number of viable bacteria. - After ayear's storage, viable bacteria had de-creased about 90 per cent. The num-ber of viable forms seemed to reach abasic minimum from which it decreasesvery slowly. Apparently those formswhich are unable to tolerate the condi-tions in frozen foods die out rapidlyduring the early period of storage.To study the effect of freezing on the

organisms themselves under conditionsas nearly like those obtaining in foods,Wallace and Tanner 26 froze 16 dif-ferent pure cultures of microorganisms,including yeasts, molds and bacteria in16 different menstrua-nutrient brothwith 5 different hydrogen ion concentra-tions, 3 different concentrations of saltand sugar, and in 2 fruit juices. Thesuspensions in ampoules were stared at

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-23.30 C. (-100 F.), and counts ofviable cells made at monthly intervals.In all cases there was a rapid drop innumbers during the early periods ofstorage. Spore forming bacteria weremore resistant. After the 8th monththe number of viable cells dropped veryslowly. These ampoules have now beenin storage for almost 3 years and stillshow many viable bacteria.

In order to determine whether verylow temperatures might not be moredestructive, bacterial suspensions werestored at -150 C. (3.20 F.), -4043C. (-400 F.) and -80° C. (-1120F.). The differences due to these tem-peratures were not pronounced. In facteven the lowest temperature employed,-80° C., did not seem to cause muchmore rapid or greater destruction thanthe higher ones.

It should be pointed out that the re-sults of freezing on micro6rganisms,expressed in percentage reduction ofviable forms may be meaningless. Theimportant problem is the number ofviable forms remaining. The fallacyof interpreting too much into per-centage reductions is well illustrated byTable III in a paper by Geer, Murray,and Smith 25 on the bacterial contentof frosted Hamburg steak. Twosamples were reported as having 1,200,-000 bacteria per gm. after freezing andstorage. One showed a 57.1 per centreduction, the other a 40 per cent. A99 per cent reduction in viable micro-organisms might leave many bacteria inthe food..The types of spoilage caused by

microorganisms in thawed frozen foodsare not unlike those in fresh foods.Whether thawed foods are more sus-ceptible to spoilage has not been ade-quately studied. Berry 27 reported thatyeasts were largely destroyed infrozen fruits and that when the canswere stored at room temperature, onlya few swelled. Wallace and Tanner 28found that frozen fruits packed in tin

swelled and in many cases burst thecans. Yeasts are quite resistant tofreezing, as shown by Tanner and Wil-liamson.28 Observations made in theauthor's laboratory during the past 3years do not confirm Berry's observa-tions. Yeasts have been so abundantin some thawed frozen fruits that theyburst the containers.The fact that frozen foods do not re-

ceive treatment which will free them ofmicroorganisms makes it necessary togive more attention to raw materialsand conditions under which they areprepared than is the case with certainother preserved foods. Attentionshould be given to quality of the watersupply and the health of those engagedin the preparation of such foods. Acarrier would be a menace. Sufficientattention is apparently given these mat-ters in the frozen meat and fish indus-tries, but this may not always be thecase in the frozen fruit and vegetableindustry. Concentration of hundredsof fruit pickers from different sectionsof the country in camps during cherrypicking time might introduce hazardswhich would be of no significance inother food industries. To secure in-formation on the possible disseminationof such diseases as typhoid, and para-typhoid fevers by frozen fruits, freshcherries and cherry juice in tin canswere inoculated with Eberthella typhi,Escherichia coli, Salmonella aertrycke,Salmonella schottmiilleri and Proteusvulgaris. The cans were closed,tipped under 26 inches of vacuunm,their exteriors disinfected, and storedat -17.8° C. (00 F.) and -400C. (-400 F.). Uninoculated con-trols were stored under the sameconditions. The results of these ex-periments were reported by Wallace andPark.29 In the cherry juice alone, theorganisms did not survive longer than4 weeks. In frozen cherries the survivalperiod was from 2 to 3 months. Suchdata justify attention to sanitary

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quality of water and medical inspectionof employees, for cherries might be usedpromptly after freezing. Heavierinoculation with more cold resistantstrains than those used might tend togreater longevity.The other problem has to do with

the presence and behavior of toxicogenicanaerobes under the conditions whichobtain in frozen foods. Some of thoseengaged in the frozen food industryhave stated that it was hard to see thatthere was the slightest danger from theconsumption of thawed frozen fruits,even under the most careless handling.It has been stated that it was common

practice to allow frozen fruits to remainon counters during the day and laterreturn them to an ordinary icebox atnight, and further, that under these con-

ditions, a large number of the cartonsmust have begun to ferment prior totheir sale. Such careless methods ofhandling a perishable food should notbe permitted, as they would greatlyaffect the quality and introduce hazardsof food poisoning which may easily beavoided by proper handling. To holdthat there is no danger is a questionableattitude. It probably originates fromexperiments with canned foods. Itis generally assumed that a food more

acid than pH 4.5 is free from botulismhazard. The growth of other organ-

isms might interfere with the protectiveaction of foods with pH 4.5, as has beenshown to be the case.

Development of sufficient toxin to killguinea pigs in a few of the fruits hasbeen explained by concomitant develop-ment of other organisms, especiallymolds, making it possible for Clos-tridium botulinum to grow. Severalreports confirm this explanation. Chiefamong them are those of Meyer andGunnison,30 and Stone.31 The formerreported an outbreak of botulism causedby Bartlett pears the pH of which was

3.86. Development of toxin in Bart-lett pear syrup was made possible by

concomitant development of a yeastand a member of the lactic acid group.This spoilage so altered the pH of thepear syrup that Cl. botulinum was ableto grow. Stone reported an outbreakof botulism caused by persimmons, thepH of which was 5. 6. Wallace andPark observed toxin formation when ayeast and acid producing bacteriumwere growing in the medium.The wide distribution of Cl. botu-

linum in the soil of the areas wherefruits and vegetables are frozen on alarge scale is an important factor. Theorganism is also known to be present inthe soil of practically all states as evi-denced by outbreaks of botulism andexamination of soil samples.

In some quarters it has been arguedthat packing of frozen foods in papercartons might eliminate the danger ofbotulism. It was suggested that airwould enter such packages and preventdevelopment of anaerobic bacteria. Thefallacy in such an argument is apparentto bacteriologists. Anaerobic condi-tions could easily be present in a foodmass exposed to the open air. The factthat a package is not hermeticallysealed is no indication that anaerobicconditions might not exist.

Effect of Freezing on BotulinumSpores-It is generally accepted thatfreezing does not steriliz a suspensionof bacteria. The spores of Cl. botu-linum have also been shown to beresistant to freezing.- They are viable,therefore, and appear in the thawedfrozen foods to compete with the otherbacteria which have survived the cold.The subject has been studied in severallaboratories with somewhat discrepantresults. Berry 27 considers the possi-bility of botulism from thawed frozenfoods to be very slight. His argumentsare apparently that the foods wouldhave taken on such appearances of de-composition that thev would not beeaten, and that frozen foods are usuallycooked before eating. These are as-

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sumptions which do not seem to be sup-ported by facts. As far as the first oneis concerned, experience with botulismfrom canned foods has been just thereverse. Geiger, Dickson, and Meyer 32discussed this question in The Ep-demiology of Botulism. They reportedthat in many outbreaks the spoilage ofthe preserved product was so striking asto attract the attention of the personopening the can; yet the foods wereserved. With regard to cooking offrozen pack vegetables, this may be therule, but it is conceivable that theymight be used uncooked.

Results of several investigations havebeen published to show that the sporesof Cl. botulinum are not destroyed byfreezing and storage in the frozen con-dition for rather long periods. Wallaceand Park 33 observed viable spores aftera year at -16° C. (3.20 F.). Jamesmade the same observation.

Effect of Freezing on Toxin of Cl.botulinum-The toxin of Cl. botulinumis not destroyed by freezing. Wallaceand Park reported potent toxin after 1year's storage at -160 C. (3.20 F.).James reported that freezing and de-frosting 15 times did not reduce thestrength of toxin.

Development of Cl. botulinum inThawed Frozen Foods-Having shownithat freezing does not destroy Cl.botulinum or its toxin, the next prob-lem is to determine the behavior of theanaerobe in thawed frozen fruits andvegetables. To be safe and of highquality, frozen foods should be keptfrozen until used. This is not done byall who are merchandising theseproducts. Several years ago frozenfruits were exposed for sale on thecounter in Chicago drug stores. Asstated, we have the word of one en-gaged in the frozen food industry thatthe cartons are frequently not keptfrozen during their sojourn in the re-tailer's establishment. Berry's experi-ence in the Northwest is somewhat dif-

ferent from that in other sections. Of20 cans of blanched peas and 10 cansof unblanched string beans inoculatedwith Cl. botulinum spores 15 to 18months previously, none proved toxiceither on thawing or on standing atroom temperature for from 3 days to3 weeks.

This has not been the experience inour laboratory as reported by Wallaceand Park. It has been demonstrated inour laboratories that occasionally Cl.botulinum may develop in thawedfrozen foods and produce its toxin.Vegetables are especially susceptiblewhether packed in glass- or tin con-tainers.The results of these investigations

correlate well with those reported byStraka and James.35 They reported thepresence of Types A and B of Cl.botulinum in inoculated frozen peaspacked in various types of containers;showed that freezing did not destroyCl. botulitum; and that frozen vege-tables (peas) which contain it may be-come toxic after thawing. Berry statedthat there is practically no danger fromfrozen foods.* James' has shown thatthe toxin is not materially reduced inpotency.Having discussed the microbial con-

dition of fresh and frozen foods, justhow far may bacteriological technic beused in their control? It is difficult tosee a place for the bacteriologist here,much as the food bacteriologist mightwish it. It would be impossible to ex-amine bacteriologically all shipments ofsuch products. Negative results wouldbe meaningless with some of our present

* Since the preparation of this paper, Berry (J.Bact., 26:459, 1933) published a report of experi-mental work in which he demonstrated survival, forat least 2 years, of lactic acid bacteria in frozen peas.Persistence of these organisms was believed to be ofpublic health significance in that they might exertan antagonistic influence on food poisoning bacteria.Frozen foods are not sterile and organisms whichfavor bacteria of the food poisoning group might bepresent as well as those which have been found toinhibit them.

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492 AMERICAN JOURNAL OF PUBLIC HEALTH

methods. Much more may be done bycareful inspection of conditions underwhich such foods are produced andhandled. This is becoming of greaterimportance for fresh foods shipped intothis country that may not be raisedunder sanitary conditions, and it is ap-parent that bacteriological methods maynot be used for control.

Certain other foods are in about thesame situation. The Bureau of AnimalIndustry does not examine the flesh ofall hog carcasses for Tricknella spiralis,because it would be too expensive, if notimpossible. In place of this it pre-scribes conditions under which the meatshall be handled to rid it of the parasite.A similar attitude must be taken towardthe foods discussed in this paper.

Bundesen 36 reached the same con-clusion in 1925 after weighing the evi-dence gathered in an epidemiologicalinvestigation of typhoid fever inChicago. He stated that bacteriologicalexamination of fruits and vegetableshad not proved of any value. In themajority of instances, Escherichia coliwas found, but it was impossible to de-termine its origin-whether it was ofhuman origin or derived from the soil.In 1 case high colon counts of a ship-ment of lettuce were traced to ice usedfor packing.

REFERENCES1. Ehrlich, B. Die Reinigung des Obstes vor dem

Genuss. Arch. f. Hyg., 41:152-176, 1901.2. Abbott, J. S. Food Sanitation. Texas Board of

Health, 4:16-18, 1910.3. Rommel, W. Wchnschr. Brauerei, 176, 1902.4. Ressel. Uber fakale Verunreinigungen auf Obst

und Gemiise. Inaug. Dissert., Berlin, 1907.5. Neumann, G. Der Nachweiss des Bakterium

coli in der Aussenwelt besondere auf Nahrungemitteln.Deut. Med. Wchnschr., 2046, 1910.

6. Clauditz, H. Typhus und Pflanzen. Hyg.Rundschau., 14:865, 1904.

7. Sartory, A., and Fillassier. Les Fruits Porteursde Microbes. Comp. rend. Soc. de biol., 67, 445-447, 1909.

8. Garcia, A. Experiment to Demonstrate theColon Bacilli and Acid-fast Organisms of TubercleBacillus MorDhology on Fruits. Ann. Rep. Comm.of Health, Penn., Pt. 2, 1022-1024, 1911.

9. Smeall, J. T. Bacteria on Fruit. Brit. M. J.,917-919, 1932.

10. Ko, R. Action of Fruit Juices Upon theTyphoid Bacillus. Taiwan Igakukai Zasshi, 179,Chem. Abst., 13:462, 1919.

11. Wurz and Bourges. Sur la presence demicrobes pathogenes a la surface des feuilles et destiges des vegetaux qui se sont developpes dans unsol arrose avec de 1'eau contenant les micro-organismes. Arch. de med. exper. et d'anat. path.,13:575, 1901.

12. J.A.M.A., 80:1628, 1923.13. Creel, R. H. Vegetables as a Possible Factor

in the Dissemination of Typhold Fever. Pub. HealthRep., 27:187-193, 1912.

14. Kurk, F. W. A Bacterial Examination ofGreen Vegetables. A.J.P.H., 8:660, 1918.

15. James, L. H. Bacterial Content of HeadLettuce. Am. Food 1., 20:302-304, 1925.

16. Murillo, F. Cholera, Typhoid and Vegetables.Plus-Ultra, 2:115, 1919.

17. Melick, C. 0. The Possibility of TyphoidInfection through Vegetables. J. Infect. Dis., 21:28-38, 1917.

18. Wary. Lancet, 1671, 1903.19. A Typhoid Outbreak Apparently Due to

Polluted Water Cress. Eng. News, 70:322, 1913.20. Pixley. New York M. J., 98, 1913.21. Morse. Rep. Massachusetts State Board of

Health, 761, 1899.22. Mills, R. G., Bartlett, C. L., and Kessel, J.

F. Penetration of Fruits and Vegetables by Bacteriaand Other Particulate Matter, and The Resistance ofBacteria, Protozoan Cyst$, and Helminth Ova to Com-mon Disinfection Methods. Am. J. Hyg., 5:559-579,1925.

23. Prescott, S. C., Bates, P. K., and Highlands,M. E. Numbers of Bacteria in Frozen Food Storedat Several Temperatures. A.J.P.H., 22:257-262,1932.

24. Fellers, C. R. Public Health Aspects of FrozenFoods. A.I.P.H., 22:601-611, 1932.

25. Geer, L. P., Murray, W. T., and Smith, E.Bacterial Content of Frosted Hamburg Steak.A.J.P.H., 23:673-676, 1933.

26. Wallace, G. I., and Tanner, F. W. Micro-biology of Frozen Foods. III. Longevity of PureCultures of Micro8rganisms Frozen in VariousMenstrua. (Unpublished data.)

27. Berry, J. A. Microbiology of the FrozenPack. The Canning Age, April, 1932, p. 251; TheWestern Canner and Packer, 25, 1932.

28. Tanner, F. W., and Williamson, B. W. TheEffect of Freezing on Yeasts. Proc. Soc. Exper. Biol.& Med., 25, 377-381, 1928.

29. Wallace, G. I., and Park, S. E. Microbiologyof Frozen Foods. IV. Longevity of Certain Patho-genic Bacteria in Frozen Cherries and in FrozenCherry Juice. J. Infect. Dis., 52:146-149, 1933.

30. Meyer, K. F., and Gunnison, J. B. BotulismDue to Home Canned Pears. J. Infect. Dis., 45:147,1929.

31. Stone, W. J. Botulism from Ingestion of RipeFruit. J.A.M.A., 92:2019, 1929.

32. Geiger, F. C., Dickson, E. C., and Meyer, K.F. The Epidemiology of Botulism. Pub. HealthBull., 127, U. S. Pub. Health Service, 1922.

33. Wallace, G. I., and Park, S. E. Microbiologyof Frozen Foods. V. The Behavior of ClostridiumBotulinum in Frozen Fruits and in Vegetables. J.Infect. Dis., 52: 1.50-156, 1933.

34. James, L. H. Effects of Freezing on theSpores and Toxin of Clostridium Botulinum. I.Infect. Dis., 52:236-240, 1933.

35. Straka, R. P., and James, L. H. FrozenVegetables. A.J.P.H., 23:700-703, 1933. See also22:473-492, 1932.

36. Bundesen, H. N. The Control of FoodsEaten Raw. J.A.M.A., 85:1285-1288, 1925.

37. Fruit Products J. and Am. Vinegar Industry,1934.

microbiological examination of fresh

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