Maintenance of Cultures of IndustriallyImportant Microorganisms1

W. C. HAYNES, L. J. WICKERHAM, AND C. W. HESSELTINE

Fermentation Section, Northern Utilization Research Branch, Agricultural Research Service, U. S. Department of Agriculture,Peoria, Illinois

Received for publication July 11, 1955

The great increase in number and size of industrialfermentations has accentuated the value of maintainingcollections of microorganisms, especially of productionstrains, assay organisms and related species. Consider-able work has been devoted to finding methods of main-taining cultures in a vigorous and stable condition. Atpresent, the chief methods of preservation of indus-trially important microorganisms are by means ofsoil culture, agar slants, mineral oil overlay, and lyo-philization. Each method has a place in any collectionof microorganisms and all are in use in the collectionof the Northern Utilization Research Branch, Agricul-tural Research Service, U. S. Department of Agricul-ture, at Peoria.The NURB Culture Collection is organized in three

parts. The first is devoted to bacteria with emphasison the genera Pseudomonas, Leuconostoc, Bacillus, andStreptomyces. These cultures are designated by theprefix "B" with the culture numbers, and total over

2800 strains. The second part is concerned with yeastsand yeast-like, organisms. These cultures, numberingover 2200, are designated by the "Y" preceding theirspecific number. The last portion of the collection con-

sists of filamentous fungi with emphasis on species ofPenicillium, Aspergillus, and the order Mucoraks,though many additional fungi of economic importanceare included. This mold collection numbers over 2400isolates and can be recognized by the fact that no letterprecedes the culture number.No list or catalogue of our cultures has been pub-

lished. The primary reason for this is that we do notwish to enter into competition with other collections.

In addition to the permanent cultures, the Collectionhas lyophil preparations of approximately 10,000strains of microorganisms in pure culture which are notintended to be permanent accessions. However, be-cause of the long viability of most lyophilized micro-organisms, nearly all are still living and represent a

reservoir of readily available material. They have notbeen incorporated into the collection because their

1 Presented in the Symposium on Maintenance of Culturesof Microorganisms at the 55th general meeting of the Societyof American Bacteriologists held at New York City, New York,May 8-12, 1955.

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usefulness has not been shown, or else they are knownto duplicate cultures already in the collection.The lyophil apparatus (figure 1) and the procedure

used is essentially the same for all three groups ofmicroorganisms (molds, yeasts and bacteria). A de-scription of the apparatus and an explanation of theprocedure were given by Wickerham and Flickinger(1946). It will not be amiss to emphasize two specialadvantages of the NURB process. The first of these isthe relative inexpensiveness of the lyophil apparatus.The main item of expense is the vacuum pump. It,however, is of a type generally found in microbiologicallaboratories. The rest of the equipment consists ofitems which either are available in all laboratoriesor may be constructed fairly cheaply. The second ad-vantageous feature is the speed of the operation. Wecan routinely lyophilize 30 cultures in triplicate in a day(3 runs with our setup). An experienced operator canincrease production to 40 cultures in triplicate (4 runs)on any day that he is free of other duties.

The Bacterial CollectionzThe bacteria in the NURB Culture Collection were

obtained, like the yeasts and molds, from other inves-tigators and by isolation by ourselves and our col-leagues. Not every strain received or isolated is kept,but those which have unique characteristics or are ofknown use in agriculture or industry are kept for preser-vation. For instance, a complete series of microorgan-isms of proven reliability in microbiological assay forvitamins and amino acids is in the collection. Strainsselected for their fermentative capacities and describedin publications by members of the USDA are retained.For example Leuconostoc mesenteroides, NRRL B-512Fand related strains useful in dextran formation; NRRLB-72, Acetobacter suboxydans which makes sorbose fromsorbitol, and NRRL B-6bs; Pseudomonas sp. whichoxidizes glucose to 2-ketogluconate and a-ketoglutarate.Strains of known utility discovered and described byothers are also sought.

If these were the only strains kept, the bacteria inthe collection would number but a few hundred. Themajority of strains are assembled because of theirrelationship to noteworthy strains. For instance over

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FIG. 1. A simple apparatus for lyophilization of bacteria, yeasts or molds

100 strains of leuconostocs which were acquired forcomparison with NRRL B-512F are included. Anotherreason for keeping large numbers of organisms of un-proven usefulness in agriculture and industry is tohave a collection of type strains helpful in identificationof isolates which prove to be interesting. Thus theNURB collection contains the N. R. Smith Collectionof aerobic sporeformers. Of course, all of these unprovenstrains constitute a reservoir from which may beselected organisms having unique and useful properties.

Maintenance of 2800 strains even if not of diversecharacteristics and requirements would be a tediousjob. In the early days of the Collection all strains werecarried on slants or in stabs and required transfer atleast every three months, some strains even morefrequently. Simultaneously with this method of main-tenance, a program of lyophilization was carried on.This relieved us in large part of anxiety about losingstrains, but we had not the courage for several years torely exclusively on lyophilization. In the course of time,however, we became convinced that lyophilized bac-teria were at least as safe as stocks kept in culture. Notmore than one strain in a thousand failed to survivelyophilization. At length, we became aware of an ap-parently direct relationship between the numbers ofcells subjected to lyophilization and the survivabilityof the preparation. Only in cases where the populationwas low did we experience failure. Furthermore, thoughwe did not investigate the incidence of variation ormutation, we became convinced that this was not aproblem with the organisms in our collection. On the

contrary, we have several times recovered from lyophila culture capable of yielding a characteristic product inquantities comparable to those recorded at the timeof lyophilization, while the progeny of the same ante-cedent culture had declined radically in productivity.For this reason it has been our custom, whenever anymicroorganism performs exceptionally well in any ofits activities, to reisolate the microorganism and torelyophilize it. By so doing we expect to preserve itsactivities unchanged.For several years now we have relied exclusively on

lyophilization for preservation of the bacteria includingthe actinomycetes. The only strains kept in culture arethose in current use, and those which are frequentlyrequested by other investigators. An exception to thegeneral routine is the maintenance of the anaerobicsporeformers. Only a few such strains are included andthey are kept in soil culture. -Inasmuch as a single at-tempt to lyophilize them failed, it was not tried again.The streptomycetes and some of the aerobic sporeform-ers maintained in lyophil are also kept in soil culture.Although we know of no Streptomyces which will

not survive lyophilization, we do know that certainspecies in the genus have two prerequisites. They arefirstly, that conidia must have been formed, andsecondly, that the conidia must have matured. Toinsure sporulation we use several media, some of whichare described in table 1. Having found one or morewhich gives good sporulation, we wait at least 14 days,sometimes longer, to allow the spores to mature beforewe lyophilize.

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TABLE 1. List of media used at the Northern Utilization Research Branch for the propagation and maintenance of the majorgroups of microorganisms

I. Molds

A. Media especially suited for growing penicilliaand aspergilli

Czapek's Solution Agar

Distilled H20 .................................... 1000 mlNaNO3 ........................................ 3.0 gK2HPO4 ........................................ 1.0 gMgSO4 * 7H20 .0.................................... O.5 gKCl ........................................ 0.5 gFeSO4-7H20 (1% soln.) ........... ................ 1.0 mlAgar ........................................ 15.0 g

Melt and addSucrose ........................................ 30.0 gpH not adjusted.

Czapek's Solution Agar With High SugarThe formula is the same as for Czapek's Solution Agar ex-

cept that 20 or 40% sucrose is substituted for the usual con-centration.

B. Media especially suited for the MucoralesPotato Dextrose Agar (PDA)

Flask IDistilled H20 ................................... 100.0 mlDextrose ..................................... 20.0 gCaCO3 ..................................... 0.2 gMgS04-7H20 .................................... 0.2 g

Flask IIDistilled H20 ................................... 400.0 mlAgar ...................................... 15.0 g

Flask IIIPotatoes-peeled and sliced ...................... 200.0 gDistilled H20 ................................... 500.0 ml

Content of flask III brought momentarily to 121 C in anautoclave. Filter through cheese cloth. Bring up to originalvolume. Simultaneously the agar in flask II is melted and thesolution in flask I is heated to boiling. Mix contents of threeflasks.pH not adjusted.

Malt Extract AgarDistilled H20....................................Malt extract....................................Peptone ........................................

Dextrose.........................................Agar............................................pH not adjusted.

1000 ml20.0 g

1.0 g

20.0 g

20.0 g

C. Media especially suited for growing Chaetomium and some

other AscomycetesYeast Extract Agar

Yeast extract.....................................Malt extract......................................Dextrose ...........................................Agar..............................................Distilled H20......................................pH not adjusted.

4.0 g10.0 g

4.0 g

15.0 g

1000 ml

II. Yeasts

One medium suitable for growing the majority of yeasts

MY AgarYeast extract......................................Malt extract.......................................Peptone ..........................................Glucose ...........................................Agar ..............................................Distilled H20......................................pH not adjusted.

3.0 g3.0 g5.0 g10.0 g20.0 g

1000 ml

III. Bacteria

A. General purpose medium suitable for maintenance of themajority of bacteria

TGY AgarTryptone..........................................Yeast extract......................................Glucose ...........................................K2HPO4 ...........................................Agar ..............................................Tap H20 ..........................................pH adjusted to 7.0.

5.0 g5.0 g1.0 g1.0 g

20.0 g1000 ml

B. A medium for the propagation and maintenance of lacticacid bacteria and anaerobic sporeformers

Deep Liver MediumLiver extract ....................................... 100 mlYeast extract ....................................... 5.0 gTryptone ......................................... 10.0 gK2HPO4 ......................................... 2.0 gGlucose ......................................... 5.0 gDistilled H20 ....................................... 900 mlpH adjusted to 7.4.

Before dispensing to test tubes, add a small quantity ofdried liver particles to each tube.

The liver extract is made by adding 2 liters of distilled waterto each pound of finely ground beef liver and heating in flow-ing steam until all the redness is gone from the liver. This willrequire about three hours during which the concoction shouldbe stirred frequently in order to break up clumps. When thesupernatant becomes yellowish fluorescent it is filtered throughseveral layers of cheese cloth. The clear supernatant is thenbottled and sterilized.

The residue which remains after the above extraction isspread on shallow trays and dried at 50 C. This constitutes thedried liver particles mentioned above.

C. A medium. designed for the maintenance of acetic acidbacteria but useful also for the propagation of

fastidious types in other genera

Acetobacter MediumTryptone ................. 5.0 gAgar................... 20.0 gLiver extract ................. 100 mlDistilled H20 ................. 900 mlAfter melting agar, addGlucose..................... 20.0 gCaCO3 ........................ 10.0gpH not adjusted.Dispense while stirring constantly to insure equable dis-

tribution of CaCO3.

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TABLE 1.-Continued

IV. MIedia upon which sporulation of Streptomyces is mostlikely to occur

Asparagine Dextrose Agar (ADA)

Tap H20 .......................................... 1000

Asparagine ........................................ 0.5 g

K2HPO4 .......................................... 0.5 g

Beef extract ....................................... 2.0 g

Adjust pH to 6.8-7.0Add agar .......................................... 17.0 g

Dextrose .......................................... 10.0 g

Bennett's Agar

H20 (distilled)...................................Yeast extract.....................................Beef extract......................................NZ amine A.......................................

Adjust pH to 7.3 with NaOHAdd Bacto-dextrose................................Agar ..............................................

(J. Bacteriol. 57: 142, 1949)

1000 ml1.0 g

1.0 g

2.0 g

10.0 g

20.0 g

It should be emphasized that there is no one culturemedium which will induce sporulation of all strains andspecies of Streptomyces. In hopes of finding at least one

that is satisfactory for this purpose with a given straina variety of media are employed including asparaginedextrose agar, Bennett's agar, Emerson's agar, potatodextrose agar, Czapek's solution agar, oatmeal agar,

yeast extract agar, starch agar, and corn steep agar.

It is wvise always to make sure cultures have an abun-dance of mature spores before storing them in a re-

frigerator.Aside from the differences in stock-culture media,

streptomycetes are handled more in the manner ofmolds than bacteria. In other words the culture ispropagated by transfer of spores rather than of vegeta-tive growth.

It is anticipated that a fourth category will be es-

tablished to contain actinomycetes and other micro-organisms such as algae and protozoa.We have mentioned the fact that with few exceptions

our lyophilized cultures have remained viable for as longas they have been under observation. Some of the tubesare 14 years of age. We no longer open tubes at specifiedintervals to check upon viability, but we record our

observations on its condition each time such a tube isopened. Thus for most lyophil cultures a record ofviability is available.Having expressed supreme confidence in the lyophil

method of maintaining and preserving stocks of bac-teria, a word of caution should be expressed concerningsterility of the suspending agent. At NURB we use

sterile bovine serum as the suspending medium formicroorganisms to be lyophilized. The serum afterseparation is sterilized by Seitz filtration and thensealed in test tubes. The sterility of each batch is

Oatmeal Agar

Rolled oats ........................................ 65 g

H20 (distilled) .................................... 1000 ml

Cook to thin gruel in double boiler, filter through severallayers of cheese cloth and bring up to volume.

Add agar .......................................... 20.0 g

pH not adjusted.

Emerson's Agar

H20 (distilled).NaCl.Peptone.Yeast extract.Beef extract.

Adjust pH to 7.0 with KOH.

1000 ml2.5 g4.0 g1.0 g4.0 g

Add bacto-dextrose ................................ 10.0 g

Agar ............ ....................... 30.0 g(J. Bacteriol. 55: 411, 1948)

checked by opening selected tubes of serum and streak-ing on agar. It is not well to rely completely on thesterility of the serum thus controlled. Some very slowgrowing or psychrophilic organisms may be present andescape detection. It has been our practice to retest eachtube of serum before it is used. This is done by spread-ing 0.5 ml of serum on duplicate plates of a nutrientagar and incubating them at room temperature forseveral days. Inasmuch as each tube holds sufficientserum for several runs, the same tube is entered re-

peatedly, and there is a danger that sterile serum maybecome contaminated. Since sterility testing uses so

much serum, we prefer not to test it before each run

but depend upon close observation. If any suspicionis aroused, the tube in question is not used until provensafe.We maintain a minimum of four lyophil tubes of

each strain, but those of special significance are madeup to 10 or more. Four tubes are put into a vial whichbears the strain number on its cap. These vials are

arranged in trays which fit into a framework inside therefrigerator as shown in figure 2. The refrigerator tem-perature is set at 5 to 10 C.No use has been made of oil-covered cultures for

keeping bacteria because we have considered themunnecessary. For long-term preservation we are sat-isfied with lyophilization; for shorter term availabilitymost of our bacteria survive for relatively long periodson artificial culture media. For instance, leuconostocsreadily remain viable and unchanged in our deep liverbroth for at least six months if kept under refrigerationfollowing good growth. Pseudomonads, coliforms,aerobic sporeformers, and so forth, on agar slants willalso survive for six months or more if kept refrigeratedfollowing good growth. If the plugs are burned off and

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depressed and rubber stoppers inserted in the testtubes, viability may be greatly extended-to two yearsor longer with aerobic sporeformers.t' Another reason for not using oiled cultures is thatthe first experience with this technique by one of theauthors was unsatisfactory. Great variation amongmicrococci such as was never experienced with othermethods of preservation was incited. Similar resultshave been reported in a collection of salmonellas (M.Caldwell, personal communication).One of the problems facing anyone who tries to

maintain a diverse collection of microorganisms is howto keep the number of media within reasonable bounds.We are fortunate in not having to face the difficultproblems in nutrition attendant upon caring for adiversity of human and animal pathogens. Neverthe-less we have organisms with different requirements andsome of them are rather fastidious. For the vast ma-jority of our strains a single medium suffices. For thelactics and anaerobes another is used while the aceticacid bacteria prefer yet another. In addition to thesethree media several others are suitable for special pur-poses or for special groups of bacteria represented byvery few strains and in little demand.When new strainsare received or isolated, occasionally

some difficulty in maintaining stability is encountered.The tendency to vary apparently is a response to newmedia and conditions to which the organism soon be-comes accustomed. The degree of variation is sufficientwith fresh isolates that cultures often are plated andcolonies picked repeatedly in an attempt to purify aseemingly mixed culture. Some strains are so reluctantto adapt themselves to new media and conditions thatthey become troublesome. An example is NRRL B-512Fwhich has been distributed far and wide for the pro-duction of clinical dextran. Most recipients of thisculture apparently experienced no insurmountabledifficulties in growing the strain and producing accept-able dextran. They probably either used the media andconditions recommended by us or, as inadvertentlyas we, were fortunate enough to select a suitablecombination to begin with. A few, however, experienceda variety of troubles ranging from no growth to produc-tion of a dextran chemically and physically dissimilarto that produced at NURB and unable to meet specifi-cations when prepared for clinical use. In most casesthe matter was quickly amended when directions weresent to describe our methods and media.As for the yeasts and molds, records are kept showing

the accession number, date of deposit, donor and do-nor's identification. This information is filed chrono-logically, and alphabetically by donor and by genusand species.The media upon which we rely most heavily for main-

tenance are given in table 1.

FIG. 2. Storage of lyophilized cultures

The Yeast CollectionYeast cultures bearing specific names number over

2200. Most of these have been studied taxonomically.More than 3800 additional strains have been isolatedfrom nature. Such isolates are lyophilized and theirability to sporulate is determined immediately afterisolation. Their cultural characteristics are recordedand probable or definite generic placement assigned tothem if possible. They are then available for develop-mental surveys or taxonomic study whenever thegenus or general taxonomic area to which each belongsis to be intensively studied. About 90 per cent of thetaxonomic work on yeasts at -this laboratory is basedon data obtained from isolates from nature.

Lyophilization of cultures was begun on a largescale in 1941, and all yeast strains have been preservedby this procedure. Each time a lyophil tube is opened,2 or 3 more are prepared, until 5 are on hand, and thenumber is held approximately at this level.

Viability of y3easts in the lyophilized state is verygood, and there is promise that investigators in thefuture may be assured that nearly all of the yeasts theylyophilize in their early years will remain alive withoutsubsequent cultivation throughout their professionallives. Rare failures do occur with Cryptococcus,Schizosaccharomyces and Saccharomycodes. Occasionally

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filamentous yeasts lose the ability to produce blas-tospores on solid media. These are grown in shake flaskswhere the mycelium develops as small dispersed ele-ments that can be centrifuged and then successfullylyophilized.

Lyophilization of yeasts has never, to our knowledge,induced variation in any of the physiological charac-teristics upon which we base species identification. Quiteto the contrary, for we believe it is the best means ofpreserving the characteristics of isolates. In fact, whenone is studying a highly variable yeast and it becomesnecessary to have certain phases as controls in eachexperiment, one may lyophilize the phases to be used asstandards, then disperse the dried pellets in 1 or 2 mlof sterile mineral oil, contained in a screwtop vial, andstreak a loop of the suspension each time a control isneeded. We are indebted to Dr. F. M. Clark of theUniversity of Illinois for this procedure.Good results have been obtained in maintaining

yeast cultures under oil. The medium used for bothmaintenance and general cultivation is MY agarwhich is described in table 1. The agar is solidified withthe tubes in an upright position. The surface of theagar is inoculated and the cultures incubated 1 or 2days at 25 C, then a layer of sterile mineral oil ap-proximately 1 cm deep is placed over the growth. Thenthe cultures are stored at approximately 5 C, andtransfers made at 2-year intervals. Some 1300 strainsare maintained this way. A few which have failed tosurvive this period are transferred at 6-month intervals.A small number of osmophilic yeasts are kept on oiledMY agar containing 20 per cent glucose.

In our experience, preservation of yeasts in drysterile soil has been very unsuccessful except for somemucoid and submucoid yeasts of the genera Cryptococcusand Rhodotorula.

The Mold CollectionWhen mold cultures are received or isolated, they are

examined first of all for their purity and identified ascompletely as possible, or, if they are already identified,the identification is checked. It is also very useful, ifthe culture is not identified at the time it is secured,to have a record of what the appearance of the culture isunder specified conditions. Thus, on a given medium ata certain age, a culture will have a characteristic sporecolor, reverse color and a soluble pigment present orabsent. These characteristics may be determined readilywith a minimum amount of labor. Later, should aquestion arise as to the appearance of the culture whenfirst obtained, the information is available.The filamentous fungi are routinely carried on agar

slants, in lyophil, and in soil cultures. However, a fewrecalcitrant to lyophilization are kept under mineraloil. Wherever possible each permanent culture is carriedby at least two of the above methods, usually in lyophil

and on agar slants. The lyophil process has the advan-tages of allowing cultures to be kept for extremely longperiods of time, the cultures are sealed and cannot be-come contaminated during storage and they requirelittle space for storage. On the other hand, when alyophil tube is opened, it may be necessary to re-lyo-philize to replace the tube. Such recultivation andreprocessing requires considerable time. In some in-stances, as among the Entomophthorales and certainmucors, in spite of abundant spore production, poorviability or no growth at all follows lyophilization.There are probably other species with spores whichfail to survive the process. For this reason, each time alyophil culture is prepared, it should be checked forgood growth within a period of one or two weeks toinsure that the preparation is satisfactory. The processcannot be applied successfully to nonsporing cultures.It is generally believed that a large proportion of cellsare killed by freeze-drying. In consequence there hasbeen speculation that selective killing might causevariation in lyophilized cultures. In our experience,however, this has not happened.The mold cultures are carried on a variety of stock

media in the form of slants. Compositions of some ofthese media are given in table 1. The aspergilli andpenicillia are carried on Czapek's solution agar, ormalt agar; certain cultures are grown also on modifica-tions of Czapek's solution agar. For example, Asper-gillus ruber which requires a high-sugar content in themedium is carried on Czapek's solution agar with 20 to40 per cent sucrose added. Members of the Mucoraleshave been carried for years on laboratory-prepared,potato-dextrose-agar with excellent success althoughthere is reason to believe that malt agar or other non-synthetic media might be just as good.

It is important to realize that some species havespecific requirements for growth. Consequently one isforced to use specific media or techniques for manyfungi. An interesting example of special techniquesrequired for maintaining unique types of microorgan-isms is illustrated by the success one of us (CWH)has had in maintaining numerous members of thegenus Syncephalis and Piptocephalis. These genera,which contain a large number of species and which arefairly common in soil, grow as obligate parasites onsaprophytic Mucorales. None has been cultivated freefrom its host. Because we are interested in members ofthis order, we have secured a number of species andare collecting additional ones which we wvish to main-tain permanently. As seen in nature, they are foundgrowing on a variety of host genera. The usual pro-cedure in securing such a parasite is to isolate myceliumof the host and parasite, and then to purify it of extrane-ous bacteria and molds by repeated subculture. A moreconvenient method is to isolate the host from the plateby picking sporangiospores from a sporangium and,

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after inoculation of three places on nutrient agar inplates, to inoculate the same spots on the agar with a

few spores of the parasite picked from their sporangia,

and usually the two then can be obtaitied in "mixed-pure" culture. However, in carrying stock cultures on

agar, one is forced to check the culture carefully, aftereach transfer, to be sure that the host has sporulatedand that the parasite's spores have germinated, at-tacked the host, sporulated in abundance, and maturedtheir spores. For some time cultures were lost becausethe host, when freed from the competition of othermicroorganisms, grew so rapidly and luxuriantly thatthe parasite could not properly grow, with resultantfailure to sporulate. Also the host might be of such a

similar gross appearance that the absence of growth ofthe parasite might be missed and the culture lost. Themaintenance of these cultures was greatly simplifiedwhen it was realized that one could use Mucor raman-

nianus as host for most species of the two genera. It is ared-colored Mucor with extremely short sporangio-phores and velvety, slow-growing colonies. When a

member of the genus Syncephalis or Piptocephalis isencountered in a soil sample or a culture is received withone of the parasites in it, we routinely inoculate plateswith M. ramannianus and infect the cultures withspores of the parasite in the above manner. In mostcases the parasitism of these genera is not so specializedthat they will not attack this new host. When the"mixed-pure" culture is established, no difficulty isencountered in maintaining it. It has the distinct ad-vantage that the host never overwhelms the parasite as

Rhizopus stolonifer may do. It also enables one to see

with the naked eye, because of the red color of theMucor, whether the host has sporulated. Invariably,the parasitic species are much taller and one can tellat a glance whether or not the surface of the host hasbeen overgrown and the parasite has fruited. Usuallythe parasite will grow over the host and far beyond on

the surface of the agar, apparently translocating nu-

trients from the host. In these areas the parasite'smycelium and fruiting can be studied without thepresence of the host mycelium and could be used forstudies on the factors required for parasitic growth.Since we started to use this technique, not a singlehost-parasite combination has been lost.

Cultures are examined prior to storage for adequategrowth and sporulation, for contamination by bacteriaand molds, for reverse color, and for the general micro-scopic morphology as seen under a dissecting micro-scope. In some species it is particularly important tocheck for sectoring or production of sterile mycelium.Once a culture has degenerated to the point where no

sporulation occurs, it is practically impossible to getit to appear again in the normal sporulating state.However, if this progressive degeneration is detectedbefore it is complete, a vigorous, heavily sporulating

culture can be reisolated which will appear as it didwhen first isolated. In some cases degeneration can bearrested by changing to a more favorable culture me-dium. Cultures however prepared are stored in refrigera-tors or cold rooms at 5 to 7 C. Each tube should bearthe date of transfer, the culture number, the culturemedium and the temperature of incubation. Beforestorage, each cotton plug should be poisoned to preventmold growth and entrance of mites. Transfers are madeat 6- to 8-month intervals.

It is also important that the chill room or refrig-erator have a low relative humidity, otherwise un-desirable molds will tend to grow on paper labels, andso forth, which makes contamination almost a certainty.A word should also be said about the care of the labora-tory. Every precaution should be taken to prevent in-festation of the work area with mites which occasionallymay be introduced with cultures, soil samples or othermaterial sent in from the outside. It is needless to saythat all materials on which fungi have been allowed togrow should be carefully sterilized before washing toprevent heavy contamination of the general work area.When working with molds, especially those with dustyspores as Rhizopus stolonifer, the transfer needle shouldbe sterilized and then moistened in sterile agar to pre-vent spores from being dispersed into the air. Also, withthese cultures it is especially important that the fewestpossible spores be used as inoculum. Whenever possible,one should transfer spore material only. In some cases alight touch of the needle on the sporulating area issufficient. However, some fungi, including many of thewood decay fungi, require the removal of a block of agarand mycelium since no basidiospores or conidia areproduced.

SUMMARY

The basic principles which guide the maintenance ofthe NURB Culture Collection are:The person in charge of a given category of micro-

organisms must be well enough acquainted with thecomponent taxa that he will usually know the mediaand conditions best suited to permit typical growth ofany strain. From more or less cursory examination of aculture, he must be able to decide whether it is typical,free from contamination, and in proper condition fordistribution. In difficult cases he must have at hisfingertips sources of information to resolve any ques-tions.The methods of preservation must be chosen from a

knowledge of the advantages and disadvantages of thevarious methods and from a comprehension of thecharacteristics and requirements of the microorganismsto be preserved. Until a group has become well known,it is not advisable to depend on a single method of cul-ture or preservation for maintenance.

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The salient hazards of maintaining a collectionmust be known and guarded against. Among these areinfestation with mites, contamination of cultures,especially with molds which grow at high humidities,misnumbering or mislabelling of cultures and so forth.The NURB collection is devoted to organisms of in-

dustrial importance, especially in the field of fermenta-tion. This specialization is emphasized in order to avoidoverloading facilties with types not adapted to fermen-tation work.The personnel of the NURB unit are required to have

thorough training in the field encompassing the micro-

organisms in their charge. Interests in taxonomy areencouraged and every effort is made to see that oppor-tunity exists to carry on a sustained taxonomic investi-gation of selected microorganisms.About 1000 cultures per person is the proper assign-

ment in maintaining the collection adequately. This,however, is dependent upon use of lyophilization forculture preservation.

REFERENCEWICKERHAM, L. J., AND FLICKINGER, M. H. 1946 Viability

of yeasts preserved two years by the lyophil process.The Brewers Digest, 21, 55-59 and 65.

Methods for the Detection and Estimation of Numbers ofSalmonella in Dried Eggs and Other Food Products'

ANNE F. BYRNE, MORTON M. RAYMAN AND MORRIs D. SCHNEIDER2

Quartermaster Food and Container Institute for the Armed Forces, Chicago, Illinois

Received for publication July 18, 1955

Dehydrated eggs are an important item of procure-ment for Armed Forces feeding. Because the presenceof Salmonella in egg products constitutes a potentialhealth hazard, it is obviously important to the ArmedForces that food infections and carrier states caused bySalmonella in eggs be prevented among troops. Thepurpose of this report is to describe improved testmethods for estimating the extent of Salmonella con-tamination in foods in order to assist in the controland elimination of this food infection hazard. Manyinvestigators (Gibbons and Moore, 1944; Schneider,1946; Gibbons, 1947; Solowey et al., 1947; Soloweyand Rosenstadt, 1948) have reported the occurrenceof these microorganisms in eggs, and McCulloughand Eisele (1951a, 1951b, 1951c, 1951d) establishedthe pathogenicity for humans of strains of Salmonelladerived from spray-dried whole egg. Outbreaks ofSalmonella infections which were traced to eggs havebeen described (Watt, 1945; Mitchell et al., 1946;Medical Research Council, 1947). Edwards et al.(1948) state that "eggs and food products containingeggs may more often be the medium of transmission ofSalmonella from animals to man than any other animal

1 This paper reports research undertaken by the Quarter-master Food and Container Institute for the Armed Forcesand has been assigned No. 547 in the series of papers approvedfor publication. The views or conclusions contained in this re-port are those of the authors. They are not to be construed asnecessarily reflecting the views or indorsement of the Depart-ment of Defense.

2 Present address: Chief, Veterinary Section, 3rd ArmyArea Medical Laboratory, SU 3004, Fort McPherson, Georgia.

food product." Hinshaw and McNeil (1951) in a reviewof Salmonella infection as a food industry problemstress the importance of animal and human reservoirsof the infection, and state that "the genus Salmonellais one of the important causes of the infection type offood poisoning."The isolation and identification of members of the

Salmonella group from food products present manydifficulties. Various methods and modifications, manyof which were originally developed for isolating patho-gens from feces and sewage, have been proposedthroughout the years (Leifson, 1936; Hynes, 1942;Galton and Quam, 1944; Felsenfeld, 1945; McCulloughand Byrne, 1952; Ayres, 1953). As a result of these in-vestigations, the use of selective enrichment media is acommon procedure and is preferred to direct culture forthe isolation of Salmonella from suspected materialswhich contain a large and varied population of micro-organisms.

MATERIALS AND METHODSWe have attempted to overcome some of the de-

ficiencies in the recommended procedures by using thedilution-enrichment-subculture method to be de-scribed. The primary purpose of the method is to deter-mine the degree of Salmonella contamination and thechanges in numbers of these organisms during process-ing and storage of eggs and egg products.Twenty grams of egg powder are weighed aseptically

in a flask containing 180 ml of sterile distilled waterand a tablespoonful of glass beads. The egg suspension

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