Maintenance of Vigorous Mold Stock Cultures H. C. GREENEAND E. B. FRED, University of Wisconsin, Madison, Wis. Methods for maintenance of stock cultures of Lain f e r m e n t a t i o n industries certain microijrganismsof the type used in the which use microorganisms carry nomical maintenance of their valuable stocks on soil as stock cultures is a n everfermentation indusfries are discussed as their present problem in work dealing a matter of standard practice with the growth of microorganr e l a t i ~ ?merits, and data are given in support of and recover them as needed. isms. All who have handled the conclusions reached. It is possible to preGood results have been obtained Serve m a n v valuable cultures with little change in from such cultures, and a study sizable stock culture collections mas undertaken to a s c e r t a i n are aware of the necessity for their desirable characteristics over long peFiods whether other still more satisusing the most efficient methods of time. Ordinary sterilized soil affords a factory m e t h o d s of carrying found applicable t o the organsuitable nzedium for carrying cultures, and such stocks could be found. .4cisms with which they are dealing. S u c c e s s f u l Earrying of a medium, can be used in commercial practice cordingly, A . sydowi and nine stocks requires that, in the since it i,y easy to *DreDare and is effective and other stock cultures were investigated on a smaller scale main, the morphological and economicu~, (in part) with a view towards physiological characters of the d e t e r m i n i n g more definitely organisms remain as constant as may be for the individuals concerned. The most simple whether methods of carrying were affecting the large-scale means possible are desirable for accomplishing the stated results. The following ten cultures were studied : dspcrgillus purpose. Methods should involve infrequent, transfer and ease of manipulation, and should be of fairly general ap- jischeri, A. Jlavipes, -4.nidulans, A . niger, A . sydowi No. 1, Penicillium aurantio-brunneum, P . citrinum, P. terrestre, d f o n plication. The growth of molds on a large scale in the laboratory has, ascus species KO.2, and Cunninghamella elegans. for a considerable period, engaged the attention of the writers. For this study four sterilized substrates of the following The mold mycelium is produced by growth on liquid medium composition were used: (1) 2.5 per cent malt extract, 2 per in tiers of pans contained in large metal incubators which have cent agar; (2) potato, 2 per cent glucose, 2 per cent agar; been described elsewhere ( 2 ) . During the past four years (3) baker's ordinary white bread; and (4) loam soil rich in about one hundred fifty mold cultures have been carried in organic matter. At each transfer, cultures were allowed to develop for one stock; the authors' standard practice has been to grow them on malt agar medium and then t o hold them in an electric week a t 28" C. on these substrates and were carried according refrigerator a t 10" C., transferring a t 6-month intervals to to the following methods: fresh tubes of the same medium, etc. Ten stock cultures 1. On malt extract medium. Transfers were made in three have been used for the large-scale production of mycelium. separate series: ( a ) At weekly intervals (continuously maintained at 28' C.). About fifty batches of mold weighing 1250 kg. (250 kg. dry ( b ) At 3-month intervals (held in refrigerator at 10' C.), weight) have been grown. I n the course of the work sig( e ) At 6-month intervals (held in refrigerator at 10' C.). nificant variations in yield of mycelium and in gross colony 2. On malt extract and potato-glucose medium, and altermorphology have from time to time appeared in the large- nately transferred between them. Transfers were made in scale cultures. A certain culture of Aspergilius sydowi has three separate series, corresponding to those on the malt extract been grown repeatedly in the incubators. It has on occasion medium. 3. On sterilized bread which was allowed t o dry down, with demonstrated much variability, with yields running from no further transfers made t o fresh bread during the year test those which are considered satisfactory to almost nothing, period. 4. On sterilized soil which was allowed to dry down, with no even when conditions have remained relatively constant and further transfers made t o fresh soil during the year test period. contamination has not been a factor apparentjly. All cultures were tested by their growth in flasks a t the METHODS FOR MAINTENANCE OF CULTURES beginning of the work and a t various intervals thereafter. Efforts to overcome this undesirable state (-if affairs have The interval depended on the series concerned-3 months included changes in the methods of handling the incubators for the standard 3-month series and for the bread and soil and a study of procedures for carrying stock cultures, es- cultures, and 6 months for the standard 6-month series. In the case of the bread and soil cultures all tests were made pecially A . sydouv'. The greatest single improvement, in so far as A . sydowi is from the original cultures which were kept without transfer concerned, has resulted from a stabilization of the inoculum. throughout the test period. Where weekly transfers were More than 2 years ago, and a t intervals since, cultures of this made, the soil cultures were alternated between malt extract and other organisms have been placed on sterilized soil, and and soil instead of being carried continuously on soil. This inoculum for the incubators has been largely drawn from was done to afford greater ease of manipulation. Five soil cultures. Sterilized soil is an excellent substrate for the cultures, A . jischeri, A. nidulans, A. sydowi, P. aurantiogrowth and preservation of various microorganisms. Barthel brunneum, and Cunninghamella elegans, were carried in this ( 1 ) has successfully maintained representative yeasts and way. The weekly transfer series was tested after 5 months, bacteria by this means. In this laboratory, cultures of the again 1 month later, and finally a t the end of a year. Tests bacteria which, in association Tvith certain leguminous plants, were made on the following basis: fix nitrogen have been carried unchanged on dry soil over a All the molds, except Monascus species and Cunninghainella 3eriod of years. To the best of the writers' knowledge, cer- elegans, were grown in triplicate liquid cultures in 500-cc. Pyrex
T
HE satisfactory and eco-
~
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INDUSTRIAL AND ENGINEERING CHEMISTRY
1298
Erlenmeyer flasks containing 100 cc. of medium. The dry weight of mycelium was obtained after 10-day incubation at 28" C. and was calculated on the average of the three cultures. The following medium, which corresponds essentially to that of the large-scale incubators, was used: ammonium nitrate, 10.0 grams; potassium dihydrogen phosphate, 6.8 grams; magnesium sulfate heptahydrate, 5.0 grams; zinc sulfate hexahydrate, 0.05 gram; ferric chloride hexahydrate, 0.16 gram; glucose, 100.0 grams; distilled water to make up to 1000.0 cc. Calcium carbonate in excess was added to the individual flasks to maintain the reaction at a point favorable to maximum growth. Growth of the Monascus species and Cunninghamella elegans was very poor on the above testing medium so that the following was employed: malt sprouts, 50.0 grams; peptone, 50.0 grams; glucose, 100.0 grams; distilled water, 1000.0 cc.
RESULTS OF THE SURVEY The results (Table I) indicate generally that, in so far as production of mycelium was concerned, i t made little difference which of the above specified methods of maintenance was used. The maximum number of tests for all substrates combined was forty, in the case of A . Jischri, and the minimum twenty-nine in the case of P. terrestre. With three exceptions the average weights showed a close correspondence. A . nidulans and P. aurantio-brunneum were adversely affected by weekly transfers alternating between malt extract and potato-glucose medium; A. sydowi in the %month transfer series alternating between malt extract and potato-glucose medium was similarly changed. The Cunninghamella and P. terrestre cultures appeared TABLEI. STOCKCULTUREGROWTHTESTS A V . WEIQHT
ORQANISM AND
MEDIUM A . fischeri: Malt extract Malt extract-potato. glucose medium Soil Bread A . pavipes: M. E. M .E.-P. G. S.
B. A . nidulans:
M. E. M . E.-P. G.
S.
B. A . niger: M. E. RI. E.-P. G. S.
B. A . sydowi: M. E. M. E.-P. G. S. B. P . aurantio-brunneum: M. E. M. E.-P. G. 6.
B.
P . citrinum:
M. E. M. E.-P. G. S.
B.
P . terrestre: M. E. M. E.-P. G. 9. B. Monascus species: M. E. M. E.-P. G. S.
B. Cunninghamello elegans: M.E. M.E.-P. G. S.
of MYCELIUM PER 100 CC. MEDITJW' A C B Grams Grams Grams
A
B
C
%
%
%
2.80
2.94
18
14
4
2.68 2.77 2.72
2.86
14 14
13 16 12
2 .. ..
1.92 2.19
..
2.39 2.24 2.60 2.68
2.18 2.23
28 12
26 15 13 13
13 10
3.17 1.63 3.02
3.23 3.10 3.00 3.22
2.84 3.02
7
..
4 93 13
16 11
2.82 2.81
..
2.80 2.85 2.97 2.90
2.89 2.87
10 5
2.38 2.31 2.51
2.19 1.78 2.35 2.31
2.29 2.02
2.98 2.28 2.99
2.96 2.94 2.93 3.01
2.99 2.98
2.49 2.43
2.47 2.54 2.60 2.60 1.86 1.92 1.94
1.97 2.01
..
3.58 3.61
..
3.60 3.53 3.52 3.60
.. ..
3.33 3.29 3.24
3.17 3.13 3.38
3.07 3.15
.. .. ..
.. ..
.. ..
2.04 2.02
.. ..
3.74 3.83
..
..
.. ..
.. .. ..
..
.. ..
..
..
..
6 10 7
10 8 11
8
18 16 21
30 41 16 23
3 45 3
4 3 7 9
2.47 2.48
11 13
14
..
..
..
..
.. ..
..
.. ..
.. ..
.. .. ..
1s 6 5
8 7
13 9
..
.. .~
.. 7
....8 10 25 .
I
.. 4 4
.. .. 13 9
.. .. 3 7
..
9
..
..
..
S 8
..
4 6 6 4
..
5 4 6
15 16 11
18 15
..
..
4 3
..
B. .. .. .. a A = weekly transfer; B = 3-month transfer, or no transfer in cases of soil and bread media; C = 6-month transfer. ~
(Tests made over 9-month period) WT. OF
CULTURE MhINTEh'AKCE Time Transferred tested Months Stock control 1-wk. intervals
.. 5
1-wk. intervals
6
6
6 3-mo. intervals
No transfer
2.65 2.78
..
TABLE11. DETERMINATIONS ON INDIVIDUAL CULTURESOF A . sydowi No. 1
6-mo. intervals
2.93
.. ..
unable to withstand desiccation on the bread medium; the former was lost within a few weeks, while the latter survived only through the first 3-month test period. It appears from the data in the last three columns that the maximum variation from the average likely to be encountered with cultures of this type is about 20 per cent. Of the molds studied, A. sydowi showed the greatest variability, with A . fiavipes running a close second. Monascus species No. 2 showed great stability, although the higher weights obtained as a result of the use of the peptone medium tend to minimize the percentage differences. Nevertheless examination of the weights showed a close correspondence.
3-mo. intervals
MAX.VARI.4T I O N FROM -4V. WEIQHT'
Vol. 26, No. 12
3 6 9 3 6 6
6 3 6
%(alternated) g:-P. G.
Soil-31. E. (alternated) 31. E. M.E.-P. G. (alternated) Soil-M. E . (alternated) M . E. 31.E. h1. E. P. G. M.E. & EI ..
P.G.
Soil Soil
NITROQEN
(DRY BASIS)
% 5.66 6.41
2.55
13.68
2.69
14.21
5.01
3.04 2.16
15.04 11.95
4.40 4.72
2.04
12.69
4.68
2.29 2.00 1.46 2.62 1.99 1.29 2.07 1.71 1.98 2.59 2.37 1.74 2.85 1.98
14.26 12.36 10.97 11.70 12.98 12.86 10.55 11 02 12.43 10.41 12.58 16.26 16.79 14.14
5.09 6.10 4.40 6.08 4.79 5.13 5.01 4.72 6.28 5.09 5.98 6.40 4.68 4.83
Soil Bread Bread Bread M. E. = malt extract; P. G. = potato-glucose medium. 9 3 6 9
a
DRY MYCELIUM PER 100 LIPID CULTURE cc. (DRY MEDIUM" MEDIEM BASIS) Grams % M. E. 2.52 11.04
Table I1 presents data obtained for individual cultures of A . sydowi over the first 9 months of the year test period. The weight of dry mycelium showed greater fluctuations from the average than did either the lipid or nitrogen content. Dry mycelium varied as much as 34 per cent, while 25 and 18 per cent were the maximum variations for lipid and nitrogen, respectively. The most consistently high yields were obtained in the case of the cultures which were transferred weekly. The yields were not, however, sufficiently high to warrant the adoption of this procedure in preference to one less time-consuming. I n general, despite some variability, the cultures on soil medium showed considerable constancy. T h a t method of maintenance involving the least labor is indicated-namely, the use of sterilized soil. N o cultures were lost on soil, except Cunninghamella elegans after 6 months, and cultures recovered were in excellent condition. YIELDSOF MYCELIUM FROM INCUBATORS The culture Aspergillus sydowi No. 1 has been used extensively for large-scale growth, and some twenty-four batches of it have been grown in the course of a 2.5-year period. The seed inoculum for the first nine batches was obtained from stocks maintained on malt agar at ice-box temperature. I n the past, standard practice in this laboratory has been to hold cultures a t 10" C., transferring a t 6-month intervals to fresh tubes of malt agar medium, etc. The seed from such cultures furnished irregular results, and the average weight of dry mycelium per liter of medium used was 16.9 grams. There was a maximum variation from this weight of practically 100 per cent, while the average variation was 47 per cent. The seed inoculum for the last fifteen batches was
December, 1931
I N D U S T R I A L A ND E N G I N E E R I N G C H E M I S T R Y
obtained by recovery from the same soil culture over a period of 1.5 years. The improvement in uniformity of yield, as well as yield per liter of medium, was great. The average weight of dry mycelium per liter of medium used was 23.1 granis. There v-as a maximum variation from this weight of 38 per cent, while the average variation was 11 per cent. Thus, for -4. s ~ d o i o ithe seed from soil cultures plainly gives superior re.sults. PREPAR-%TIOS O F S O I L STOCK CULTURES
bstrate for mold stock cultures a. uyed in this r e r y simply prepared : To air-dried orchard loam soil (hfiami silt, loam) sufficient water is added to bring it to a moisture content of about 20 per cent,. The soil is then transferred in convenient amounts (about 5 grams on a dry basis) to ordinary half-inch (1.27-em.) culture tubes. The tubes are plugged Jvith cotton and given four 3-hour sterilizations at 15 pounds per square inch (1 kg. per sq. em.) pressure on alternate days, and tested for sterilit,y by addition of yenst-water-glucose broth to tubes selected at random. The tubes :%rethen inoculated with 1 cc. of a heavy spore or mycelium suspension of the desired mold and kept a t room temperature. That there is appreciable growth and sporulation on the soil can usually be ascertained without. difficulty by direct microscopic observation. While the addition of nutrient to the soil may bring about somewhat grwter growth, it does not seem t o enhance the keeping qunlities of th. cultures.
1299
It has been found possible to preserve on soil mold stocks used for large-scale gronth-namely, dspergillu,s fischeri, A. sydowi, and Penicillium chrysogenum-for over 2 years without loss of their essential and desirable characters. The results of testing such cultures of A. jscheri and A . syldowi are included under the soil averages in Table 1. Moreover, the gross colony characters have remained much more constant than did those of the corresponding cultures maintained in the usual way on agar slants. The soil cultures can be recovered as required simply by streaking some of the soil particles on fresh agar slants.. It is not in all cases advisable to depend on soil alone for the preservation of valuable stocks, but reserve stocks may without difficulty be prepared on soil, and the writers believe that in many instances soil will be found to be a n excellent medium for maintenance, with a minimum of change over long periods of time.
LITERATURE CITED (1) B a r t h e l , C., Centr. Bakt., 11, 48, 340-9 (1918). (2) Peterson, W. H., Pruess, L. -M, Gorcica, H. J.. and Greene, H. C., ISD. EKG.CHEY.,25, 213 (1933).
RECEIVED August 11, 1934. This work !vas supported in part by a grant f r o m t h e Wisconsin klumni Reeearrh Foundation.
Saturated Solutions of Urea in Liquid Ammonia Vapor Pressures and Compositions from - 26.4" t o 101.0" C. WALTERSCHOLL AND R. 0. E. DAVIS, Fertilizer Investigations, Bureau of Chemistry and Soils, Washington, D. C.
U
REA is lrno~xmgenerally to in
T h e solubility of urea in liquid ammonia has ~ ~ ~~ ~ ~ ~ ~ ~ l $ ~ ~ been determined f o r the temperature range -26" or pressure gage and compressed ~ of the air for balnncing, 1 1 aid to 1010 C, T h e break in the solubilify curce mercury columns, the vapor presprobably indicates the transition f r o m co(ivH2)2.- sure of the material in A against YHS to CO(!VH2)2 U E 45.6" C. The solubility an air pressure. The volume of tube A and connection and the temperature-pressure curces indicate that to the mercury U is 6.09 cc. The is formed within the range of mercury manometer employed no was capable of measuring up to 5 temperature inzjestigated. The solubility deteratmo3pherea pressure. This will be minations trgree closely, in general, with those of A e?$!l& n(;t ~ ~ ~ h ~ a ~ ~ Junecke; there is a slight zariation for the s d u ing point of 132.7' C. vas used. bility near [he transition point, and at the higher S?.nthetiC ammonia was d r a ~ n
a n h y d r o u s liquid ammonia, but t'here have been no rneasurenlents of its solubility until recently. The only published data so far encountered are those by Jdnecke ( 2 ) in hi5 study of the carbon dioxideammonia-v-ater s y s t e m . The lnetllod enlployed b!, llilll is described in a n earlier paper ( I ) . New interest attaohes to solutions of urea in liquid ammonia temperature,s, since the successful operations of commercial processes for the synthesis of urea. Deternlinations of the solubility of llrea in anhydrous liquid ammonia over ,.he temperature range -26" t o 101" c. have been carried out by the authors employing a method whereby the solubility of the urea and the vapor pressure of t'he saturated solution are obtained in one apparatus.
SOLUBILITY DETERMIX'ATIOSS The apparatus employed is shown in Figure 1. The isoteniscope was constructed t o withstand pressures up t o 60 atmospheres. The Pyrex tube, A , 7 em. long, 9 mm. i. d., is soldered t o an inner cap and attached t,hrough a Rezistal valve, B , and tube to one arm of a U-shaped apparatus containing mercury. The two arms of the U are of capillary Pyrex tubing with 18 em. visible, incased in slotted steel cylinders and conuected a t the bottom with each other through a Rezist,al valve, L, .~PPARATUS.
from a cylinder, dried, and condensed by liquid air, and all noncondensable gases were p u m p e d off. Mixtures of the composition represented in Table I n-ere confined in chamber A of the isoteniscope. The required amount of urea was neighed and transferred t o this chamber, the vessel attached to the isoteriiscope, and the whole completely evacuated. The required amount of ammonia was measured in a volume buret and condensed into chamber A at liquid air temperature. The isoteniscope was placed in a water bath thermostatically controlled, The temperature mas adjusted to that temperature a t which all solid just passed into solution. The approximate temperature was first determined with similar mixtures in sealed glass tubes. The liquid mixture was continually agitated by a magnetically operated stirrer to prevent supersaturation. The heat from the magnetic coil surrounding chamber A was completely dissipated into the bath as found by actual measurements and did not affect the temperature of the mixture. The bath temperature was measured by a thermometer graduated in tenths of a degree and calibrated by t'he Bureau of Standards. -4fter establishing equilibrium by maintaining t'he mixture a t
