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tions which are reproducible and easily prepared. From photoelectric color, I. C. I. data can be calculated if desired. Photoelectric color can be correlated with the Union system on a statistical basis.
Acknowledgment The authors’wish to acknowledge the invaluable cooperation of Hellige, Inc., in preparing the experimental equipment required during the course of this work. The instrument developed for the present purpose is known as the Hellige-Diller photoelectric colorimeter, Model 405.4. They also wish to acknowledge the assistance obtained by frequent reference to a private report by 1‘. -4.Kalichevsky and B. IT. Story.
Literature Cited (1) Am. Soc. Testing Materials, Standards on Petroleum Products
and Lubricants, Designation Dl55-39T. (2) Ibid., Designation D156-38.
(3) Chevreul, M. E., “Color Chart” (publisher unknown). (4) Diller, I. M., J. Biol. Chem., 115,315-22 (1936). (5) Diller, I. M., paper presented before the rlmerican 011 Chemists’ Society, fall meeting, 1941. (6) Ferris, F. W., and McIlvain, J. M.,IND.ENG.CHEM., ANAL. ED.,6,23-9 (1934). (7) Gardner, H. A., “Physical and Chemical Examination of Paints, Varnishes, Lacquers and Colors”, 9th ed., Washington, D. C., Institute of Paint and Varnish Research, 1939.
Vol. 14, No. 8
Hardy, -4. C., “Handbook of Colorimetry”, Cambridge, Mass., Technology Press, 1936. Harris, Moses, ”Prismatic Chart” and “Compound Chart”, 1811. Judd, D. B., J . Opticd Soc. Am., 23,369-74 (1933). Lovibond, J. W., “Light and Color Theories and their Relation to Light and Color Standardization”, London, E. and F. X , Spon, 1916. Maerz, A. and Paul, M .P., “Dictionary of Color”, New York, McGraw-Hill Book Co., 1930. Munsell, A. H., “A Color Notation” and “A Color AtlaJ”, Baltimore, Md., Munsell Color Company, 1933. Kelson. 15’. L., Oil Gas J . , 37, No. 3, 74 (June 2, 1938). Ostwald, W., Chem.-Ztg., 43, 681-2 (1919). Parsons, L. W., and Wilson, R . E., J. IND.EX. CHEM.,1 4 , 269-78 (1922). Ridgway, R., “Color Standards and Somenclature”, London, Weslev. 1912. Rogers, T. H., Grimm, F. V.,and Lemmon, S . E., IND.ENG. CHEM.,18, 164-9 (1926). Scofield, F., Judd, D. B., and Hunter, R . S., A . S.T. .If. Bull., 110,19-24 (May 1941). Societe Franpaise des Chrysanthemistes, “Repertoire de Couleurs”, designed by Dauthenay and others. Story, B. W., and Kalichevsky, V. 9 . , IND.ENG.CHEX,A N LL. ED., 5,214-17 (1933). C. J. Tagliabue Mfg. Co., Brooklyn, N . Y., “New and Revised Tag Manual for Inspectors of Petroleum”, page 57, 25th ed., 1939. Vinoch, H., Refiner Natural Gasoline M f r . , 16,601 (1937). Weir, H. M.,Houghton. W. F., and Majewski, F . SI., P r o c rlm. Petroleum Inst., XI, No. 75, 60-72 (1930).
Determining the Deterioration of Cellulose Caused by Fungi Improvements in Methods GLENN A. GREATHOUSE, DOROTHEA. E. KLERIME, AND H. D. BARKER Bureau of Plant Industry and Bureau of Home Economics, Department of Agriculture, Washington, D. C.
A
CCURATE practical methods of determining the factors
involved in the deterioration of fabrics and other fiber products are widely needed a t the present time. Numerous investigations have shown that cellulose deterioration may be a result of the action of fungi, bacteria, ultraviolet radiation, chemicals, or a combination of these factors. Nost of the effort has been directed toward a comparison and appraisal of preventive treatments rather than toward a better understanding of the causes and mechanism of deterioration. Methods of evaluating deterioration have varied so greatly that it is extremely difficult to make accurate comparisons. The present paper deals largely with (1) experiments on the refinement of technique for testing the amount and rate of cellulosic decomposition that might be caused by various types of microorganisms, and (2) suggestions for the establishment, within certain limitations, of a standardized quantitative technique for testing the effectiveness of mildew-resisting treatments. For the latter purpose certain modifications of the preparatory treatments for the fabric would probably be required.
Literature Review Several excellent reviews (6,6,7, I d , 13) on the microflora associated with fabrics and raw fibers composed principally
of cellulose have been published. They show that most of the reports on the microbiology of cotton and its manufactured products give only the identification of microorganisms found on these materials. Only a few investigators have attempted to secure quantitative data that may represent the ability of these organisms to destroy cellulose. For example, Searle (10) determined the wet breaking strength of mildewed fabrics to estimate deterioration. He developed a method in which 37.5 x 3.75 cm. (15 X l . 5 inch) strips of cotton fabric were wound on filter candles which had been coated previously with a soil suspension. These strips were then incubated for 3 or 6 weeks by placing each candle in a test tube containing a small quantity of \rater. A loss of 55 to 93 per cent in stren th occurred during 6 weeks’ incubation. Searle’s method k e quently failed to give close agreement between replicates under apparently identical conditions. Thom. Humfeld, and Holman (18) determined the breaking strength of mildewed duck after conditioning in a standard temperature and humidity room for 2 or more days. They abandoned mixed cultures because of the difficulty in obtaining comparable results, and after testing pure cultures of many organisms, selected Chaetomium globoszlm Kunze. A mineral agar was used to support the cellulose sample which was incubated 14 dags at 28” t o 30” C. Rogers, Wheeler, and Humfeld (9) made quantitative chemical and physical analyses of mildewed duck. They measured the activity of Ch. globosum and Spirochaeta cytophaga Hutchison
ANALYTICAL EDITION
August 15, 1942
615
and Clayton,.grown an cotton duck on a mineral salts agar medium and incubated for 1 t o 18 day8 a t room temperature (about 28' CJ. They used square 0.5-liter (16-ounce) screw-cap, closed bottles. Furry, Robinson, and Humfeld (4) studied mildew-resistant treatments on fabrics using the procedure described by Rogers, 'O' Wheeler. and H UYlrl" ~ $ *,Y,. '~ Other'investiaators ( d , 3, 11) have used soil burial tests, or
check of the casu& ahencies t g a t are operative in a ^given test; because of so many unknown or uncontrolled factors, close agreement between results from different tests-if between samples in the same test-would not be expected. Such pracedures have, however, resulted in valuable contributions and undoubtedly will continue t o be useful for checking and improving laboratory methods.
Materials and Methods Bleached, 8-ounce Army duck was used throughout these investigations. Before exposure to the attack of the fungi, the fabric was treated to remove sising finishes, any residual waxes, pectins, and other substances that might serveasaddednutrients. For the degrertsing procedure the material was extracted twice for 2 hours each in two changes of carbon tetrachloride, followed by a treatment with0.05 per cent starch and protein-solubilizing enzyme preparation a t 60' C. for 1 hour. Finally, the cloth was thoroughly rinsed in distilled water (I). The fabric was divided into large blocks so that each block would furnish one of the ten replicates used for each determination. The blocks were cut into strips measuring 15 em. (6 inches) in the warp direction by 3.1 em. (1.25 inches) and by raveling the width was reduced to exactly 2.5 em. (1 inch) ( 1 ) . The ten replicates used for each determination were selected by a series of random numbers. Individual incubation chambers were 0.5-liter (16-ounce), square bottlesmeasuring 16.25 cm. (6.5) inches to theneck. Metal screw t o m to these bottles were modified to Dermit a uniform ex;hinge of gases but to exclude any contakinating organisms. Sterile nutrient media in containers with these modified caps
~~~
~~
~
FIGURE 1. INDIVIDUAL INCUBATION CHAMBER ADAPTED TO THE USEOF LIQUIDMEDIUM
~
was cut out of the center of both the cap and the waxed-pa&; filler. A round piece of glass filter fabric, the diameter of the filler, was inaerted between the cap and the ring of waxed filler (Figure 1). I n order t o keep the strip of cotton duck in contact with the liquid medium and yet prevent it from being submerged, a mat of glass fabric was placed in each bottle to support the strip and serve as a wick. These mats, which were approximately 5 x 15 X 0.25 em. (2 X 6 X 0.1 inches), are made from a mater i d similar to Pyrex and thus are not attacked by organisms. (Detailed description of the alass fabrics that were found to be
.~ ~
~~~
&¬ ravel. U n s e k d s t r i p s , however, were re-used several times in these experiments with little difficulty from raveling.) Sufficient liquid medium was pipetted into each bottle to come up to the upper surface of the glass fabric when in the horimntal position. The liquid medium may be conveniently measured into the bottles by means of measuring or an automatic zero pipet (8). The liquid in the culture chamber should not submerge the cotton test strip. The bottles after being capped and stacked horizontally were autoclaved for 20 minutes a t 6.8 kg. (15 pounds) pressure. With only slight modifications this test system could be changed to one of continuous renewal of mineral solutions. This might be useful and advisable in testing treated fabrics when leaching of toxic chemicals is suspected. Stock cultures of the various fungi used in these investigations were maintained in test tubes on filter paper placed on the surface of an agar slant prepared according to the desired mineral formula. About 10 to 14 days before the spores were required for inoculating the fabric, transfers were made to Petri dishes containing sterile filter paper on mineral salts-agar. The inoculum was prepared by harvesting the fruiting bodies and mycelium on the surface of one piece of 7-cm. filter paper and suspending them in 50 ml. of sterile distilled water in Erlenmeyer flasks containing small glass beads. The flask was shaken until an even suspension was obtained. (This system of preparing the inoculum has since been sim-
by shakinlg the flask until the inoculum is dispersed by {he sction of the glass beads, adding sterile distilled water, and pipetting off the suspension. Agar and labobor are saved and opportunity for contamination is minimized.) While it appears t h a t within reasonable limits the amount of inoculum far each strip is not very important, it is recommended that 1 ml. of the resultant suspension be transferred by meam of a sterile pipet. The strips of fabric were incubated in a darkened, air-conditioned room which was maintained a t 85' to 86" F. (29.4" to 30.0" C.) and 90 to 92 per cent relative humidity
The liquid nutrient that appeared to be well adapted t( the cellulose-decomposing fungi so far tested is representec by Formula A, and is prepared as falllows: C./L
For comparison of formulas a n d techniques t h e method described by Rogers, Wheeler, a n d Humfeld (9) also was followed. In each experiment the standard procedure was followed except for t h e variables under s t u d y or when specific mention is made for a certain procedure. T h e mineral
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nutrient agar, designated as Formula B, used by Rogers. Wheeler, and Humfeld (9) was prepared LMfollows: T a p water, ml. NaNOs, g r a m s XaHPO4, g r a m M so4, g r a m ~ 8 1gram , Agar, g r a m s
1000.0
3.0 1.0 0.25
0.25 10.0
Both formulas were used with and without agar. After 7 days’ incubation the strips were removed from the bottles and carefully rinsed in runnin water to remove the fungal growth. The air-dried strips were $aced in an oven at 80” to 85” C. for 1 hour. These samples were conditionedin constant temperature 21.11’ C. (70’ F.) and humidity (65 per cent) for at least 4 hours previous to making the breaking strength measurements by means of the motor-driven Scott tester (1). The average loss in breaking strength of the inoculated strips, expressed as departure from the average of the controls, was calculated and recorded as the degree of deterioration. The pH of the media was determined by the Beckman glass-electrode apparatus before autoclaving and again after harvest. Adjustment of the liquid media to a given pH with hydrochloric acid or sodium hydroxide before autoclaving appears, as judged from several experiments, to be valueless. After autoclaving, the pH of adjusted and nonadjusted media becomes approximately equal.
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such as Ch. globosum, could not be safely incubated much beyond 7 days if reliable breaking strength tests were to be obtained with the Scott tester. I n this period of time approximately three-fourths of the original strength is destroyed. Consequently, in succeeding experiments, harvests were made a t the end of 7 days, unless otherwise specified. A subsequent experiment was designed to evaluate the deterioration of cotton duck when no mineral salts were added. lnoculated strips and controls were incubated with distilled water and on water agar, using Ch. globosum as the test organism. The procedure, except for mineral nutrients, was standard. The data obtained are presented in Figure 2.
COMPARISON O F S O L I D AND L l O U l D SUBSTRATES ( 7.DAY INCUBATION P E R I O D ) ^ ^
U N I NOCULATED
INOCULATED
EFFECT O F DURATION O F INCUBATION PERIOD
Experimental Results
It was thought advisable to determine experimentally the length of incubation period ar.d the concentration of Ch. globosum inoculum required to yield the most consistent losses in breaking strength. In this experiment the percentage of loss in breaking strength a t the 7-, 12-, and 14-day harvest periods was 73, 93, and 97, respectively. Control strips that received identical treatments, except for inoculation, were harvested a t 0, 7, and 14 days. The breaking strength values for the corresponding harvests were, 57.6, 53.5, and 52.2 kg. (127, 118, and 115 pounds), respectively. The average for the controls of all other experiments was 59 kg. (130 pounds) after 7 days’ incubation. This value has been used in all subsequent calculations. The inoculum, prepared as described under Materials and Methods, was centrifuged a t 2500 r. p. m. for 20 minutes, and 4 ml. of the resultant dense deposit were then suspended in 50 ml. of sterile distilled water. This dilution was designated as concentration &. This concentration was further diluted to l to 10 and l to 50 and these suspensions were designated as Kz and K3, respectively. S o significant difference in loss of breaking strength was found between inoculum concentrations K 1 and K z for the 7-day incubation period. (As used in this paper “significant” refers to statistical odds of 19 to 1 and “highly significant” t o odds of 99 to 1. Since the mean values for the results obtained are largely presented in graphical form, the differences required for significance accompany each graph. Significance of main effects may be judged by differences in height of the bar graphs or points on the curve that indicate the means that are to be compared. Significance of interactions may be judged by the degree of failure of the curves to extend in a parallel direction.) Although the growth resulting from concentration Ka developed somewhat less rapidly and gave a significantly lower breaking strength for the 7-day harvest, this difference was not detectable for the 12- and 14-day harvest periods. Thus, except for extreme dilutions, it appears that with Ch. globosum the amount of inoculum is not of great importance when favorable nutrition and conditions of incubation are provided. These initial experiments also indicated that under favorable conditions active cellulose decomposers,
E
301
I
CoPIrolr o n 1tquidSvbilra:e
DAYS
FIGURE2. Loss IN BREAKINGSTRENGTH WITH DISTILLED WATER AND WATER AGAR SUBSTRATES Difference required for significance a t odds of 99 to 1 and 19 t o 1: between means for inoculated us. uninoculated and solid u s . liquid, 8.80 a n d 6.56 pounds. D u r a tion of incubation period, 8.80 and 6.38 pounds
When no salts were added, growth of the fungus was very slow on the strips of cotton duck and deterioration was slight. The strips on water agar yielded a highly significant loss in breaking strength when compared with those incubated with distilled water. This is interpreted as indicating that the agar itself is of some nutritive value. Since it wa5 difficult to determine what was being furnished, and since it is general knowledge that agars from different sources vary somewhat in their composition, it seemed desirable to remove this variable. Furthermore, several tests with ether extracts of agar gave no indication of any growth substance that could be detected with the Chaetomium. The uninoculated controls gave no evidence that the presence of constituents in agar had any effect on the loss of breaking strength. It does seem important, however, to note that in 7 days about 1.8-kg. (4 pounds) loss in breaking strength was shown for the controls, and that about 6.8 kg. (15 pounds) was registered for the controls that were incubated 14 days. For this reason it seemed important to record the results of the various experiments in terms of the control rather than as actual breaking strength. Since many investigators of cellulose deterioration, and especiauy those using Ch. globosum as a check on the efficiency of rot-proofing treatments, have used Formula B and closed
ANALYTICAL EDITION
August 15, 1942
rs -
INTERACTIONS
““:r a FIRST
MATTER
LERATION
FORMULA X ‘ATE ff MLlTEl
ORDER
FORMULA X AERATION
SECOND ORDER
AERATION X STATE OF M 4 m l
8ol 70
&ERATION X F O R M U L A X STATE OF M A T T E R
I
90
A-Formula A B- Formula B S-Solid L- Liquid Ae- Aerated N-Ae -Non-Aeraled
80
70
617
A significantly greater loss in breaking strengti was obiaiied from the aerated than from the nonaerated cultures. Although this difference was not great, the trends were so consistent that its failure to attain high significance suggested an examination of the sampling error obtained in the analysis of variance. The results were striking and furnish additional evidence of the need for providing aeration. The graphic presentation of meansquare sampling error or residual variance among replicates for various stratifications of data is given in Figure 4. The sampling-error variance for Formula A is only about half that for Formula B. The solid state had less within variance than did the liquid state. When sampling error is
o c
and for closed is 88.1583. One practical implication of the difference is that, to give Differences required for significance a t odds of 99 t o 1 a n d 19 to 1 : between means for the same precision in a comparison between formulae. s t a t e of m a t t e r , a n d aeration. 4.13 a n d 3.11 pounds means, approximately 9.5 times as many replicates would be required with a closed RS with an open system. bottles, an experiment was designed to compare Formulas Many modifications of Formula A were tested with Ch. B and A. The effects resulting from the bottles being closed globosum, but as no promising leads were obtained the data with metal caps and with aerated glass fabric tops were are not presented. However, no differences were found on evaluated. By using a 2 x 2 x 2 factorial, this experiment halving or doubling the amount of nitrogen, using Washingalso permitted the further testing of solid vs. liquid media. ton, D. C., tap or distilled water, or in the presence of iron, I n Formula A ammonium nitrate was selected as the source manganese, zinc, etc. Despite these results the use of disof nitrogen, in view of the possibility that all organisms may tilled water and the addition of the trace elements have been not be capable of utilizing effectively only the nitrate or only retained in Formula A, since these elements appeared to do the ammonium radical, and also because the presence of the no harm for the organisms tested and are known to be required ammonium and the nitrate radicals increases the buffer for certain fungi. capacity of the solution. Since fungi difier in their nutrient, respiratory, and moisA comparison of the data secured with Formulas A and B ture requirements, an experiment was performed to determine used in the liquid and solid state, with and without provision favorable conditions for six fungi that previously had been for uniform acration for the culture bottles, is presented in found to be active cellulose decomposers: Ch. globosum Figure 3. Kunze, Chaetomium elatum Kunze and Schmidt, Stachybotrys The nitrogen contents of Formulas A and B mere 0.350 papyrogena Sacc., Metarrhizium sp., Alternaria sp., and and 0.495 gram per liter, respectively. Since statistical Hormodendrum sp. (After this manuscript was prepared analysis of the data from a subsequent experiment showed the attention of the writers was called to the fact that, acno significant differences for nitrogen levels ranging from cording to the usage establish by Charles Thom, this fungus 0.175 to 0.700 gram per liter, it seems logical that the differshould be designated as Cladosporium sp.) The numbers ence in nitrogen equivalent may be largely ignored in evaluunder which these specific cultures are maintained in this ating the results for this experiment. laboratory are 1042.4, 1043.4, 1331.1, 1334.1, 1022.2, and Ch. globosum yielded approximately equal losses in breaking 1236.1, in the order listed. Herbarium specimens have been strength, irrespective of the formula or the phase of substrate deposited with the Division of Mycology and Disease Survey, employed. These results were particularly encouraging since, as is brought out in the discussion, the liquid technique has many advantages over M A I N EFFECTS INTERACTIONS the agar method. FIRST O R D E R ‘ S E C O N D OROER X i t h “closed” bottles the degree of exF O R M U L A S:t:!&F AERATION FORMULA x FORMULA X AERATION X AERATION x FORMULA FIGURE 3. EFFECTOF AERATIOKAXD OTHERFACTORS STRENGTH
ON
BREAKIKG
STATE O F MATTER
AERATION
STATE O F H A l T E R
X STATE O F M A l T E R
INDUSTRIAL AND ENGINEERING CHEMISTRY
618 MAIN EFFECTS
FORMULA A I N H l r N O l )
z
120.
FORMULA 0 ( N B N O l I
3 Hormodendrum sp. 4 Allernaria S P , 5 C h i e t o m i v m ploborum
6.0
70
65 pn
75
8.0
8.5
AT HARVEST
FIGURE 5. EFFECTS ON BREAKIXG STREXGTH OF SIXORGANSXS WITH Two FORMULAS 19 LIQUID MEDIA Differences required for significanoe a t odds of 99 t o 1 and 19 t o 1: between means for organisms, 17.09 a n d 12.93 pounds; formulas, 9 i S and 7.17 pounds
U. S. Department of Agriculture, Kashington. D. C. A comparison of the influence on breaking strength of these six fungi when grown on Formulas A and B, modified to 0.350 gram per liter of nitrogen, is presented in Figure 5. In addition to this factorial design, five of the organisms were further tested as to their behavior on liquid and solid media (Figure
Since the results presented in Figure 5 showed a highly significant interaction for organisms with nitrogen sources, it seemed desirable to determine the relation between sources and levels of nitrogen on the two Chaetomium species. The influence of three sources and three levels of nitrogen on the deterioration activity of Ch. globosum and Ch. elatum is presented in Figure 8. The basic solution was Formula A and equivalent quantities of nitrogen were used for each source. Ch. globosum produced a loss in fabric breaking strength of 53.5 kg. (118 pounds) with sodium nitrate, 49 kg. (108 pounds) with ammonium nitrate, and 42.5 kg. (96 pounds) with ammonium dihydrogen phosphate as sources of nitrogen. On the other hand, the growth of Ch. elatum under similar conditions resulted in losses of 30.4, 40.4, and 39.9 kg. (67, 89, and 88 pounds), respectively. Levels
INTERACTIONS
MAIN EFFEC'
1
ORGANISM
Vol. 14, No. 8
TATE OF
LIQUID
Y&ITFD
I
4
SOLID
~5
0
~
I
6).
The interactions between Organisms and formulas show that all organisms except Ch. globosum caused a greater loss in breaking strength on Formula A than on Formula B. These differences are highly significant. The loss in breaking strength is associated with change in pH. -The pH values a t harvest for FIGURE6. EFFECTS O N BREIKISGS T R E S G T H O F FIVEORQANIS&fS WITH the fungi grown on Formula B ranged from LIQUID4 N D SOLID M E D I 4 7.7 to 8.5. The nitrogen source of the formulas Source of nitrogen, ammonium n i t r a t e (Formula A) Differences required for significance a t odds of 99 t o 1 a n d 19 t o 1: between means for seems to influence the p H of the medium. organisms, 15.39 and 11.62 pounds: s t a t e of matter, 9.74 a n d 7.35 pounds All fungi studied, except Ch. globosum, yielded a greater loss in breaking strength with a pH of 7.0 or less. of nitrogen were of little importance. From the graph it Each of these organisms, however, is influenced by other appears that pH, including the effect that levels and sources factors, as indicated in the interaction between liquid and of nitrogen have on pH, is a very important factor in detersolid states. Alternaria sp. gave a greater loss in breaking mining the response of cellulose-decomposing fungi. strength on liquid medium than on solid. In contrast, the Previous to autoclaving the solutions containing ammonium other organisms, particularly S. papyrogena, produced more dihydrogen phosphate, ammonium nitrate, and sodium nitrate loss on the solid medium. Subsequent experiments, using the pH values were approximately 5.5, 6.6, and 6.8, respecdouble and single wicks with identical amounts of liquid tively. After autoclaving, they were approximately 6.0, 6.7 substrate, indicated that this mas not a response to the solid and 6.9. As the fungus utilized the phosphate salt the pH agar but was due to a difference in available moisture. decreased to 4.7 for Ch. globosum and to 5.3 for Ch. elatum, Certain of these organisms differ markedly in the rapidity whereas with utilization of the sodium salt the pH increased with which they bring about cellulosic deterioration. In to 8.1 and 7.2, respectively. The ammonium nitrate source Figures 5 and 6 the time of harvest was 7 days for all organappeared to have good buffering capacity, the pH a t harvest isms except Alternaria sp. and Hormodendrum sp., which were varying between 5.6 and 6.8 for both organisms grown at harvested a t 14 days. Both these organisms are good celluthe three nitrogen levels. lose decomposers but develop very slowly. Metarrhizium sp., however, develops so rapidly that harvest should be made prior to 7 days. Discussion A photographic comparison of typical response of MetarAs an approach to ft standardbed teohique applicable to rhizium sp. on the two formulas, after 7 days' incubation, is testing mildew resistance of cellulosic materials and comshown in Figure 7 . Under a favorable controlled environparing the ability of microorganisms to decompose cellulose, ment, Metarrhizium sp. caused 94.9 and Ch. globosum 81.5 the data from these studies indicate clearly that the glass per cent loss in breaking strength a t the end of 7 days' wicks for the support of the test sample in a liquid medium incubation.
August 15, 1942
ANALYTICAL EDITION
619
and the glass fabric for permittingexchange MAIN EFFECTS INTERACTIONS I DRrlN l0"RCL O r LWrL o/ O I C A l t l l W x S 0 " R C ~01 N x L i Y i l O F N of gases in culture chamber tops have im8%" portant advantages over the solid-agar, closed-system technique. 1. Less time and work are involved in securing equivalent data. With the solidmedium procedure the mineral salts-agar and the strips of fabric are sterilized separately and the sterile strips are then placed in the incubation bottles on the agar under aseptic conditions. With the glass wick-liquid medium system only one sterilization is required. There is also much less risk of contaminating the sterile strips since they are not transferred from one container to another after they have been sterilked. In addition, the glass wickliquid medium system could be readily OF Chuetomium globosum AND Chaetomium datum O N FIGURE8. EFFECTS modified toa continuous or periodic medium BREAKING STRENGTH renewal technique. 2. Experimental results have demonDiKerenoes required for significance st odds of 99 to 1 and 19 to.1: between means for organilms. 2.42 nud 1.91 pounds; ~ o m r e eof~ mtrogen and levels of nitrogen. 3.09 and 2.34 pounds strated that the aerated system yields reliable results with fewer replicates, and consequently saves testmaterial, time, andwork Mixtures of microorganisms have been largely abandoned over thenonaerated agar system. The mean-square sampling because of the difficulties involved in getting comparahle error between replicates for the nonaerated system with Ch. results with successive samples (12). Pure cultures of the alobosum was found to be 88.2 as comvared to 9.3 for the individual fungi were used in these investigations. Results aerated system. with a single organism may not yield the same results as 3. Aear itself w&s found to S U D O ~ V sufficient mineral those produced under natural conditions where many ornutrients for slow growth of fungi. S h c e it is difficult to ganisms are involved, but the same condemnation applies determine what is being furnished and agars from different to any controlled method. Thus the choice lies between a sources vary in their usable constituents, it would seem carefully standardized test and the actual judgment of exadvisable to eliminate this variable. posure to natural conditions, which is costly in time and 4. It has been found essential to change the sodium rarely produces results that are capable of being duplicated. nitrate to some other nitrogen source, because the utilization The selection of the test organism will be influenced by of the nitrate radical by most fungi resulted in a medium too many factors, depending on the problem to be solved and alkaline for their development. Ammonium nitrate, as the materials in question. Ch. globosum has been recomnitrogen source, has proved superior to sodium nitrate probmended by Thorn et al. (12) after an extensive survey of ably because of its buffering capacity and by supplying both possible test organisms, presumably on account of its ability the ammonium and the nitrate radicals for the fungi. to decompose cellulose, its frequent occurence on cotton, and its ease in handling in culture. These studies have shown that Metanhizium sp. is superior to Ch. globosum in producing rapid losses in breaking strength, especially on neutral to acid media. This, along with its equal ease of handling, indicates that Metarrhizium sp. might be a useful organism for test purposes. It is a common soil-inhabiting organism, and thus it may be an active agent in deteriorating sandbags or the fabrics exposed to the burial test. In evaluating cellulose deterioration by the activity of fungi, it is desirable to use standardized media in order to facilitate duplication of the work. The media should be buffered so that the original pH is not changed materially by the utilization of any radical or ion. From the results here reported for six fungi it appears that ammonium nitrate fills more of these requirements than does sodium nitrate or ammonium dihydrogen phosphate if but one nitrogen source is to be used.
Summary and Conclusions
OF Metarrhiriurn SP.TO FORMULAS FIOURE 7. RESPONSE A AND B
Incubated ior 7 days. Leit. uninoouiated oontrol. Center, inoeulated s&mpIewith Formuln 1. Right. with Formula B
A standardized quantitative method for the evaluation of the ability of fungi to decompose cellulose is described. Certain features of this procedure may also prove useful in developing a standardized method for testing ro6proof treatments for fabrics. This technique, employing glass fabric and liquid nutrient instead of solid mineral agar, possesses advantages with respect to accuracy and speed over previously reported methods. Provision for uniform aeration
INDUSTRIAL AND ENGINEERING CHEMISTRY
620
of culture chambers is highly important, especially in reducing sampling error variability. Metarrhizium sp. and Chaetomium globosum cause very rapid decomposition, are easily handled, and have other features that make them satisfactory test organisms. They are definitely superior to Chaetomium elatum, Alternaria sp. Hormodendrum sp., and Stachybotrys papyrogena. Ammonium nitrate was found to be a better source of nitrogen than sodium nitrate for the development of most of the fungi. The pH of the su6strate appears to have an important influence on the activity of cellulose-destroying fungi.
Acknowledgment Grateful acknowledgment is made to Ruth E. Rogers for suggestions in connection with the textile phase of the investigation, to L. M. Ames for identifying the species of Chaetomium, to Vera K. Charles for determining the identity of Stachybotrys papyrogena, to Charles Thom for the tentative identification of Metarrhizium sp., and to Katharina Bollenbacher and Helen G. Wheeler for valuable laboratory assistance
Vol. 14, No. 8
with the transfer of cultures and breaking strength measurements.
Literature Cited (1) -4m.SOC. Testing Materials, Standards on Textile Materials
(1941). (2) Armstrong, Chemistry & Industry, 60, 668-74 (1941). (3) Bur. Agr. Chem. Eng., U. S. Dept. Bgr., personal communication (1942). (4) Furry, Robinson, and Humfeld, ISD. ENG.CHEM.,33, 538-45 (1941j , ( 5 ) Levine andveitch, Ibid., 12, 139-41 (1920). (6) Prindle, Tertile Research, 4, 413-28, 463-78 (1934). (7) Ibid., 5, 11-31 (1934). (8) Rigler and Greathouse, Science, 92, 363-4 (1940). (9) Rogers, Wheeler, and Humfeld, U. S. Dept. Bgr., Tech.Bull. 726 (1940). (10) Searle, J . TeztileInst., 20, T162-74 (1929). (11) Thayson, Bunker, Butlin, and Williams, Ann. A p p l i e d Biol., 26, 760-81 (1939). (12) Thom, Humfeld, and Holman, Am. Dyestuf Reptr., 23, 581-6 (1934). (13) Veitch and Levine, Science, 49, 618 (1919). PRESENTED before the Division of Cellulose Chemistry a t the 103rd Meeting of the AMERICAN CHEMICAL SOCIETY, Memphis, Tenn.
Rapid Volumetric Method for Determination of Sulfate Ion MERLE RANDALL AND HENRY 0. STEVENSON, University of California, Berkeley, Calif.
B
ALAKHOVSKI and Ginsburg ( I ) suggested in 1931, but did not investigate, a volumetric method for the determination of sulfate ion, which consisted of adding a known amount of barium chloride to the solution containing sulfate and titrating the excess with sodium pyrophosphate. The following reactions occur:
+ +
Ba++ SO4-- = BaSOn(s) 2Ba++ PZO,---- = BalP,O,(s)
(1) (2)
,4n excess of pyrophosphate ion hydrolyzes and the end point is determined by using some such indicator as phenolphthalein. The barium sulfate is found by difference. Barium pyrophosphate is soluble to the extent of only 0.01 part in 100 parts of water a t 20" C. (Hodgman and Lange, b), and by using a water and alcohol medium, it was believed this solubility could be further decreased. The sodium salt of the pyrophosphate ion was chosen, since barium pyrophosphate is soluble in ammonium salts. Since the reaction of barium toward the sulfate ion is known and understood, the problem concerned the reaction of barium ion with pyrophosphate ion. Actually, the method was found to be experimentally unsatisfactory because (1) the alkalinity of the pyrophosphate ion depends upon its hydrolysis in water, and this hydrolysis is too slow t o give a satisfactory and reproducible end point. (2) At low alcohol content, the solubility of barium pyrophosphate is great enough to allow some hydrolysis of the pyrophosphate, thus giving a premature end point. The amount of sodium pyrophosphate needed to react with a given amount of barium a t various proportions of alcohol is shown in Figure 1. The results are erratic. As the alcohol content increases, the amount of pyrophosphate ion required to titrate the barium increases rapidly. Since no constant value of barium could be ascertained, the method was abandoned.
Disodium Hydrogen Phosphate Method Although the sodium pyrophosphate method was unsatisfactory, the authors felt that some other salt might be substituted, and disodium hydrogen phosphate was found satis factory. The extreme alkalinity of monohydrogen phosphate, HP04--, is well known. The presence of alcohol should have little or no effect upon its hydrolysis, and a t the same time alcohol serves to cut down the solubility of barium hydrogen phosphate, BaHP04. The reaction is: Ba++ HPO,-- = BaHPOp(s) (3) rlmmonium compounds must not be present, because of the solubility of barium hydrogen phosphate in the presence of ammonium ions.
+
Percent alcohol FIGURE1. STO-D.4RD SODIUM PYROPHOSPHATE SOLUTION REQUIRED TO CHANGE POINT OF METHYLRED IN ALCOHOL-WATER MEDIA
