Microbiological Deterioration of

Organic Materials:

Its Prevention and Methods of Test

United States Department of Commerce

National Bureau of Standards

Miscellaneous Publication 188

UNITED STATES DEPARTMENT OF COMMERCE • Charles Sawyer. Secretary NATION AL BUREAU OF STANDARDS • E. U. Condon, Director

Microbiological Deterioration of

Organic Materials:

Its Prevention and Methods of Test

by Edward Abrams

National Bureau of Standards Miscellaneous Publication 188

Issued November 1, 1948

For sale by the Superintendent of Documents, U. S. Government Printing Office, Washington 25, D. C.

Price 25 cents
PREFACE

The deterioration of textiles, leather, and other organic materials by mildew

and rot is a major problem in the tropics, and is also important in many parts

of the continental United States. Accordingly, it has seemed worth while to present the information on the subject that has been accumulated by the

National Bureau of Standards in the past several years, and to describe the

methods currently used to ascertain the susceptibility of materials to mildew

growth and to evaluate protective treatments. Part I has been written primarily for persons looking for general information on the cause and prevention of

mildew damage. Part II is addressed more particularly to the technologist and

the laboratory worker.

E. U. Condon, Director.
Contents

Part I. Review of the Literature

Page

Preface n

I. Introduction 1 II. Microbiology ^ 1 III. Enzyme act|on 4

IV. Organic materials utilized by microorganisms 5

1. Cellulosic materials 8

2. Proteinaceous materials 10

3. Leather 10

4. Miscellaneous materials 10

(a) Rubber 10

(b) Plastics 11

V. Prevention of deterioration 11 VI. Testing for fungicidal efficiency 14

Part 2. Investigation of Microbiological Tests

VII. Development of mildew testing at Bureau 18 VIII. Representative problems 22

IX. Test methods _ 25

1. Pure-culture biological methods 25

(a) Rot-resistance tests with Chaetomium globosum 25

(1) Direct-inoculation—sterilized specimen method. 25

(2) Mycelial mat method. _ 26

(b) Mildew-resistance tests with Aspergillus niger 26

(1) Mycelial mat method 26

(2) Sugar-free medium method 26

2. Mixed-culture biological test methods 28

(a) Soil-burial test 28

(b) Soil-suspension test 28

3. Accelerated weathering test 28

X. Experimental results and discussion 29

XI. Summary and conclusions 38

XII. References 39

Microbiological Deterioration of Organic Materials

Its Prevention and Methods of Test

By Edward Abrams *

This paper presents a detailed review of the literature on the microbiological deterioration of organic and fibrous materials. Also, representative problems incidental to the development of test methods for the evaluation of mildew

resistance and the development of mildew resistant materials are given. The most widely recognized test methods

for the evaluation of mildew-resistance and rot-resistance are listed. A wide variety of fungicides for different uses

is classified according to effectiveness. Plastics and plasticizers and related materials are rated according to their susceptibility to support mold growth.

Part 1. Review of the Literature

I. Introduction

The deterioration of organic materials by

microorganisms in nature is of vital importance

to man. Everyone is familiar with the accumulation of leaves and plant material in the fall. However, by the following spring practically all of this debris becomes a part of the soil. In this way nature renews her store of energy, the microorganisms completing the carbon, nitrogen, and

sulfur cycles. Unfortunately, microorganisms are not discriminating in selecting their nutriment

and are, therefore, a source of great loss when they

attack organic materials of economic value.

From ancient times man has been plagued by

mildew. As no method of control was known,

it has been regarded as a necessary evil. A list of materials attacked by mildew would cover the

entire field of organic materials. Because of ignorance and inertia a great waste of materials

has been tolerated through the ages. Under

favorable conditions of moisture and temperature

many substances are attacked by molds. The

present paper is restricted to the consideration

of cellulosic and proteinaceous materials, rubber,

and plastics. Foodstuffs are not included because

their treatment presents an entirely different

problem.

II. Microbiology

The terms microorganisms, molds, and mildews

have been used interchangeably. Strictly speaking, in this field the term microorganisms should

also include bacteria. However, this discussion

is restricted to the effects of molds and mildews

(the terms are synonymous) as the moisture level required for deterioration by bacteria is rarely

encountered except in specialized fields, such as

the maritime industry.

Molds are filamentous, branching forms of

plant life. They belong to the subphylum Fungi

of the phylum Thallophyta. According to defini- tion they are plants that do not have roots, stems,

or leaves. They are free of chlorophyll and require

preformed organic materials and certain inorganic

substances for their growth. Most molds are

prolific spore producers. The spores serve to

' Present address: Institute of Textile Technology, Charlottesville, Va.

propagate the individual as do the seeds of higher

plants. These spores are formed either at the

tips of mycelial branches or are borne in special

bodies known as sporangia or perithecia. They

are liberated readily in vast numbers and float easily in the air. In this condition they are able

to survive for long periods of time and under

extreme conditions of humidity and temperature.

Figure 1 shows the sporehead of Aspergillus niger

magnified 1,000 times.

The number of genera and species of molds that

have been isolated and classified runs into the

thousands. They vary greatly in then morphology, physiology, and biochemical activities. Most

of them are aerobic. Thev thrive at temperatures

between 10° and 37° C and in the pH range from

2 to 8, with optimum growth at pH 4 to 7.
FiGUBic 1, Spore head of Aspergillus niger.

Many of the delicately attached spores can be seen floating in the field. Magnification, 1.000X. Molds attack a great variety of organic compounds, including simple carbohydrates, fats, waxes, oils, cellulose, lignins, proteins, and even

synthetic plastics and elastomers. In the process

of growing they produce a variety of organic

acids, including oxalic, citric, fumaric, gluconic,

and carbonic. When protein materials are the

source of nourishment they usually liberate

ammonia. However, not all molds have the same capacity for attacking these different substances. Some prefer cellulosic materials, whereas

others prefer protein materials. With regard to food utilization, molds are extremely efficient. It has been reported that as much as 50 to 60 percent

of the decomposed substrate is converted to the

cell substance, which includes the mycelium and

the spores. The soil is a rich source of molds. Each gram

of soil contains between 10,000 and 200,000 spores

and bits of mycelium. Dry soil, in the form of

dust, carries the spores everywhere. When the

temperature and humidity are favorable the

2 Miscellaneous Publication 188 of the National Bureau of Standards
spores germinate and develop a branching mycelium. The food supply in the spore becomes

exhausted rapidly, and the further development

of the mycelium is controlled entirely by the

amount of available nutrients in the substrate. In some cases, the mycelial threads of some of the

molds can extend laterally for long distances away

from their food supply over normutritive surfaces,

such as glass or metal. This type of growth may

result in surface etching due to the excretion of

acids or alkalies by the mold.

Mold growth causes "rot" in the case of the

cellulose destroyers, or "mildew" in the case of the surface growers. Rot is measured by loss in

breaking strength, whereas mildew is measured

by the visible amount of mold growth on a test specimen.

Holzinger [83] in 1909 studied the influence of

osmotic processes in a medium on the growth of

microorganisms. In 1910 Artari [9] continued

work along the same line and studied the influence

of the concentration of nutrient media on the

growth of algae and fungi. The most rapid

growth took place in 10 percent glucose solution,

which he found to be the optimum concentration.

Solutions prepared with less glucose but with

other material sufficient to establish an osmotic

pressure equivalent to that of the 10-percent

solution did not support equally rapid growth.

Kellerman and McBeth [95] in 1912 reported

that, contrary to Omeliansky's observation that

cellulose-destroying bacteria do not grow upon

solid media, most of the species isolated by them

grew readily upon such media as beef agar,

gelatin, starch, potato, and dextrose. Some, in

fact, liquefy gelatin. Molds were found to be as

effective as bacteria in destroying cellulose. They

claimed to have isolated 75 species of molds

representing a large number of genera. Species of

Penicillium, Aspergillus, and Fusarium were most

numerous. They pointed out that in the destruction of pure cellulose either by bacteria or molds

in synthetic media, the associative action of

organisms that presumably have no cellulolytic

enzymes frequently stimulate the growth of the

cellulolytic organism and increase its destructive

power. This has been borne out by much recent work in which it has been shown that many

organisms require special growth substances for proper development. These substances are nor- mally obtained from other organisms in a more or

less symbiotic relationship. To illustrate, the

Australian workers in this field have proposed

the use of an active cellulose destroyer, Memnoniella echinata, as a test organism. Although this organism is assuredly an excellent cellulose destroyer, it will not grow upon synthetic media

unless it is contaminated by another organism or

it is supplied with biotin. It is believed that the

contaminating organism supplies biotin.

Scales [139] using McBeth's methods, continued

the work of isolating cellulolytic organisms and

isolated 30 species of Penicillium and 10 species of

Aspergillus.

In 1917 Linossier [101] studied the growth

characteristics of Oidium lactis. He found that the

yield of 0. lactis was distinctly proportional to the

weight of the food. The exception to this was a group of mineral substances that seemed to act as

catalysts, exerting their maximum action at minimum dose. However, when food was given

in increasingly greater amounts than could be

assimilated during the time of the experiment,

the yield increased less and less, finally reaching

a maximum. Increasing the food concentration

beyond this point a harmful effect on the yield was

noted, especially with reference to nitrogenous

food.

Roberg [134] in 1928 studied the growth characteristics of Aspergillus niger, A. fumigatus, and

A. oryzae. He found that these organisms would

not grow in the absence of Fe or Zn. In the

absence of Cu there was mycelial growth but little fruiting. Also, in the absence of Cu, A niger did

not become black.

Galloway [64, 65] in 1930 and 1931 isolated

molds from mildewed cotton fabrics and found

that they were mainly Aspergilli, Penicillia, and

Fusaria. He attempted to explain the cause of

what was commonly called "diamond spot"

stains. These he believed were caused by the

growth of organisms that would begin at the

intersection of two fibers and spread from this

point.

Karnicka and Ziemiecka [91] in 1935 reported

that molds were the principal agents of cellulose decomposition in soils with a low humus content,

especially when the pH was less than 6. Fertiliz- ation with nitrogen stimulated fungal activitv,

whereas applications of lime diminished it. Thirteen molds, including four of the genus Ohaetomium, were isolated from an acid soil. Their study

revealed that in acid soils cellulose is first attacked

by fungi, followed by actinomycetes, and finally

by bacteria. In neutral soils bacteria start the

degradation.

In further studies Galloway [66] in 1935 showed

that the rate of spore gei'mination is governed

primarily by the atmospheric humidity, rather

than the moisture content of the substrate.

Considerable variation is shown in the moisture

requirements of the more common groups of

molds. The minimum relative humidity varies

from 75 to 95 percent. Aspergillus glaucus, A.

candidus, and A. versicolor can develop at 75- to 80-percent relative humidity.

Molds have been found capable of growing

under extreme conditions of temperature and pH.

Thorn [164, 165] cites evidence of mold growth at low temperatures, as in refrigerators. He reports

Microbiological Deterioration 3
the presence of molds in nickel electroplating

solutions. Porges [129] in 1938, reported a fungus

growth in a copper-plating bath containing a saturated solution of C11SQ4 and 6.8 percent of

HoSO,.

Mulder [115] in 1939 verified Roberg's [134]

findings with regard to the necessity of copper

for the growth of Aspergillus niger. He found

that the presence of cadmium increases the copper

requirement. Many other species of Aspergillus

and also Acetobacter aceti also require copper for growth. Mulder suggests that A. niger may well

be used to estimate the copper content of soils. Corroborating the work of Karnicka and Ziemieeka [91], Skinner and Mellem [152] in 1944

added finely divided filter paper to acid soils that were 60 percent saturated with water. They

found no evidence of the activity of cellulolytic

bacteria, although mold growth increased greatly

in spite of the excessive moisture. In soils with

a pH above 5.0, both molds and bacteria showed

an increase. The conclusion of Dubos [51] that

both aerobic bacteria and molds take part in the

decomposition of cellulose in nonsaturated soils, unless they are distinctly acid, is shown to be

correct. In a study of a wide variety of molds, with the

object of finding a more suitable cellulolytic test organism, Pope [128] in 1944, working with Greathouse, isolated and identified a new organism,

Metarrhizium glutinosum, from a bale of deteriorated cotton. It was found to have extraordinary

cellulolytic power and was used in the evaluation

of mildew-proofing agents. White and Downing

[172] in 1947 have shown this organism to be

Myrothecium verrucaria.

Zuck and Diehl [179] in 1946 found that certain

angiocarpous fungi, which had heretofore been

unrecognized as cellulose destroyers, were significant agents in the slow cellulosic breakdown of

fabrics during aerial exposure. Among these

organisms are Diplodiella cowdellii, Hendersonia

sarmentorum, Leptosphaeria sp., Diplodia sp., Phoma herbarum, and Ophiobolus sp. These fungi

have tended to escape detection because of the

employment of standard plating techniques that

do not support the growth of these slow-growing

fungi.

The retting of bast fibers, such as jute, flax,

and ramie, is also a form of microbiological deterioration. Here, however, man has been able to derive some benefit from the activities of these

organisms. Formerly [93] it was believed that

the process of retting was merely the breakdown

of pectin and other cementing substances with

the ultimate separation of individual fibers. It was not believed that these organisms could be

responsible for cellulolytic activity. As the process had developed from ancient times, all information relating to it was of a more or less empirical

nature. However, by 1940 Ruschmann and

Bartram [138] were able to show that the best

quality of bast fibers are obtained when the conditions of retting are carefully controlled. They

found that the aerobic organism Alternaria tenuis

destroys both pectin and cellulose rapidly. If the process is permitted to go to completion, the

bast fiber is completely destroyed.

III. Enzyme Action

Xo discussion of biological activity can be complete without investigating the role played by

enzymes. To understand the role of enzymes in

cellulose deterioration a knowledge of cellulose is required. The molecule of pure cellulose is composed of beta-glucose units linked through the

1-4 positions to form long chains. Before it can

be utilized by microorganisms as a source of

energy it must be broken down to water soluble

molecules. This degradation, previous to oxidation by the cell respiratory system, is enzymotic,

probably mainly hydrolytic, and is believed to be

the work of more than one enzyme.

De Bary [47] in 1886 was the first to make a

careful study of the dissolution of cell walls by

plant fungi. The formation by these organisms

of a cellulose-dissolving enzyme, later designated

as cellulase, was thus indicated. In 1888 Ward

[171] reported, from microscopic observations,

that the fungus, Botrytu sp., secreted an enzyme

capable of hydrolyzing the cell walls of the host

tissue. Von Euler [55] obtained from the wooddestroying fungus Merulius lacrymans an enzyme

that acted upon the hydrolytic products of cellulose but not on cellulose itself. Pringsheim

[130] in 1912 reported a cellulase from thermophilic cellulose bacteria. The decomposition of cellulose by these bacteria was believed to occur

in two stages: (1) the reduction of cellulose to

cellobiose, and (2) cellobiose to glucose. The

enzymes were considered to be endoenzymes as the cell-free extracts had no action on cellulose. Schmitz [140], in 1920, obtained cellulase from a

great number of molds. Karrer, et al. [92], in 1923, showed that cellulase is present in the

digestive system of the edible snail. Selhere

[144, 145, 146], from 1905 to 1910, examined the

hydrolysis of cellulosic materials by enzyme

action, the end product being glucose. He

found that cellulose was attacked more rapidly

if it was first treated with 25 percent caustic soda

or with ZnCl2 or precipitated from Schweitzer's

reagent. He concluded that the physical condition of the cellulose was an important factor in determining the rate of hydrolysis under enzyme

4 Miscellaneous Publication 188 of the National Bureau of Standards
action. Grassmann and Rubenbauer [72] in 1931 stated that a dialysed and concentrated

enzyme extract from Aspergillus oryzae hydrolyzed

filter and parchment papers at a slow rate. Grassmann, et al. [73] in 1933 found that a crude

extract of this fungus hydrolyzed many polysaccharides. It hydrolyzed a cellulose prepared

from beechwood at a slow rate but a hydrocellulose

prepared from the same source and one prepared

from cotton was hydrolyzed 25 times as fast. The optimum pH was 4.5. Clayson [41] in 1942

found that the ease of decomposition of naturally

occurring cellulosic materials appears to be determined largely by whether the encrusting substances are readily attacked or are resistant.

Schonleber [141] believed that the orientation of rows of micelles parallel to the longitudinal axis

of rayon fibers gives the fibers greatly increased

resistance against the penetration of cellulose- dissolving enzymes.

Various workers have claimed to separate cellu- lase and cellobiase. Either these are different

substances (pH optima varied from 5.9 to 4.7), or they are influenced by contaminants. According

to Norman and Fuller [120], in 1942, the existence

of an hydrolytic enzyme system capable of producing either glucose or cellobiose from cellulose may be considered proved but what is lacking is any information of the intermediary steps. In

view of the fact that growth of some ceUulolytic

organisms is inhibited by low concentrations of

glucose, it may well be that the disintegration does

not normally proceed through glucose.

Depending on concentration, acids, bases, and

salts will either stimulate or inhibit enzyme action.

The concentration of salts of heavy metals neces- sary to inhibit enzyme action depends on the

nature of the enzyme and the metal, according to Hata [81] in 1904. The activity of many catalase

preparations is reduced 50 percent by 0.005 mg of

HgCl2 per liter. However, Kehoe [94] in 1922

stated that high concentrations of certain neutial

salts of the alkalies and alkaline earths will reactivate enzymes inactivated by the salts of heavy

metals. Robbins [133] in 1916 found that salts not only affect the activity of enzymes but also

their formation by microorganisms. Some or- ganic chemicals also poison enzymes. Among

these are HCN, HCHO, alcohols, and chloroform.

Light may either destroy or inhibit enzymes

especially in tbe presence of oxygen and fluorescent

substances.

Ziese [176] in 1931 worked with cellulases from

various sources and found them to be relatively

thermostable. In a dilution of 1 to 2,000, they

may be heated 5 to 10 minutes at 100° C. without

complete loss of activity. A 1 percent solution of CuS04 and 3 percent of HCN separately have

practically no effect, but when mixed they are strongly inhibitory. Glutathione and cysteine,

but not H2 S or Na2 S2 3 , are inhibitory when

phosphate buffer is used, but not when citrate

buffer of the same pH is used. The complexity

of this subject may be a result of the fact that

microorganisms that attack cellulose may be

separated into three groups, depending on oxygen

intake, according to Dubos [51]: (1) strictly aerobic

forms which are specific to cellulose, (2) strictly

aerobic forms which attack both cellulose and

starch, and (3) facultative anaerobes which attack

cellulose. In 1945 Siu and White [151] reexamined the

evidence for enzymotic break-down of cellulose. They decided that the enzymotic break-down of

cellulose is an often emoted but unconfirmed

theory. The theory assumes the presence of two

enzymes. Cellulase is believed to hydrolyze cellu- lose to cellobiose and then cellobiase converts

cellobiose into glucose. They found that claims

have been made that these two enzymes have

been separated by the selective adsorption of

cellobiase on alumina. The crude cellobiase thus

obtained presumably is capable of hydrolyzing

oligosaccharides of six anhydroglueose units or

less, while the crude cellulase preparation can

attack only those of greater complexity. In reviewing previous work in this field they found

that most of the preliminary work was done with

crude enzymotic preparations from the digestive

tract of the edible snail, Helix pomatia, and the

ground mycelium of Aspergillus oryzae. Ground

filter paper or wood was exposed to attack by the

preparation in question, and the increasing reducing ability of the resulting mixture was used as the index of enzymotic action. They conclude by

saying that despite all of these studies, up to the

present time there has been no confirmed isolation

of a cell-free preparation capable of hydrolyzing

cellulose. They found no ceUulolytic activity in

the metabolic filtrate of Cytophaga D or Myrothecium verrucaria (Metarrhizium glutinosum) con- centrated 30 times.

IV. Organic Materials Utilized by Microorganisms

1. Cellulosic Materials

Among the materials which have been reported

as being subjected to microbial degradation, cotton and other cellulosic materials rank highest.

Figure 2 shows cotton fabric heavily invaded by

Aspergillus niger. The earliest reports in this

field are, more or less, observations of mold

activity. In 1912 Osborn [125] reported that

mildew of cotton cloth appears to be due to many

common saprophytic molds which, he believed,

fed upon the starchy matter in the sizing. He

Microbiological Deterioration 5
Figure 2. Camouflage fabric with inferior fungicide showing the invasion of the weave by the mycelium and spore- heads of Aspergillus niger.

pointed out that the fungi could grow only in the

presence of sufficient moisture. Therefore, if cotton materials were not permitted to become

damp, they were not likely to become moldy.

In 1913 Kroulik [97] reported that microorganisms, which he believed to be bacteria and actinomycetes, decompose cellulose even at temperatures

as high as 60° to 65° C. In the same year, McBeth

and Scales [111], unlike the other workers of the

period, were actually working at the causes of

cellulose degradation by microorganisms. They

found that a selective medium like cellulose agar was superior to Omeliansky's selective culture

method [100], and the filter-paper method of

Van Iterson [100]. They found that molds and

bacteria, capable of destroying cellulose, are present in all productive soils. The cellulose- destroying types attack cellulose most rapidly

in the presence of oxygen and often lose their

power to do so when grown on artificial media.

The truth of this fact accounts, in part, for the

great difficulty in finding suitable test organisms

that will maintain their growth characteristics

from generation to generation over long periods

of time. In the same year, Mutterlein [116]

found that aerobic organisms were more important than anaerobes in the decomposition of

cellulose in nature. In 1915, Guegen [78] reported

that the black spots and patches that tend to develop on tent cloth and sails when exposed to damp air, and that cause weakening of the fibers as if by sulfuric acid, are due to the growth of

molds. He listed two organisms as being especially destructive

Pleospora infectoria and P.

herbarum. He believed that the spores of these

molds were rarely airborne but were in the tissues

of the textile fiber. In 1916, Otto [127] made as thorough a study

of the cellulolytic activity of microorganisms as had been made up to that time. He used a wide

variety of cellulosic materials as substrates in- cluding blotting paper (true cellulose), various

natural celluloses, and modified celluloses such

as oxycellulose, hydrocellulose and hydrated

cellulose. He found that the response of molds to these substrates varied considerably. But he was able to report that soil molds were capable

of "dissolving" cellulose. He believed that "solution" of cellulose was effected by means of en- zymes which caused hydrolytic cleavage. The

cleavage of various types of cellulose was effected

by the same molds, with hydrolytic cleavage

taking place in those molecular groups which

were common to all the forms of cellulose tested. Hutchinson and Clayton [85] in 1919 isolated a new cellulose-destroying bacteria, Spirochaeta

cytophaga, which was to be studied later by

Thom and his coworkers for possible use as a test organism. Hutchinson and Clayton studied the

growth habits of this organism and reported that

it was aerobic and had great cellulolytic power.

For the next few years various authors were

reporting in the same vein. This was the period

following the First World War, and it is probable

that as a result of the war much practical information had been gathered. Armstead and Harland

[4, 5], Fleming and Thaysen [58, 59], and Thaysen

and Bunker [158] reported on the effects of microorganisms on textile fibers, especially on the fact

that cottons of different origins had varying re- sistance to destruction by microorganisms. By

1924 Thaysen [159] was able to sum up his observations with the following: Moist cellulose- containing materials, when not incrusted with

lignin, constitute a very suitable food for many

types of microorganisms. In the presence of about 10 percent of moisture the spores develop

a mycelium that may be entirely on the surface

of the fiber, perforate it in places, or may even

develop within the fiber. Certain bacilli also develop colonies that gradually penetrate the

fiber. As these spores are normally present on raw cotton, the chances of infection in finishing

processes are great. These organisms produce

both chemical and physical or morphological

6 Miscellaneous Publication 188 of the National Bureau of Standards
changes in the fiber, involving loss of strength

and usually a marked discoloration. The penetration of the fiber is aided by the enzyme, cytase,

which converts the cellulose into glucose. The

glucose then is used by the organisms as a source

of energy and on oxidation yields C02 , H, and

organic acids. A primary stage in the production

of glucose is the hydration of the cellulose,

wherein the fiber is more readily stained by dyes

and retains the color better. The destructive

action of the microorganisms on cotton may be

followed, almost from the start, by the swelling

action of a solution containing equal parts of CS2 and 9 percent of NaOH. This reagent gives

the normal fiber a microscopic appearance similar

to that obtained with Schweitzer's reagent, that

is, swelling with constrictions. Fibers with microbiological damage swell more quickly and present an almost uniformly swollen appearance without

constrictions, possibly because of the destruction

of the cuticle of the fiber. He confirms the

findings of others by stating that acetylated cellu- lose is resistant to bacterial attack and that

bleached cotton is more resistant than unbleached

cotton. Finally, he agrees that some types of

cotton are more resistant to degradation than

others. American cotton is most resistant, whereas Indian cotton is least resistant. In 1925 Thaysen and Bunker [160] reported further on their

work with various cellulose derivatives. They

found that cellulose acetate was most resistant

to microorganisms, whereas cuprammonium rayon was least resistant.

In 1927 Serrano [147] showed that the deterioration of abaca fibers (manila hemp) was due to the

action of cellulose-digesting organisms of the genera Aspergillus, Penicillium, and Chaetomium.

The growth of these organisms, he found, was

favored by dampness, poor ventilation and ele- vated temperatures. Winogradsky in 1929 [174]

made a careful study of cellulose decomposition

and reported that cellulose undergoes rapid oxidation in the soil and the product closely resembles

oxycellulose. The biological oxidation of cellulose, he said, is differentiated from the purely

chemical process in that the product does not

reduce Fehling's solution. Assimilable nitrogen,

preferably inorganic, is essential to this process, the consumption amounting to approximately 2 parts of nitrogen for each 100 parts of cellulose decomposed. The consumed nitrogen is trans- formed into organic combination, but there is evidence of some partial reduction to ammonia

even with unrestricted access to the air. The

optimum reaction for cellulose decomposition is pH7. Slight changes of reaction occur during the

process, depending on the form in which the

nitrogen is supplied.

In 1930 Thaysen and Bunker [162] were the

first to report on controlled field exposures in

which cotton, wool, hemp, and flax fabrics were

exposed to the action of microorganisms at Ceylon Cyprus, Kenya, Trinidad, the Malay States,

and England. The materials were hung in the

air, both in and out of sunlight; buried in the

soil; and submerged in the sea at Ceylon. In marine waters disintegration was most rapid,

and was mainly the result of bacterial action. In

soils the deterioration was caused by bacteria,

fungi, and termites. In the air the damage was

produced by molds and actinomycetes. They

found that hemp and flax were most resistant to degradation.

Up to 1937 the most thorough study of cellulose destruction in the soil was made by Madhok [107].

He found that the most rapid decomposition of cellulose in the soil took place at an initial pH of

5.5 with NaX03 as a source of nitrogen. This

indicated that the first rapid attack was by fungi,

followed later by bacteria as the reaction tended

toward neutrality. Decomposition started most

rapidly at 37° C, but later the rate at 27° and 30°

C approached the 37° C rate. Fertilization of the

soil over a period of 25 years had no appreciable

effect on the microorganisms decomposing cellulose. This latter observation was later verified by

Batson, Teunisson, and Porges [16.] In the same

year Galloway [67, 68] published a general review

of the microbiology of textiles. He pointed out

that the microbes were useful as well as harmful,

as mold enzyme products were used in many

textile procedures.

In 1938 Barker [12] made a study of \-arious bast

fibers, including flax, ramie, hemp, pita grass, jute,

and sisal. In such fibers other polysaccharides

are closely associated with cellulose and are conconsidered to be an integral part of the cellulose aggregate. Xylan is the most common of the

carbohydrates retained by the fiber. The molecular structures of cellulose and xylan are similar,

and the action of fungi is to hydrolyze the latter,

breaking down its molecular chains into shorter

lengths and thus causing loss of strength or rotting.

He emphasizes the accelerating effect of moisture on the deterioration of fibers by microorganisms.

In another paper [13] in the same year, Barker

considers the use of antiseptics for the protection

of cellulosic fabrics. He finds salicylanilide to be

excellent for this purpose. The effectiveness of the

Thaysen acetylation process [159] is again affirmed.

He believes that it renders a cotton surface unsuitable as a nutrient source for microorganisms.

Shults [150] in 1938 reported that the development of Aspergillus niger can be inhibited by

drying cotton fabrics to 20-percent moisture content followed by storage at 71 to 73-percent

relative humidity. This is at variance with

Thaysen and Bunker [158], who claim that 10

percent of moisture is sufficient to cause germination of spores. Shults points out that viscose

products are easily degraded by microorganisms.

In 1939 Galloway [69] found that the minimum

Microbiological Deterioration 7
moisture regain for the development of microorganisms on jute fibers was 17 percent.

At about this time the trend in this field was

toward the study of individual organisms that

could be demonstrated to grow on a cellulose- containing medium. In 1939 Hooper [84] isolated

from the soil an aerobic micro-organism that was

capable of growing on an inorganic medium, with

such cellulosic materials as filter paper or cotton

as the sole source of carbon. Although the

organism was studied in detail, it was not identified. By following the disintegration of cotton

fibers under the microscope, Hooper found that

the cuticle is removed from the fiber during the

early stages of attack, and then both the cementing material and cellulose are slowly digested.

The particulate nature of the cellulose fibrils of

the cotton-fiber cell wall is clearly visible in the

partially disintegrated fibers. With the outbreak of war in Europe in 1939, the

tempo of research in this field increased. Thaysen,

et al. [163] found that in microbiologically active

soil cellulose acetate rayon was completely resist- ant whereas cellulose, wool, silk, and cellulose rayon fabrics were disintegrated. Bryson [30] pointed out the need for preserving jute fabrics for sandbags. He believed the naphthenic acid

derivatives and particularly copper naphthenate

to be most promising.

In 1940 Waksman [170] recognizing the need for up-to-date information on cellulose deterioration

published a review of the microbiology of cellulose decomposition. He pointed out that while cellu- lose was resistant to ordinary chemical reagents

and the digestive juices of higher animals, it was

readily decomposed by a great variety of microorganisms.

In 1941 Armstrong [6, 7, 8] and Barker [14] studied the problems of jute-sandbag preservation.

They found that damp jute bags were subject to

rotting. Molds and bacteria of soils are responsible for this degradation. Molds attack jute that

contains at least 17 percent of moisture, while at

least 22 percent of moisture is required for attack

by some strains of bacteria. Armstrong recom- mends a variety of preservative treatments and

points out that treated jute bags can be expected

to have a useful life of 18 months to 2 years, whereas untreated bags usually last a few weeks

at best. In 1945 Larnach and Wyke [98] investigated the

problem of mold growth on books and paper.

They observed that there was no growth of fungi

when the relative humidity was below 75 percent.

Therefore, to control mold growth they recom- mended the use of silica gel in book cases. In the same year Fargher [57] made a study of the

growth substances present in cotton. He found

that raw cottons contain appreciable quantities

of K, Na, Ca, and Mg, all of which are essential

to vigorous growth of molds. The principal trace

metals, Fe, Cu, and Zn, which stimulate the

growth of certain organisms are also present.

Most of the metals are present as water-soluble

salts of organic acids, which are consumed readily

by micro-organisms. Also present are sulfate,

chloride, phosphate, glucose, and more complex

carbohydrates, and nitrogenous substances. Flour

used in sizing affords an additional source of

nitrogen as well as of carbon, and supports more

prolific and rapid mold growth than starch.

Modified starches are utilized more readily than

are unmodified starches. The consumption of the

naturally occurring food materials can be followed

by measurements of reducing power (copper

number) and pH of the cotton during exposure to damp conditions. When the water-soluble constituents are removed, cotton becomes more

resistant to micro-organisms. The resistance be- comes still greater after scouring with alkalies. For this reason it is believed that linen appears to be more resistant than cotton to microbiological

degradation.

Bayley and Weatherburn [17, 18] in 1945 draw

attention to the fact that while copper preservatives appear to be excellent in laboratory tests, they have been able to isolate copper-tolerant

fungi from copper-treated fabrics. Goldthwait, et al. [70] in 1946 reported that

cotton fibers, yarns, or even fabrics could be

partially acetylated in such a manner that the

cotton retains its outward appearance but ac- quires several new properties. The resulting

modified cotton is highly resistant to mildewing

and rotting and has a lower moisture regain.

2. Proteinaceous Materials

Although the documentation of the microbiological degradation of proteinaceous materials

is not as extensive as that for cellulosic materials,

the problem is equally serious. The organisms

involved are, in general, not the same as those

which degrade cellulose [60, 153]. Organisms

that attack proteinaceous materials may cause

rotting of the material, as in the case of wool, or they may attack important substances in the protein material, as the oils in leather. Figure 3

shows a circle of vegetable-tanned leather, heavily

overgrown by molds.

In 1924 Burgess [32] published a study of the

types of molds and bacteria found on damaged

wool. He believed that molds caused discoloration, whereas bacteria caused rotting. In 1926

Hayes and Holden [82] made the same observations with regard to silk. In further studies on

wool, reported in 1928 and 1929, Burgess [33, 34]

found that mildew on stored woolen goods was

entirely due to molds rather than to bacteria.

Soaps and other conditioners enhance its development. The alkalies combine with the wool render8 Miscellaneous Publication 188 of the National Bureau of Standards
Ficure 3. Untreated vegetable-tanned leather on a sucrose agar plate uninoculated and incubated for 7 days a 28° C. The growth, which completely covers the leather, is mainly Aspergillus niger. The ease with which leather supports a growth of mildew is thus demonstrated

ing it more easily attacked. Finishing materials

that are hygroscopic in nature raise the moisture

content to the optimum level for mold growth.

Thaysen and Bunker [162] in 1930 exposed

woolen fabrics to the action of microorganisms at Ceylon, Cyprus, Kenya, Trinidad, the Malay

States, and England. The fabrics were hung in the air, both in and out of sunlight; buried in the

soil; and submerged in the sea at Ceylon. Except

in ah exposure the wool was rapidly degraded,

especially by bacteria where moisture was rela- tively high.

Burgess [35] in 1930 reported that wool could

be made mold-resistant by the use of chrome in

the dye bath, and in 1934 [36, 37] that the amount

of nutrient substances hi wool greatly influences

its mildew susceptibility, especially in a damp

atmosphere. He noted that intense chlorination

and treatment with hydrogen peroxide or ultraviolet light increases susceptibility to attack by

molds and bacteria and that bacterial development requires actual wetness. At this time he

recommended the use of salicylanilide as a preservative. Finally, in 1934, in an effort to study the

mechanism, of bacterial degradation of wool.

Burgess [38] found that wool fibers could be dis- integrated into their component elements of

epithelial scales and cortical cells by treatment

with an appropriately buffered solution of trypsin

or pepsin. The action of the trypsin solution is similar to that of proteolytic bacteria, but is more

rapid. He suggested this treatment as a laboratory method for determining the resistance of

wool and other animal fibers to bacterial action.

In 1939 Thaysen, et al. [163] reported that wool

and silk were completely disintegrated in microbiologically active soil. Harold [80] in 1941

pointed out that damage due to molds is of greater

magnitude than that due to bacteria because of

the greater amount of moisture necessary for bacterial growth. Wool in the grease or wool

treated with oil emulsion tends to mildew at lower temperatures than scoured wools.

Schonleber [141] in 1941 found that casein fibers are more easily deteriorated by bacteria than by

molds. She found two organisms to be especially

active in this respect, namely, Proteus vulgaris

and Micrococcus sp. In 1943 Ayres and Tobie [10] found that molds

and bacteria that have the power to hydrolyze

proteins usually give very active preparations of proteases on appropriate treatment. The paper industry, which requires large quantities of wool felt for use in paper-making machines,

has been put to great expense by the short life of

these materials due to the rapid rotting of the

felts. The conditions are severe as these felts are completely wet for long periods of time. There

Microbiological Deterioration 9
are many papers dealing with this subject but few

workers have been able to solve the problem.

Binns [24] in 1934 measured the extent of bacterial degradation of woolen felts by the loss in

strength. He found that attack by bacteria

depends in part on the source of the water used in

paper manufacture. It is generally increased by

the presence of phosphates and is inhibited by acid

conditions. Binns suggested the use of various

antiseptics but expressed the opinion that comparatively large concentrations are needed for

protection.

3. Leather

Although leather is a protein substance, its degradation by microorganisms is quite different

from that of other proteinaceous materials.

Although many reports have been received from

tropical battlefields to the effect that leather rots and falls apart rather rapidly, none of the speci- mens that were received were observed to be in

this condition. Furthermore, it has not been possible to rot leather in the laboratory, even in

soil-burial beds and in tropical room exposure

tests. To be sure, leather articles such as shoes

and equipment cases have been observed to fall apart, but on examination it was found that the

stitching, usually cellulosic in nature, bad rotted.

No rotting was ever observed when nylon thread was used for stitching leather. As shall be shown,

the primary damage to leather by microorganisms

is the stiffening and shrinking caused by the re- moval of the oils and greases. Figure 4 shows

Figure 4. Two cases of Army shoes received from storage on the island of Saipan illustrate the importance of 'packaging. The package on the right was in a badly deteriorated state when received, whereas the package on the left was in excellent condition. On opening

the cases, the shoes in the package on the right were heavily mildewed.

mildew growth on new Army shoes stored under

extreme conditions of temperature and humidity.

In 1925 Wilson and Daub [173] reported that

after hides are tanned the greatest damage is caused by molds. Untanned hides, on the other

hand, are easy prey for bacteria.

In regard to the fats and greases commonly

found in processed leather, Vaubel [167] in 1928

found that molds attack free fatty acids to a

greater extent than glycerides. Unsaturated fatty

acids are more readily utilized than saturated acids

and the cis form more than the trans form. Thus,

oleic acid is more susceptible to mold growth than

elaidic acid. In 1932 Grimshaw [77] found that

both olive and lard oils are good sources of food for

molds but that mineral oil is not as good a source. With reference to the effects of mold growth on

leather, Lloyd [102] in 1934 observed that molds

may produce stains, destroy tannins, hydrolyze

fat, produce spues, and under certain conditions

may pit the surface.

A study of microbial growth on 55 leathers by

Colin-Russ [43] in 1940 showed damage when the

moisture content was about 30 to 35 percent of the

total weight.

In 1945 Dempsey [48] found that chrome-tanned

leather does not mold readily. Semichrome

leathers mold more easily, whereas vegetabletanned leather is most susceptible to mold growth.

The direct effects of mold growth include damage

of the grain, formation of stains that cannot be

removed without damaging the grain, and, finally,

destruction of pyrogallol tannins. Threads and

fabric linings in leather articles may be affected

by the mold growth on the leather. Free acid

resulting from hydrolysis of triglycerides by molds

may corrode metal fittings.

Kanagy, Charles, Abrams, and Tener [90] in

1946 made a study of the effect of mold growth on

vegetable-tanned leather. They found that the

growth of mildew on leather increases its stiffness,

decreases its tensile strength and stretch at the

breaking point, and weakens the grain surface.

Chemical tests revealed a loss of grease, watersoluble materials, glucose, tannins, andnontannins.

No appreciable deterioraton of hide substance was

found. It was concluded that the molds, in assimilating the grease, bring about decarboxylation of

the fatty acids, as shown by decrease in saponification number.

4. Miscellaneous Materials

(a) Rubber

The deterioration of rubber in the latex stage

has been well documented and has been the subject of study for many years. However, the

microbiological deterioration of cured rubber has

not received equal attention. In 1914 Sohngen [155] showed that microorganisms do not grow on air-dried rubber because of

10 Miscellaneous Publication 188 of the National Bureau of Standards
the low water content, which he found to be approximately 0.5 percent. However, in the pres- ence of large amounts of water, common India

rubber furnishes a medium for the growth of bacteria and molds, inasmuch as it contains proteins, sugars, and resins. Scott [142] in 1920 also showed that vulcanized

rubber, especially pure gum compounds, develops

growths of Stemphyliurn macrosporoideum when

exposed to moist air. This growth was described

as resembling dust. Some threads were seen to penetrate the surface and were probably responsible for the loss in strength that occurred. In 1938 Kalinenko [89] dispersed rubber in a

flask and inoculated it with Aspergillus oryzae. In 1 month he found that 32 percent of the rubber

had been destroved.

ZoBell and Grant [177] in 1942 studied the degradation characteristics of various types of rubber.

Their criterion of rubber degradation was in- creased oxygen consumption and C02 production.

They used inocula from sea water, garden soil, old garden hose, and cracks in tires. All of these

inocula increased the oxygen consumption 5 to

10 times. The rate of oxygen consumption increased exponentially with time.

Dimond and Horsfall [49] in 1943 pointed out

that wherever rubber is to be used either in contact with water or under conditions of high moisture, as in the tropics, a mildew-proofing agent

should be used.

(b) Plastics Plastic materials are of such recent origin that

aside from the experience gained in the recent war,

there has been no documentation of microbiological deterioration. On the contrary, for a time

it was believed that because these materials were man made or synthetic they could not possibly

be the source of microbiological nutrition. This

fallacy was disproved early in the recent war, when it was found that in the tropics many types

of plastics supported copious growths of microorganisms. Brown [29] in 1946 sums up a good

portion of the information gained during the war

by reporting the work of several laboratories.

V. Prevention of Deterioration

The early use of mildew-proofing agents was

haphazard and was based on empirical rather

than on experimental information. Fargher [57] in 1945 stated that prior to 1926, ZnCL, ordinarily

in a concentration of 0.8 percent, was the principal

mildew-proofing agent for fabrics but that it was

not entirely effective. Other agents used were

salicylic acid, sodium fluoride, sodium silicofluoride,

phenol, cresylic acid, or formaldehyde.

Corrigan [44] in 1920 recommended the use of heat, light, and such antiseptics as copper sulfate,

potassium nitrate, zinc sulfate, zinc chloride, boric- acid, formaldehyde, and phenols for the destruction of fungal growths on finished goods.

In 1924 Thaysen [159] reported that acetylated

cellulose was resistant to fungal attack. It was

found that bleached cotton was more resistant to decay than unbleached cotton. O'Brien [121, 122], working with rubber, re- ported in 1926 that a 0.1 percent solution of

paranitrophenol was an effective mildew-proofing

agent. In the same year Stevens [156], working

with crepe rubber, also found paranitrophenol to be an effective fungicide.

The earlier observations that bright fight tends

to inhibit fungal growth and that ultraviolet light

is actually fungicidal was explained by Bedford

[22] in 1927, who found that destruction of microorganisms by ultraviolet light is due to the production of peroxide and is directly proportional to the quantity generated.

In 1928 a group of workers at the Shirley Insti- tute in Leeds, Great Britain, investigated a large

number of compounds for possible use as mildewproofing agents. They found that salicylanilide was the most efficient of all compounds examined.

In honor of the Shirley Institute they named it Shirlan. Fargher [57] in 1945 describes Shirlan

as having all the desired properties:

1. Lack of volatility during fabric processing.

2. High degree of solubility to insure uniform distribu- tion in fabric.

3. It is unaffected by heat, metal surfaces, or by sizing

and other finishing materials.

4. It is colorless and odorless.

5. It has no undesirable action on textiles.

6. Xo undesirable action on subsequent bleaching or dyeing.

7. Low cost.

8. Harmless to man. By 1930 Shirlan had been used rather widely, and

Hall [79] recommended its use in place of ZnCL.

In the same year Burgess [35] pointed out that the

use of chrome in the dye bath imparts a considerable mold resistance to wool. With 0.5 percent

of chrome, either as Cr03 or Cr2 3 . he obtained

good mold resistance, and with 1.0 percent of

chrome the results were even more satisfactory.

In the same year Thaysen and Bunker [162]

suggested the use of mixtures of iron and chromium salts for both woolen and eellulosic fabrics

and recommended copper oleate. Fargher, et al. [56] summed up their long search for an ideal

antiseptic for the cotton industry by reiterating

that Shirlan was the best. Other excellent ma"-

terials were stated to be ortho chloromercuriphenol, para acetoxymercuriacetanilide. thallium

carbonate, para nitrophenol, and trichlorophenol.

Xakazawa and Takeda [117] in 1934 reported

Microbiological Deterio ratio

11
on their efforts to control mold growth on military

instruments. They found that wax containing

1 .0 percent of paranitrophenol was effective for long

periods. Other effective compounds were chloropicrin and para dichlorobenzene.

In 1934 McCallan and Wilcoxon [112] studied

the toxicity of various compounds of many

elements on various organisms. Toxicity was

measured by the concentration of a particular

compound that would permit only 50 percent of

the spores to germinate. This concentration of a

fungicide has come to be known as its minimum

lethal dose (MLD). The data are interpreted in terms of the periodic system of the elements.

They found that the toxicity increases toward the

center of the periodic table and is less at either end.

Toxicity within a group increases with increase of

atomic weight. Compounds of positive elements

show nearly the same toxicity, regardless of the

compound used, but the hydrides of negative

elements are all toxic, whereas highly oxidized

forms show only slight toxicity. There is a tendency, with many exceptions, for an element

that is toxic for one organism to be toxic for others.

Compounds of Ag and Os are the most toxic. Other toxic elements are Hg, Cu, Ce, Cd, Pb, Tl,

Cr, and As. Lloyd [102] in 1934 found that paranitrophenol

could be used to prevent mold growth in tanning

liquors used in the leather industry. In the same

year Burgess [37, 38] brought attention to the

fact that the amounts of nutrient substances

present on untreated materials greatly influences

their susceptibility to mold growth. Depending

on the presence of these substances, more or less of a particular fungicide is required. He recom- mends the use of Shirlan for cotton and wool.

For wool he also recommends the use of chrome.

In regard to the use of chrome. Armand [2, 3] believes that chroming removes protein degradation products from wool and thus removes the source of mold nutrition. He does not believe

that chrome is fungicidal but that the wool is now

"clean." He found that none of the dyes

commonly used conferred antiseptic qualities on

wool.

In 1937 Jarrell, Stuart, and Holman [87] found

that mineral khaki dye could be made mildew re- sistant by the addition of a relatively insoluble

copper compound. The precipitation of insoluble

copper compounds on cotton duck by means of Na2 C03 , the inclusion of copper compounds with

other metal compounds in mineral-dyed cotton

duck and the addition of basic CuC03 to a waterproofing solution are effective means for mildewproofing cotton duck, provided the copper content

is equal to at least 0.11 percent of-metallic copper,

based on the weight of the finished fabric. However, for the most rigorous use a copper compound

containing at least 1.7 percent of metallic copper,

based on the weight of the finished fabric should

be precipitated on the fabric. When chromium

and iron are also used in the treating compound,

somewhat less copper is required.

Neill and Travers [118] in 1938 found that

waterproofing treatments on tent fabrics acceler- ated their deterioration by microorganisms. They

recommended the use of the iron-chromium process but thought it was too expensive for general

use. For practical purposes they recommend the use of a 1-percent solution of Shirlan. Schults [150]

suggested that Aspergillus niger could be inhibited

by drying materials to 20-percent moisture content and storing them at 71- to 73-percent relative

humidity. If fabrics are to be kept wet for any

length of time, they are best preserved with

formaldehyde, Na2S03 , or salicylic acid.

By 1938 the use of copper naphthenate had

become so widespread, especially for treating

wood, that Smith [154] responded to the need for

information regarding its origin. Copper naphthenate, he stated, is a combination of copper

with naphthenic acids found in petroleum oil. It is soluble in kerosine, toluene, turpentine, and

similar organic solvents. It is effective in the

control of wood fungi, termites, fungi that attack

jute and other cellulosic fibers, and even as an

antifouling agent for ship bottoms. In 1939

Bryson [30] found the naphthenic acid derivatives,

particularly copper naphthenate. to be most

effective for preserving jute fabrics. Everitt and Sullivan [54] in 1940 tested a large

number of compounds, using five test organisms.

Fungistatic activity was shown by phenyl thioarsenite, 4-chloro-2-nitrobenzene sulfonamide, so- dium l,2,naphthoquinone-4-sulfonate, and sulfa- nilamide. The best of the fungicidal compounds

were phenyl benzothiazole and mercaptobenzothiazole. In the same year Colin-Kuss [43], working with leather, found that the most efficient antiseptics, considering cost, were beta naphthol

(0.1 to 0.4%), HgCL (0.1%), and especially phenyl

mercuric nitrate (0.0075%). Among those that were effective but uneconomical were paranitrophenol, glycocarvolene, para ehloro-meta-xylenol,

and salicylanilide. To this list Morrow and

Richardson [114] added sodium pentachlorophenate. In the same year Richardson [132] tested

chrome-tanned calf leather treated with various

phenolic compounds. It was found that lower

concentrations were needed than for vegetabletanned leather. The best compounds were sodium

trichlorophenate (0.02%), paranitrophenol (0.1%)

sodium tetrachlorophenate (0.1%), sodium 2- bromo-4-6-dichlorophenate (0.1%), and pentachlorophenate (0.1%).

Furry, et al. [61] in 1941 made a study of 135

chemical treatments for the prevention of mildew

in cotton fabrics. They found that fairly good

protection is obtained by acetylating cotton fabric for 21 hours at 20° to 25° C, but the treatment

tends to weaken the fabric. In regard to this

12 Miscellaneous Publication 188 of the National Bureau of Standards
point. Thaysen. et al. [163], by varying the conditions and. concentrations of his aeetylating mixture, has been able to treat cotton without loss of strength. Among other excellent treatments they

list salicylanilide in wax or resin, orthophenylphenol, 2-chloro-orthophenylphenol, and pentachlorophenol. The sodium salts of these compounds, however, are too soluble for use on fabrics.

Armstrong [6, 7] in 1941 was concerned with the

mildew-proofing of jute sandbags for military

purposes. For this purpose he recommends the

use of (1) tar distillate or creosote (25% in fiber);

(2) copper compounds, such as the napthenate,

oleate, or stearate (O.S to 1.0% of copper to be in

the fiber); (3) cuprammonium solution (1.0 to 1.5% of copper to be in the fiber), (4) copper salts,

such as copper chromate. aqueous colloidal copper,

and copper carbonate; or (5) cutch-cuprammonium solutions in a two-bath process (0.8 to 1.0% of copper in the fiber). A minimum of

0.35 percent of copper must be present to prevent

microbiological action.

In 1942 Marsh and Duske [110] found that

fabrics treated with a 1 to 2,000 aqueous dilution

of phenyl mercuric acetate showed effective

mildew resistance even after several washings.

The treatment has no apparent effect on the feel or nature of the fabric. The cost of processing is small, and tests indicate that it is nontoxic at this concentration.

Although the use of copper compounds had

gained steadily, it was recognized that some were

better than others. Jamison [86] in 1942 showed

that copper was easily adsorbed by many soils. Consequently, if the copper ion was the only

fungicidal element of a particular compound, its adsorption would leave a treated fabric or other

material unprotected. On the other hand, if the

copper was coupled to a radical, such as the

naphthenate. which has been demonstrated to possess fungicidal capacity [108], the loss of copper

by adsorption would stiU leave the treated

material protected to a degree. However, Osnitskaya [126], in the same year, differs with this concept. She finds that naphthenic acids may

serve as a source of carbon for some species of

microorganisms.

Dimond and Horsfall [49] in 1943 found that,

although mercaptobenzothiazole was an excellent

fungicide, it was inactivated by zinc oxide. However, where it was not possible to eliminate zinc

oxide from a material, tetra methyl thiuram

disulfide was an excellent substitute for mercaptobenzothiazole, being entirely compatible with zinc

oxide. This is especially important in the rubber

industry, where zinc oxide is essential to the compounding of rubber. In the same year, Ter Horst

and Felix [157] found that 2,3-diehloro-l,4-naphthoquinone was effective in the control of 22

important fungi with no harmful effects to cotton

fabrics. At the same time Ordal and Deromedi

[124] were reporting that two synthetic wetting

agents, lauryl sulfonate and the dioctyl ester of

sodium sulfosuccinate, enhanced the germicidal

action of buffered solutions containing 2,4-

dichlorophenol or 2,4,6-trichlorophenol. They

attributed the enhancement of germicidal action

to a synergistic action between the wetting agents

and the undissociated phenols. Finally, in the same year Robertson [135] showed that slight

concentrations of organic mercury compounds in leather had a stimulant effect on mold growth.

He produced this effect with concentrations as low as 1 to 20,000 and 1 to 40,000

The year 1944 showed a sharp increase in the

number of publications in the field of materiel

preservation. The war in the South Pacific area had intensified research in this field. Old compounds were reexamined in the hope that improved

methods for using them could be developed.

Greeves-Carpenter [76] recommended the organic

mercury compounds, for which Shiraeff [148]

had proposed new methods of chemical determination. Lollar [103, 104] [105, 106], in a series of papers, reexamined all the available fungicides for use on leather. He tested 40 compounds and

found the best were paranitrophenol, para chlorometa-xylenol, para chloro-meta-cresol, salicylanilide, pentachlorophenol, mercaptobenzothiazole, dihydroxy dichloiodiphenylmethane, tri- chlorophenol, and tetrachlorophenol. He was one

of the first to recommend the use of mixtures of fungicides. He found that copper and zinc com- pounds gave little or no protection on leather. However, copper compounds were receiving the

attention of many workers in this field. Azevado

and Maffei [11] reported that the most effective

treatment for cotton canvas consisted in immersing

the canvas in a 10 percent solution of copper

sulfate, followed by immersion in a 10 percent

sodium hydroxide solution. Woodford [175]

warned that military textiles of cellulosic origin

in the India climate required copper in some form.

To increase the effectiveness of the copper compounds, he proposed the use of such binders as

plasticized pitch, gear oil, and bituminous emulsion. Carter [39] was of the opinion that metallic

naphthenates give greater protection to cellulosic materials against microorganisms than other

metallic organic or inorganic fungicides, with which

Jones [88] agreed. Jones also recommended the use of cutch followed by potassium dichromate.

Finally, Marsh, et al. [108] made a study of various

organic copper compounds, including copper

naphthenate, copper oleate, copper tallate, and

copper resinate. They showed that copper naphthenate prevents rotting of cotton fabrics by

mildew at a lower concentration than any of the

other compounds. They held that this was due,

in part, to the fact that naphthenic acid itself is a potent fungicide. Factors that influence the

fungicidal properties of the four compounds were

Microbiological Deterioration

835770—49 3

13
claimed to be leaching, adsorption, and chemical

deactivation by means of hydrolysis, and the

subsequent formation of insoluble copper compounds in the soil. In 1945 the only important new fungicides to be examined were the highly effective pyridyl

mercuric compounds. Appling and McCoy [1] found that pyridyl mercuric acetate and pyridyl

mercuric chloride were as effective as phenyl

mercuric compounds. However, their toxicity

toward Aspergillus niger was not as great. By

this time the excellence of dihydroxydichlorodiphenylmethane (G4) had become so well es- tablished that Shiraeff [149] worked out a chemical

test for its estimation. Borghetty [25] found

dihydroxydichlorodiphenylmethane, phenyl mercuric acetate, and phenyl mercuric nitrilotri- ethanolamine to be effective fungicides. Neish

and Ledingham [119] developed a new fungicide,

copper dimethylglyoxime which they claimed was more effective than copper naphthenate.

The compound was made by reacting dimethylglyoxime with copper acetate. Furry [63] made a study of the fungicidal properties of natural dyes

and found that osage orange, quercitron, divi divi,

fustic, logwood, and quebracho were all effective. Fargher [57] sums up the work of many years by

saying that salicylanilide in 0.01 to 0.2 percent

solution has inhibiting power equal to that of an

0.8-percent solution of ZnCl2 , and paranitrophenol

is almost as effective. To produce great resistance

in materials highly sensitive to mildew, two or more antiseptics, generally contrasted in chemical

constitution and partition coefficient between

water and oil should be used together, as for in- stance salicylanilide in combination with paranitrophenol or mercaptobenzothiazole. The rot- proofing treatment should provide protection

against deterioration by chemical tendering by

air, light, and moisture as well as by molds and

bacteria. The acetylation of cellulosic materials

and the reduction of water adsorption to a very

low figure by coating with plastics are applicable

only to a limited extent. The success of the latter method depends upon obtaining plasticizers that

resist the growth of microorganisms and deterioration by exposure to the weather as strongly as the

plastics with which they are applied. The organic

antiseptics used in mildewproofing are not suit- able for rotproofing, since only fungicidal substances that are substantially insoluble in water

are effective. Bayley and Weatherburn [19] found that the ability of cotton fabrics containing

copper naphthenate to resist microbiological

attack in the soil was impaired when the fabrics were leached in water for 24 hours. Chemical

analysis showed that this effect was not caused

by a loss of copper during the leaching but rather

by a change in the copper compound resulting in a greater proportion of the organic solvent—insoluble form. The effect did not appear to be dependent on the pH of the leaching bath. In this

respect it is interesting to note that in 1939,

Rabanus [131], while studying the cause of the

ineffectiveness of copper carbonate as a fungicide

for telegraph poles, found that oxalic acid,

formed by wood fungi, converts the copper car- bonate into insoluble CuC2 4 , which is ineffective as a fungicide.

As their value came to be appreciated, copper

compounds received increased attention. However, it was rumored that these compounds, in- cluding the highly regarded copper naphthenate,

contribute to the deterioration of fabrics on which

they are deposited. Bartlett and Goll [15] found

that copper sulfate under certain conditions has

a tendency to promote the oxidation of cellulose. Copper naphthenate, on the other hand, does not

have any deteriorating effect on cellulosic materials.

They had exposed their treated fabrics to artificial ultraviolet light. Bayley and Weatherburn [20,

21], however, exposed their treated fabrics to weather conditions for a period of 7 months.

They, too, found that there is no increase in the

rate of deterioration because of the presence of

copper naphthenate or copper oleate. In the attempt to improve the tenacity of copper

naphthenate Bayley and Weatherburn [21] in 1946 suggested the use of waterproofing materials over the treated fabric. Curwen [45] suggested

that the slow-leaching out of copper naphthenate on exposed parts of treated materials could be overcome by the part-formation of aluminum

naphthenate during the process of manufacture.

This mixture permits the use of about 80 percent

less naphthenate than is normally recommended.

VI. Testing for Fungicidal Efficiency

Strictly speaking, the ideal method of testing

the efficiency of a fungus-proofing compound is exposure to severe field conditions. However, this

is time-consuming and in most cases impractical

for other reasons. Therefore, accelerated methods

of test have been sought. The first workers in this

field used empirical methods with uncontrolled

conditions. Levine and Veitch [99], [168] in 1920

reviewed the subject of mildew resistance of

textiles and proposed a method of testing the

relative effectiveness of various treatments. They

leached treated samples in running tap water for

2 or 3 days in order to wash out easily removable

fungicides and fermentable matter. Then the

damp samples were placed in Petri dishes containing nutrient agar jelly, and were incubated for 7 to 10 days in a dark chamber at 20° to 25° C.

After this preinoculation period the samples were

14 Miscellaneous Publication 188 of the National Bureau of Standards
inoculated with pure cultures "of several species of

molds and reincubated for 3 weeks to a month.

They were examined each week for mold growth,

and the observed conditions were rated on a scale of 10. The test was considered to be a severe one. The only treatment found to survive the full period of the test was that with copper ammonium

hydroxide, the "cuprammonium process." Correlating their test with service conditions, they observed that treated canvas which gave a rating of

at least six did not mildew on exposure to Washington, D. C, weather during the summer and

fall months. In the same year Fleming and Thaysen [58] developed a test of mildew damage in

which they treated the cellulosie material with

NaOH and CS2 . Under the microscope they were

able to observe and count the damaged fibers. In 1926 Bright [28] attempted to study the

extent of mildew damage by examining the fibers under the microscope. He used the Congo red

test and the swelling test of Fleming and Thaysen

[58]. In the following year Morris [113] tested

"fabric antiseptics" for toxicity to at least eight

species of molds. The efficiency of each antiseptic was compared to phenol as a standard. He found

that although one mold may resist 10 times as much of one antiseptic as another species, the

situation may be entirely reversed when another

antiseptic is tested. Therefore, he concluded, a

considerable number of species must be used in

testing.

In 1929 Searle [143] described a new test method. To compare the resistance of fabrics to molds and bacteria, strips were wound around

filter candles that had been covered with a thin

layer of soil, and were incubated for as long as 6 weeks in a moist chamber at 30°C. They were

tested for strength at periodic intervals. Searle was the first to observe the interesting phenomenon that cotton fabric deteriorated by molds

has the same cuprammonium viscosity as normal

cellulose. Thaysen and Bunker [162] in 1930 relied on

outdoor exposures followed by microscopic exa- minations. They exposed fabrics in various

parts of the world, under a wide variety of conditions. In the following year Ellis [52, 53] and

also Hall [79] proposed a new microscopic test method which involved the use of lactophenol

and Cotton Blue. By this method mold hyphae

can be differentiated from cotton fibers. In 1934 Burgess [38] proposed a laboratory

method for determining the resistance of wool and

other animal fibers to bacterial action. He found

that wool fibers could be disintegrated into their

component elements of epithelial scales and corti- cal cells by exposure to an appropriately buffered

solution of trypsin or pepsin. He showed that

the action of the trypsin is similar to that of

proteolytic bacteria but is more rapid.

In 1934 Thorn, et al. [166] examined all of the

available test methods and found that they fell into

two classes: (1) the simulation of the field hazard

under conditions more or less closely observed and

recorded, and (2) accelerated methods in which the

natural agents were given what were assumed to be special, favorable conditions to attack the

fabric and thus to concentrate into a short period

the destructive effects normally extending over

long periods. In both procedures, after incubation, it was necessary that some kind of laboratory

examination be applied to give comparative

values to the measurement or estimation of the

stage of deterioration. They considered only the

effect of cellulolytic organisms. After investigating the possibilities of a test based on a mixed

culture inoculum they came to the conclusion

that only a pure culture inoculum could be ex- pected to give reproducible results. This decision

necessitated the selection of a suitable test or- ganism and an appropriate medium. They set up the following requirements for a suitable test organism:

1. Capable of rapid destruction of cotton fabrics.

2. Must be easy to cultivate.

3. Must be adaptable to a fairly wide range of conditions.

4. Must be easy to identify in order to maintain in pure

culture or to free from contaminants.

Thorn, et al. selected a modification of Czapek's

agar medium similar to that used by Dubos [51] in 1928 but differing in that 3.0 g of NaN03 per

liter was used in place of 0.5 g per liter and a pH

of 6.8 in place of 7.5. Chaetomium globosum [40] was found to be the most destructive of the

molds tested. It had been reported previously

as a cellulose destroyer by Galloway [64] in 1930,

by Thaysen and Bunker [161] in 1927, and by

others. When tested against other species of

Chaetomium, it was found to have the greatest

cellulolytic power. This was confirmed by

Greathouse and Ames [75] in 1945. In figure 5 can be seen C. globosum growing on lacquerimpregnated filter paper.

In 1940 Rogers, et al. [136] made an intensive

study of the effects of fungal growth on cotton

fabrics. As test organisms they selected Chaetomium globosum and Spirochaeta cytophaga. They

reaffirmed the excellence of the choice of Chaetomium globosum as a test organism and observed

that C. globosum is unique in that the pH of the

medium is 6.8, both before and after completion

of the incubation period.

Everitt and Sullivan [54] in 1940 tested fungicides by adding them directly to the growth

medium. They distinguished between fungistatic

and fungicidal compounds. In the same year

O'Flaherty and Doherty [123] reported that there was no standard accelerated test for evaluating

fungicides for leather. Several pages farther in

the same journal, Richardson [132] reported that

he had sprayed treated squares of leather with a

Microbiological Deterioration 1.5
Figuhe 5. Growth of Chaetomium globosum on filter paper impregnated with a lacquer containing a fungicide. The growth of Chaetomium globosum on the specimen indicates an inferior treatment. (Note bacterial contamination of agar.) (Unsteriiized specimen used.)

suspension of mixed mold spores. These were

stored damp at room temperature for 50 days.

The time required for visible mold growth on

untreated squares was 6 to 9 days.

In 1941 Downing and Whitmore [50], speaking

for the leather industry, observed that laboratory

tests are not altogether reliable because molds

gradually acquire immunity to fungicides. It should be pointed out that this objection does not

apply to pure culture tests in which the organisms come in contact with fungicides only once and

are then discarded. Downing and Whitmore are

referring to mold chambers that are used constantly for testing fungicides. To prevent the

molds from acquiring immunity, they recommend

that different fungicides be tested alternately.

Greathouse, et al. [74] in 1942 made some note- worthy contributions to the pure-culture test method. Rogers, et al. [136] had recommended

the use of square 16-ounce screw-cap bottles for incubation chambers. Greathouse, et al., greatly

improved them by modifying the metal cap. A

hole was cut in the cap and was covered with a

piece of closely woven glass fabric. This served

two purposes: (1) It became easier to maintain

sterility, and (2) better aeration of the incubating

specimen was possible. Another modification was the substitution of a glass wick for the agar

that had formerly been used. They also held

that the adjustment of media to a given pH with

HC1 or NaOH before autoclaving was valueless, as after autoclaving the pH of adjusted and nonadjusted media becomes approximately equal.

From results obtained with a variety of dilutions

of inoculum, they concluded that the concentration of inoculum, within rather wide limits, does

not seem to affect the degree of growth after 14 days of incubation, and for this reason it is not

necessary to specify the concentration of a spore

suspension with any great exactitude. In addition to Chaetomium globosum, they worked with

a new organism, Metarrhizium sp., which was

later named Metarrhizium glutinosum by Pope

[128] and which has been identified as Myrothedum

verrucaria by White and Downing [172]. They

considered Myrothedum verrucaria to be superior

to Chaetomium as a test organism. Their out- standing contribution, it seems, was their in- sistence on buffered media.

By 1943, soil-burial tests had achieved a degree

of popularity. Many workers were realizing the

limitations of the pure-culture test methods and

10 Miscellaneous Publication 188 of the National Bureau of Standards
were seeking a more severe test method. Furry

and Zametkin [62] developed the soil-suspension

test. Test specimens were inoculated by immersion in a soil suspension made from composted

soil. The inoculated specimens were incubated on agar medium, glass wick-nutrient medium, or even cotton-batting in nutrient medium, all in 16-ounce square bottles. They claimed excellent

differentiation of fungicides, approaching the soil- burial test in potency. Borlaug [26] recom- mended the use of the soil-burial test for synthetic

fabrics in preference to pure-culture methods.

And Bunker [31] suggested the use of aluminum

frames for mounting soil-burial test specimens.

These were buried vertically in typical light

garden soil and kept at 30° C and 20-percent

water content. They were tested weekly by at- tempting to tear them by hand. Finally, Bertolet [23] decided that as a result of extensive work

in testing the mildew resistance of jute and

osnaburg fabrics used in sandbag construction,

the laboratory-accelerated mildew test should be

abandoned in favor of the soil-burial test as a measurement of the service life of this material.

In 1944 Lollar [103, 104] proposed the use of

mixed cultures of molds for testing mildewproofed leather. ZoBell and Beckwith [178] made

a similar recommendation for testing rubber compounds. Fabric testing was also showing a return to less controlled test procedures. Azevado [11] described an accelerated rotting test in which test strips were interlayered with strips of rotten canvas. The incubation period was 60

days under tropical conditions. However, such

workers as Marsh, Greathouse, and Barker were

still carrying on the fight for standardized, controlled test methods. Marsh, et al. [108] in

testing a variety of copper compounds, concluded

that laboratory test methods had not yet developed to such a stage that then- results alone

could be used as accurate predictions of the

resistance to mildewing or rotting of a treated

fabric under all of the varied service conditions

to which it might be subjected. Therefore, they

tested treated fabrics by simulating field conditions before subjecting them to pure culture

tests. These modifying tests included: leaching,

adsorption by bentonite, and chemical deactivation by means of hydrolysis. They also made use

of a copper-tolerant mold, Aspergillus niger.

They emphasized the futility of attempting

evaluation by the use of mixed cultures.

Klemme, et al. [96] in 1945 examined 43

organisms for cellulolytic activity. Of these 29

caused more than 50 percent loss in breaking

strength of cotton duck when incubated for 7 days on a liquid mineral salt medium that contained

NH4NO3 as the nitrogen source. Also, they

studied the effect of using different sources of

nitrogen. Whereas most of the organisms grew

best when NH4 N03 was the nitrogen source,

Chaetomium globosum was unique in that it grew

best on NaN03

. This was later verified by

Greathouse and Ames [75], who studied the

cellulolytic acticity of 16 species of Chaetomium.

They found that C. globosum was the most active

member of this genus. The ability of this group

to decompose cellulose was found to vary with the source of nitrogen, the pH, the temperature, and

with the fabric preservative.

Up to 1945 the requirements for leaching

specimens before a mildew test were more or less haphazard. Thorn, et al. [166] had suggested

that fabrics be soaked in several changes of water

or in running water for 2 days. Several Government procurement specifications called for a

leaching period of 30 hours in running tap water. Furry and Zametkin [62] suggested a 24 horn

leaching period and specified that for each ounce

of fabric there should be 1 gallon of water with

three complete changes during the 24 hours.

Goodavage [71] described two types of tests-, a spray test for water repellent materials, and a

leaching test. For the leaching test he specified

that there be 1 gallon of water at 65° F for each

specimen 6 inches by 12 inches and suggested that

there be three changes of water in 24 hours. In

1945, Marsh, et al. [109] developed an apparatus

that could deliver water at a temperature of 30° C

to quart Mason jars. The flow was so regulated

that there were 10 changes of water per hour.

Also, they considered the effect of steam steriliza- tion on organic fungicides and concluded that it

is, perhaps, an unnecessarily severe test for

these fungicides. When a nonsterile technique is required, as in the case of the steam-distillable

organic fungicides, they found that C. globosum was more satisfactory than certain other forms as a test organism as it is less apt to be contaminated.

They recommended a filter paper-mat technique

in which the test fabric was planted ou a mat of

mycelium. The mycelium was induced to grow

on a filter-paper strip supported on a mineral

salts-agar medium. This type of test was found

to be much more severe than the previously

described pipette inoculum procedure. They believed that the choice of the best test or combination of tests to determine mildew resistance

of a fabric depends on the service conditions

under which the fabric is to be used.

In the same year Dean, et al. [46] reported on

extensive studies of the soil-burial test method.

They used only composted soil and carefully

controlled the temperature and moisture. To

obtain reproducible results they found that it was

necessary to use fresh soil for each test. The soil- burial resistance for cotton osnaburg treated with

copper naphthenate was measured for 10 identical

tests and was found to be reproducible with a

coefficient of variation of 8.5 percent.

In 1946 Romano 1437] reexamined the role of

pfie rob io logical De ten oraHon 17
nitrogen in the growth of Chaetomium globosum.

Waksman and Hutchings [169] had shown that a

ratio exists between the activity of cellulose decomposing microorganisms and the amount of

available nitrogen present. The higher the cellu- lose content of soils, the greater is the need for

nitrogen to maintain cellulolytic activity.

Romano used NH4N03 because it maintained a more stabilized pH than NaN03 or KN03 . He

found that the optimum concentration of nitrogen was 1.4 to 1.75 grams per liter of medium. When

the concentration of nitrogen was increased as high as 7.35 grams per liter, there were no toxic or inhibitory reactions. Varying the other ele- ments in the medium had no effect on growth.

He concluded that the nitrogen concentration of

the nutrient media in general use was insufficient for maximum growth.

Part 2. Investigation of Microbiological Tests

VII. Development of Mildew Testing at Bureau

The primary problem of testing for mildew

resistance at the National Bureau of Standards

has been the development of tests that are reproducible and sufficiently severe to give that margin

of safety that is required for tropical use. Considering the great number and variety of

fungicides, it is obvious that they cannot be equally

effective, especially as it has been shown that they

act in a variety of ways. In order to determine

their relative effectiveness, it is necessary to have

methods of bioassay. The term "bioassay" is stressed because chemical analyses alone are not

sufficient. It can easily be ascertained that a

particular fungicide is effective in a particular

concentration. Why, then, is it not sufficient to make a quantitative analysis of a treated material

to learn whether the desired concentration has

been deposited? First, chemical analysis cannot

determine the degree of penetration of a fungicide

or its distribution over the treated material.

Second, as has been pointed out previously,

different materials react in decidedly different

ways to fungicides. For example, fungicides that

are effective for cotton may not be effective for

leather. Furthermore, it is important to test a

material with regard to its ultimate use. A

material that is to be protected only during storage

need not have as permanent a fungicide as a

material that is to be exposed to the elements and

which may come in contact with the ground.

Many workers have attempted to develop test methods for the evaluation of fungicide-treated

materials. The first to tackle this problem were

those interested in the preservation of cotton

textiles. Fabrics to be tested were inoculated

from rotted fabrics or from composted soils. No effort was made to determine the nature of the

organisms used or to accelerate the test by adding

nutrients. Within the past decade Thorn and

his coworkers have reexamined the question of

mildew tests and have decided that any test that

makes use of mixed cultures of organisms does

not lend itself to scientific accuracy. Thereupon,

they studied the growth habits of many cellulose- destroying organisms. They chose an exceptionally stable organism Chaetomium globosum

Kunze which, during 5 years of continuous subculturing in the Microbiological Laboratory at the National Bureau of Standards, has not been

observed to change its growth characteristics.

It grows well on a mineral salts-agar medium

when a cellulosic carbon source is provided.

When growing the organism for stock cultures,

filter paper is the usual source of carbon. When

testing a cotton fabric, the test material itself supplies the carbon. If the specimen does not

contain a fungicide, C. globosum will utilize the

cellulose as a carbon source. If, however, the

fabric is adequately mildew-proofed, the organism

will either be poisoned or the cellulose will not be

available as a source of carbon, depending on the

mildew-proofing agent used.

As originally proposed by Thorn and his co- workers, the culture medium was a modification

of that proposed by Czapek:

NaN03 3.0 g. K2 HP04 1.0 g. MgS04 . 7H2 0.25 g. KCl 0.25 g. FeSO-4 0.01 g. Agar 10.0 g. Distilled water 1.0 liter. Adjust pH to 6.8 with NaOH or HC1 as required.

This medium was originally developed for mycologists in order to study the growth characteristics

of various organisms. For this purpose a rather

sparse growth is preferred. Therefore, by using

Thorn's medium, which does not give maximum

growth, C. globosum was not being used to fullest advantage as a test organism. In brief, the test 1 method was as follows: strips of treated fabric of a size suitable for determining breaking strength were sterilized and laid on a surface of mineral

salts-agar in bottles under aseptic conditions, after which they were inoculated with an aqueous

suspension of the spores of C. globosum. Un- I treated fabric was given the same test to serve as a control. Next the bottles were incubated for | 14 days at a temperature of 28° C, after which

the specimens were removed and washed, and,

j when dry, their breaking strength was determined. J Of course, the untreated fabric was completely i

18 Miscellaneous Publication 188 of the National Bureau of Standards
degraded. If a fabric retained 90 percent of its original strength, it was considered to be adequately mildew-proofed.

Shortly before the outbreak of the recent war it became necessary to test the mildew-resistance of

sandbag fabrics. As a better method was not

available, Thorn's test was used. However, its shortcomings soon became apparent. The optimum pH of 6.8 could not be maintained and it was found that certain fungicides inhibit C. globosum at concentrations far less than that

necessary for effective mildew-proofing. For

example, it is known that copper naphthenate, for

effective mildew-proofing, must be in fabric to the

extent of 0.8 percent of metallic copper, based on

the finished weight of the fabric, yet 0.3 percent

will inhibit C. globosum in this test. By observing the visual appearance of growth

from day to day, it was noted that the degree of

growth, as seen with the unaided eye, was a good

measure of the physical break-down of the fabric.

If there was no growth by the seventh day, there was none by the fourteenth day. Therefore, after 35,000 strips had been observed, the test was

shortened to 7 days, and breaking strength determinations were discontinued. However, this did

not increase the severity of the test. It could not

be used for the evaluation of new fungicides. It was used mainly in procurement testing of approved fungicides for army materiel. A chemical

analysis of the treated fabric was first made and if there was sufficient fungicide present, a Chaetornium test was given to determine the uniformity

of treatment application.

While this test was being used a concerted

effort was being made to improve the cellulolytic

activity of Chaetomium. By experimenting with a

variety of media, some of which had been sug- gested by Greathouse and Marsh, a medium,

double-buffered with K2 HP04 and KH2 P04 was

selected (see page 25).

According to Romano [137] this medium, containing 3.0 grams of NH4 N03 , has the optimum

nitrogen content (1.6 gy liter) for C. globosum.

Furthermore, the pH is close to 6.8 and requires no adjustment. Although Greathouse and Ames

[75] have shown that NaN03 gives slightly more

breakdown of cellulose with C. globosum, it has

been considered advisable to use NH4 N03 because

of greater stability of pH due to buffering action

and also because this medium can be used for Aspergillus niger by adding 30 grams of sucrose per liter as a carbon source. Inasmuch as tap

water has been specified, no trace elements need

be added. However, in localities where the tap

water is not close to neutrality, or where it contains large quantities of minerals, distilled water

should be used with the addition of the necessary

trace elements.

Although this medium improved the reproducibility of the test, it was still not the ideal test.

On the basis of work by Greathouse and Marsh,

the test known as the Chaetomium mat method

was standardized. It is more potent than the

Chaetomium method of Thorn but is not quite as severe as the soil burial test. The test makes use

of the double-buffered salts-agar medium (page

25). A strip of sterile filter paper is placed over

the medium and is inoculated with C. globosum

spores under aseptic conditions. After 3 days of

incubation, a dense white mycelium develops over

the surface of the filter paper. This is the mat

that gives the test its name. At this point an

unsterilized test specimen is dipped in a fresh

inoculum and placed over the mat, which is in- cubated again for 14 days. Even copper naphthenate-treated fabric loses some of its strength

in this test. The loss is roughly comparable to that of a 6-week soil-burial period.

Up to this point the emphasis has been on

cellulosic materials. Historically, cellulosic materials were the first to be considered from the

point of view of microbiological deterioration.

Early in the recent war, however, many reports were received from tropical battlefields that not

oidy cellulosic materials but a wide variety of

materials was being attacked by fungi—woolen

blankets, leather shoes, and instrument cases,

all types of plastic materials, rubber (synthetic

and natural), and even the lenses of binoculars

and cameras. As cellulose was rarely a component

of any of these products, C. globosum was obviously unsuited as a test organism. Furthermore,

the organisms that attacked these materials in the

field were usually surface growers that caused

damage in other ways than loss of strength.

Consequently, an entirely new philosophy of

mildew testing was developed. The measure of

mildew resistance in this type of testing is the

degree of growth of the test organism rather than

the determination of its effect on some desirable

property of the test specimen as, for example,

breaking strength in the case of cellulosic materials.

Mildew-resistant camouflage paints were the

first noncellulosic materials to be tested in this laboratory. Inasmuch as Aspergillus niger is such an ubiquitous and prolific organism, several

strains were obtained from the United States Department of Agriculture, and the one that is now a standard test organism was finally selected. The medium chosen was the double-buffered modification of Czapek's containing 30 grams of sucrose per liter. The organism was able to grow on this without obtaining nourishment from the test specimen. However, if the specimen did not

contain a fungicide, the organism would in- variably overgrow it. Figures 6 and 7 illustrate

the A. niger test. Aspergillus niger has been found to be a very

useful test organism. Although it was originally

selected for testing the mildew resistance of camouflage paints, it was found that it could be

Microbiological Deterioration 19
Figure 6. Mildew-resistant camouflage fabric on Aspergillus niger -plate. Specimen completely inhibits growth of organisms.

used as a test organism for all materials which, in the field, supported a growth of the prolific surface

growers, as for instance leather. Vegetabletanned leather contains all the necessary nutrients for the growth of A. niger. Although it

is not necessary to use a mineral-salts-sucrose

agar medium in testing leather, a greatly accelerated test is obtained when it is used. Other

materials that have been tested for mildew re- sistance by the use of A. niger are woolen fabrics,

rubber (natural and synthetic), plastics, and

plasticizers.

The medium used for all tests in which A. niger

is the test organism is the same as the doublebuffered C. globosurn medium (page 25) with the

addition of 30 grams of sucrose per liter. The

test is as follows: A sterile culture plate is inoculated with the spores of A. niger and is incubated

for 48 hours at 28° C and a relative humidity of

90 to 95 percent. At the end of this time the sur- face of the agar is completely covered with white

mycelium. Thereupon the treated specimen is moistened and placed over the center of the mat

and incubated again for 7 days. At the end of this time an adequately mildew-proofed specimen will

be entirely free of growth and may even have a white zone of inhibition around it. Beyond the

specimen, however, the white mycelium has

turned to black spores. The untreated control on the other hand will be completely covered by

the black spores. The A. niger test has been modified slightly to

test the mildew susceptibility of materials such

as rubbers and plastics, which do not necessarily

contain fungicidal substances. The object is to determine whether these materials can support

a growth of mildew. Therefore, the sucrose is omitted from the medium. As the organism

cannot grow on this incomplete medium, the test sample is first placed on the agar medium and

then is inoculated by pipette, as in Thorn's

Chaetomium test. As stated previously, fungicides cannot be

tested apart from their existence on organic

materials. Although many compounds are ex- tremely toxic to microorganisms in solution, their

usefulness on fabrics or other organic materials

may be limited by then excessive solubility, their

activity as catalysts in the photochemical deterioration of cellulose, or by then instability to ultra violet light. Therefore, any evaluation

test method must include the resistance of the

treated material to micro-organisms under a variety of conditions. To be more explicit, a

treated fabric may be tested with a pure culture of a particular mold that is known to have cellu20 Miscellaneous Publication 188 of the National Bureau of Standards
Fioure 7. Camouflage fabric without fungicide on Aspergillus niger plate. Specimen heavily invaded by organisms.

lolytic power. In a test of this nature the treated

fabric may show no growth of mildew and may

retain all of its strength, whereas an untreated

control specimen may be completely degraded.

This merely indicates that the particular fungicide

is of sufficient concentration to prevent the test organism from developing. It tells nothing about

the permanency of this fungicide. A second test which then suggests itself is one that seeks to determine the solubility of the fungicide as it exists on the treated material. Washing the

treated material in running tap water for 24 hours

has proved to be adequate for most treated materials. As a rule, however, treated leathers are leached for 48 hours because the fungicides are usually combined with oils and greases, which tend

to decrease their solubility. Copper sulphate,

which is an admirable fungicide, is easily leached

out in a test of this 'nature. Fungicides that successfully pass a mildew test after 24 hours of leaching are next subjected to

artificial weathering conditions in which the combination of ultraviolet light and moisture are brought to bear on the treated material for a

definite period of time, such as 360 hours, depending on the type of weathering machine used.

Certain chemicals, s\ich as manganese compounds,

are known to act as catalysts in the photochemical deterioration of cellulose. Others, such as the chlorinated phenols, and especially pentachloro phenol, break down in the presence of

ultraviolet light and liberate HC1, which attacks

fabric and weakens it considerably even before

being subjected to the action of molds. In order

to determine whether a fungicide has weakened a

fabric during ultraviolet light exposure, breakingstrength determinations must be made after ex- posure and before mildew tests are attempted.

A loss of more than 10 percent in strength is usually indicative of photochemical degradation.

However, a fungicide may break down as a result of an artificial weathering test without damaging

the fabric. An illustration would be a buffered

pentachlorophenol treatment in which the HC1 is neutralized as it is formed. A subsequent mildew

test would then show the lack of fungicidal

activity.

Although a series of tests such as has been

outlined would seem to be sufficient to rank

fungicidal treatments according to their efficiency,

that, unfortunately is not the case. The use of

artificial weathering followed by pure-culture in- oculation tends to divide fungicides into two

groups: those that resist the effects of light and

water and those that do not. Furthermore, it does not rank the effective treatments in order of

Mierdbiological Deterioration 21
excellence. By this method a fungicide is either

good or bad. But experience has shown that all good fungicides are not equally good, and some

of the bad ones have their uses. Inasmuch as the military materiel, for which

these investigations were undertaken, were to be

used under the most rigorous conditions, a very

severe test was desired in order to assure a wide

margin of safety. Field exposures were timeconsuming and otherwise impractical. A practical solution to this problem has been the adoption

of the soil-burial test. This is the most potent

microbiological test method in use at the present

time. Top soil or compost is kept in small containers in a cabinet in which the temperature and

humidity are carefully controlled. Originally,

the results obtained with this test were unusually

variable, even for a biological test. However, it has been found that if the temperature and

mositure are carefully maintained, the results are much more reproducible. As in all other biological tests, untreated material of the same type as the treated test sample is tested at the same time

to show that organisms responsible for deterioration are active. Inasmuch as this test was so

variable, another control was considered necessary. It was decided to choose one fungicide

that was invariably effective and to use it as a

reference standard for each new fungicide tested

in the soil beds. In this way if there were any

variations in results due to variations in the conditions of the soil bed, they would be reflected in

the standard samples. The unknown could then

be evaluated in terms of the standard. This

standard was, and still is, copper naphthenate in a concentration of 1 percent of metallic copper,

based on the finished weight of the fabric.

Strangely enough, many of the fungicides that

broke down during artificial weathering proved

to be excellent in the soil-burial test. Among the

best of these were the chlorinated phenols, especially pentachlorophenol and dihydroxydichlorodiphenylmethane. Either of these compounds might

be recommended for materials that were not to be

used in direct sunlight, as, for example, in stored

materials. On the other hand, many compounds

that had successfully passed the accelerated

weathering test plus a pure culture test were

quickly degraded in the soil. These were chiefly

mercury compounds and the more soluble chlor- inated phenols. Such compounds might well be

used for materials that would not normally come

in contact with the ground as, for example, stored

articles. Again, this is a test that divides treat- ments into two distinct classes. Although there

is a slight tendency for ranking of the superior

treatments, it is not entirely correlated with the

ultimate use of the material. The main virtue of

this test is the wide margin of safety it permits

when one relies on its results, especially for

treated materials that are to receive most severe exposure as sandbags and tents in tropical regions.

VIII. Representative Problems

The following investigations are representative

of the microbiological research and development

conducted at the National Bureau of Standards

during the past 5 years. The problems are numbered from 1 to 10. This same numbering

scheme is followed in the discussion starting on

page 29 and in the summary beginning on

page 38.

1. In developing the Chaetomium test, which

had been first suggested by Thorn and other

authors [166] it became necessary to know the

rate at which cellulosic materials deteriorate

under standard conditions, and whether cellulosic materials of different origins break down in the same manner. For experiments to obtain this data, untreated cotton osnaburg (a coarse, un- bleached cotton fabric), having an average breaking strength of 73 pounds for 1 inch-wide specimen, and jute burlap, having a breaking strength

of 121 pounds for a 2-inch-wide specimen, were

used. To reduce the inherent variability of jute

burlap, which has only 12 threads per inch, 2-inch

raveled specimens were used. Eighty specimens

of each type of fabric were sterilized for 1 hour in

the autoclave. Thereupon, 10 specimens of each

sterilized fabric were set aside for breakingstrength controls. The remaining specimens were

laid on double-buffered mineral salts agar and

were inoculated with 2 milliliters of inoculum pre- pared in accordance with the method outlined

under Chaetomium test procedures (page 25).

The specimens were then incubated at 28° to 30° C and a relative humidity of 90 to 95 percent.

Ten specimens of each fabric were removed after

3, 4, 5, 7, 8, 9, and 10 days. After conditioning

at 70° F and 65-percent relative humidity, they were broken and were compared with the controls. In table 1 the results are listed as strength retained

in percentage of the original strength.

2. Inasmuch as cotton fabrics of different

thicknesses, treated with equal concentrations of

the same fungicide, showed varying resistance to degradation in the soil-burial test, it was decided

to investigate the effect of thickness of fabric on

the rate of deterioration in the soil-burial test. For this investigation four fabrics were selected. They were in order of increasing thickness: a 4- ounce lightweight sheeting with a breaking

strength of 46 pounds per inch width, a cotton

osnaburg having a breaking strength of 84 pounds

per inch width, and a 20-ounce duck having a breaking strength of 219 pounds per inch width.

22 Miscellaneous Publication 188 of the National Bureau of Standards
Inasmuch as these fabrics were all fairly tightly

woven, it was considered advisable to select a

loosely woven fabric in order to determine the

effect of tightness of weave on microbiological

deterioration. For this purpose a 37.5-ounce

loosely woven duck, having a breaking strength

of 499 pounds per inch width, was chosen. Fifty

specimens of each fabric were buried in the soil beds in a randomized pattern to eliminate the

effect of possible variations in the soil bed. After

6, 10, 14, 21, and 28 days, 10 specimens of each

sample were removed, conditioned and broken.

The results in table 2 are listed as strength

retained in percentage of the original strength.

3. The manner in which specimens are buried

in the soil-burial test has varied from laboratory

to laboratory. Bertolet [23] favored horizontal

burial at a depth of approximately % inch. On

the other hand, this laboratory preferred vertical

burial to a depth of 4 inches. To test the effi- cienev- of these two methods, cotton osnaburg having a breaking strength of 79 pounds per inch

width was used in comparative tests. Sixty

specimens were buried vertically and sixty speci- mens were buried horizontally. Ten specimens

of each were removed after 2, 4, 5, 7, 8, and 9 days and were conditioned and broken. The

results in table 3 are listed as strength retained in

percentage of the original strength.

4. Although relatively small concentrations of

copper compounds were known to inhibit Chaetornium globosum under laboratory conditions, tbeir

resistance to leaching from fabric in the field was

not known. In order to recommend the concentrations of each fungicide that would not only be

effective from the start but would have a safety

factor for prolonged effectiveness in the field, it became necessary to determine the permanence

to leaching of the superior copper fungicides.

For this investigation cotton osnaburg strips were

impregnated with copper naphthenate, cuprammonium, and copper ammonium fluoride, all in a concentration of 1.0 percent of metallic copper,

based on the finished weight of the fabric, except

that for cuprammonium the copper requirement was 1.5 percent. Lower concentrations had

previously been eliminated by failure in the soil- burial test. Twenty specimens of each of the

treated fabrics were extracted with distilled water

in a Soxhlet apparatus at a temperature of 70° C.

After 24, 48, 72, and 96 hours of extraction, speci- mens of each fabric were removed and given a

qualitative Chaetomium test. The results of this experiment are listed in table 4.

5. From various sources there had been conflicting information regarding the activity of

copper compounds as catalysts in the photochemical degradation of cellulose. Inasmuch as copper napbthenate was the most important of

the copper fungicides, an experiment was set up

to determine the effect of various concentrations

Microbiological Deterioration

of this compound on cellulose in the presence of

light. Cotton osnaburg, having an original

breaking strength of approximately 85 pounds, was impregnated with copper naphthenate in

Stoddard's solvent in five concentrations. The

concentrations (based on metallic copper, in the

copper naphthenate, as percentage of the finished

weight of the fabric) were 0.5, 1.0, 2.0, 3.0, and

4.0 percent. Although copper naphthenate is rarely used in fabrics in greater concentration

than 1.5 percent, for the purposes of this experiment it was considered advisable to work with as wide a range of concentrations as possible.

Two types of light exposure were used. In one

(see table 5, a) the samples were hung in a weathering machine equipped with Sunshine carbons.

The apparatus consisted of a vertical carbon arc mounted at the center of a vertical cylinder. The

panels of Corex D filter glass, which normally

surround the carbon arc, were removed for this

experiment to increase the intensity of the ultra- violet rays. The alternating-current arc was

operated on 60 amperes and 50 volts. The samples were mounted on a rotating rack inside the cylinder. The distance from the center of the axe to each specimen was 18% inches. The rack rotated

about the arc at a uniform speed of about 1 revolution in 2 hours. The samples received a fine spray

of water once during each revolution of the rack.

Specimens of each sample were removed after 50,

100, and 150 hours. The specimens were raveled

to an equal thread number approximating 1 inch

in width and were broken. The copper content

of the broken strips was determined. The data

are reported in table 5, a. Each figure for residual

strength represents an average of 10 specimens.

The figures for copper content are based on three

determinations for each average.

In the second type of light exposure, 10 strips of each concentration and 10 strips of untreated

fabric measuring 8 inches in the warp direction

by the full width of the fabric, which was 36 inches, were mounted loosely on wooden frames on the

roof of the National Bureau of Standai'ds in Washington, D. C. Each month thereafter* one

strip of each concentration and a strip of untreated

fabric were removed from the racks and given the same analyses as the samples that had been

exposed to ultraviolet light. The results appear

in table 5, b.

6. Practically all of the better known mildewproofing agents have been evaluated by a large

number of laboratories by the use of one or more

test methods. In the effort to determine the

relative effectiveness of the most popular test methods an ambitious investigation was undertaken. Ten-ounce cotton duck, having an original

breaking strength of approximately 145 pounds, was impregnated with 35 of the most commonly

used mildew-proofing agents. The compounds

used are listed in table 6. Each compound was

23
applied to the fabric in three concentrations. The

middle concentration of each compound is either

the one that is most generally used, or where this

information was lacking, it represents the concentration that is most likely to give good protection

under some but not all of the test conditions.

The test methods (see page 25) selected for

this investigation include

a. Aspergillus niger—mycelial mat test. b. Chaetomium—direct inoculation; sterilized specimen,

qualitative test.

c. Chaetomium—mycelial mat test. d. Accelerated artificial weathering—360 hours.

e. Chaetomium—mycelial mat test after 360 hours accele- rated artificial weathering.

f. Pencillium 66 (USDA)—mycelial mat test.

g. Soil-suspension test.

h. Soil-burial test—3, 6, and 9 weeks. Marsh and Greathouse recommended the use

of Pencillium 66 for two reasons: (1) it is an

active cellulolytic organism, and (2) there were

indications that it is not inhibited by mercurial

fungicides. Therefore, it was decided to test it in

comparison with Chaetomium globosum, which is inhibited by such fungicides.

In the visual tests 5 specimens were used for each operation, and 10 specimens were used for the breaking-strength determinations. The results, as they appear in table 6, are thus the

average of 5 observations in the qualitative tests and 10 determinations for the quantitative tests.

7. In order to determine whether molds derive

nourishment from the protein substance of vegetable-tanned leather or from the oils and greases

that are added during manufacture, a large sample

of vegetable-tanned leather was degreased by

extraction with Stoddard's solvent. By this

method none of the water-soluble materials was

removed.

The degreased leather was divided into speci- mens measuring 2 inches by 2 inches. Five speci- mens were tested for resistance to Aspergillus

niger. Of the remaining specimens five were

treated with fatty oil, five with leather grease,

five with fatty oil plus 1 percent salicylanilide,

and five with fatty oil plus grease pins 1 percent

salicylanilide. All of the modified specimens were also tested for resistance to Aspergillus niger.

The results appear in table 7. 8. In a series of cooperative tests of leather in which three other laboratories participated, it was shown that the Aspergillus niger test, developed

at the National Bureau of Standards, was as

reliable as the mixed-culture tests developed by

other laboratories. In some instances the A.

niger test was more severe than any of the other

tests. The test is outstanding for its reproducibility and for its speed, requiring only 7 days for completion. This test was used in the evaluation

of a large number of leather treatments of which 1 the most satisfactory, 1 1 in number, are presented a in table 8. Vegetable-tanned leather squares measuring

2 inches by 2 inches were impregnated with four

concentrations of the 11 compounds dissolved in Stoddard's solvent. Although the concentrations

of each compound cover a wide range, they are not evenly spaced. The great absorptive power , of leather makes control of application very 1

difficult. However, it is believed that the con- I centrations within each treatment are spaced far enough apart to demonstrate the level at which

mildew resistance is effective.

9. The entire program of research in the field of the mold susceptibility of plastics, which has 1 been reported by Brown [29], was initiated at the

National Bureau of Standards as a result of an

accidental observation. Before the need for

mildew-proofing of plastic materials was recognized, it was observed that different samples of vinylite-coated cotton fabrics showed varying ! responses to test organisms. Some supported

a vigorous growth, whereas others did not. To i! | the eye there was no apparent difference in these j materials. As they were supplied by different i manufacturers, and as there was no variation in the i results of any one manufacturer's product, the

basic components of these vinylites were obtained

from each processor. It was then found that the

components from the various plants differed in I only one respect, namely, the plasticizer. All t used the same vinyl chloride-vinyl acetate co- [ i polymer, but there were two plasticizers in use. \ t Some used tricresyl phosphate, which was demon- I strated to be mold resistant. The coated fabrics,

[ which had consistently supported a growth of i mildew, were found to contain a derivative of

J ricinoleic acid that was extremely susceptible to 11 mold growth. Thereupon, a study of 15 pure ] plastics (polymers) and 83 plasticizers was under- I taken. The polymers were prepared in the form 1

of 2 inch by 2-inch squares without plasticizer I by the members of the Plastics Section, National

Bureati of Standards. These specimens were i tested both in the complete medium-A niger test j

1 and the sugar-free A. niger test. As the plastici- i zers were, in all cases, liquids of varying consist- i i ency, a modified technique was developed. Glass j rings, ji inch in length by % inch in diameter were

fitted to the center of Petri dishes. The sterile I I agar was poured into the Petri dishes in such a 1 manner that the rings were surrounded, but not I i invaded, by agar medium. In the case of the t sugar-free test, the plasticizer was poured into the t cylinder before inoculation. In the complete ! culture medium test the agar around the cup was i permitted to develop a mycelial mat, after which

24 Miscellaneous Publication 188 of the National Bureau of Standards
the cup was filled with plasticizer. The results are reported in table 9, a and b, and are averages

of five observations for each test. 10. The study of the susceptibility of plastics

to mold growth stimulated the investigation of the

mold susceptibility of a group of related materials.

These included unvulcanized rubber (natural and

synthetic), nylon fabric, glass fabric, and mineral

wool. The results are reported in table 10 and

are averages of five observations for each test. IX. Test Methods

In the foregoing description of investigations,

the test methods are outlined briefly. At this point it is desirable to set forth in greater detail

the techniques of the methods used. The types

of biological test methods fall into two general

classes, namely, pure-culture and mixed-culture

methods. In the pure-culture methods, Chaetomium globosum is used to measure the rot resistance

of cellulosic fabrics and Aspergillus niger is used

to test the mildew-resistance of all noncellulosic

materials. The tests are as follows:

1. Pure-Culture Biological Methods

(a) Rot-Resistance Tests with Chaetomium globosum

(1) Direct inoculation—sterilized specimen method.—As this test can be used for either visual

(qualitative) or breaking strength (quantitative)

determinations, two types of test specimens were

used. For visual examination, squares measuring

2 inches by 2 inches were used. Breaking strength

specimens were cut 6 inches long in the warp

direction by 1% inches in the filling direction.

The specimens were raveled to an equal thread

count which was approximately 1 inch in width

in the filling direction before breaking strength was

determined. As a control, untreated material,

similar in all other respects to the treated material, was tested in the same manner as the test sample

to verify the viability of the test organisms.

A double-jacketed autoclave was used at a pressure

of 15 pounds per square inch and an exhaust temperature of 121° C for steam sterilization. For

leaching the specimens before the mildew tests, an apparatus was developed which consisted of a series of quart Mason jars so arranged that tap

water was delivered to the bottom of each vessel

at a rate of flow to permit five changes per hour.

The water was maintained at 27° to 30° C and

the pH was approximately neutral. Specimens

were leached for 24 hours for most materials and

48 hours for treated leather. For incubation

chambers, Petri dishes (10 cm) were used for visual

tests. For quantitative tests, 16-ounce square

bottles with modified screw caps were used. The

modified screw caps were prepared by cutting

circles 2 cm in diameter from the centers of the

caps. Circular pieces of glass filter fabric (OwensCorning Fiberglas—CS. 30A-20) were inserted on the inside of the caps. The culture medium was made up as follows:

XH4NO3 3. g. K2 HP04 2. g. KH,P04 2. 5 g.

MgS04 . 7H,0 2. g. Agar 20. g. Tap water 1. liter. The culture medium was melted in the autoclave,

and 40 ml was poured into each bottle or sterile Petri dish. The Petri dishes were then left undisturbed until the agar had hardened. The bottles

with culture medium were sterilized for 20 minutes,

after which they were placed on their sides to harden

.

In preparing the inoculum, scrapings from a

ripe, fruiting culture of Chaetomium globosum

USDA 1042.4, which completely covered a 10-cm

Petri dish, were transferred to an Erlenmeyer flask containing 100 ml of sterile water by means of a

sterile tungsten loop. The black spore clusters were squeezed against the sides of the flask with

a sterile pipette until the tiny spores could be

seen to go into suspension. Specimens that had

been leached for 24 hours were sterilized for 1 hour

in the autoclave. When the sterilized specimens were cool, one specimen was placed in contact with the agar medium in each bottle under aseptic

conditions. One or two milhliters of inoculum,

depending on the size of the specimen, was then

distributed evenly over each specimen by means

of a sterile pipette. The inoculated specimens were incubated for 7 days, for the qualitative test, and for 14 days, for the quantitative test at a temperature of 28° to 30° C and a relative humidity of 90 to 95 percent. In the quantitative test the breaking-strength specimens were removed

from the bottles after 14 days of incubation, if not

completely degraded, and were gently washed to remove any growth of mildew. The specimens were air-dried and then conditioned at 70° F

and 65 percent relative humidity for 24 hours,

after which they were broken. AU averages of residual strength reported in this paper are based on 10 specimens for each test. In the qualitative test the specimens were examined for growth of Chaetomium globosum.

Any growth which could not be seen with the

unaided eye was not considered to be significant.

The average degree of growth was based on five specimens. The specimens were graded as follows: No mildew.—No visible growth of mildew.

Very slight mildew.—The smallest amount of mildew growth which can be seen with the

unaided eye—usually one or two isolated spots of growth. This is usually indicative of spotty

treatment.

Slight mildew.—Small patches of growth, which

Microbiological Deterioration 25
also indicate spotty treatment or perhaps differential leaching.

Moderate mildew.—Either many small patches of

growth or a thin uniform growth over the entire

specimen.

Heavy mildew.—Specimen is completely covered

with mildew, but the underlying fabric structure is faintly visible. Very heavy mildew.—Specimen is completely

covered with a heavy growth of mildew, and the

underlying fabric structure is no longer visible. Aseptic conditions were maintained by carrying on all work in a dust-free room with filtered air. As a further precaution, the room was equipped

with Sterilamps.

(2) Mycelial mat method.—Inasmuch as this

test cannot be used for making qualitative visual

observations because a specimen may show little or no visible growth on the upper surface and yet

show a marked decrease in breaking strength, the

test specimens were prepared for breaking-strength

determinations as described in the previous test method (page 25). It was not necessary to use

additional viability controls for this method, since

each culture bottle contained its own control. Only culture bottles that had a well-developed

3-day mycelium were used. The apparatus, cul- ture medium, and inoculum for this test were the same as described for the previous test (page 25),

except that only 16-ounce square screwcap bottles were used. The culture medium was melted in

the autoclave and was poured into 16-ounce

square screwcap bottles with modified caps. The

bottles containing agar were sterilized for 20

minutes, after which they were placed on their

sides to harden. For the source of carbon, strips

of filter paper measuring 1% by 5K inches were

sterilized in bottles for 1 hour. When cool, one

strip of sterile filter paper was transferred to the

surface of the hardened agar medium in each agar

bottle under aseptic conditions. Two milliliters of inoculum were distributed evenly over each strip of filter paper in the agar bottles by means of a

sterile pipette. The inoculated strips were then

incubated for 3 days to develop mycelial mats.

Bottles that failed to develop vigorous mats were

discarded. At this point, unsterilized, leached

test specimens were inoculated by immersing them

in a fresh inoculum in a large beaker. Excess

moisture was drained off by holding each specimen

against the side of the beaker. The specimens were then laid out smoothly over the 3 -day-old

mycelial mats and Were incubated at 28° to 30°

C and a relative humidity of 90 to 95 percent for 14 days. The data were obtained as described in the preceding quantitative test method (page

25). Figure 8 shows a specimen of wood over- grown by C. globosum in the mycelium mat test.

(b) Mildew-Resistance Tests with Aspergillus niger

(1) Mycelial mat method.—Inasmuch as breaking strength had no significance in a test in which

the test organisms were surface growers, the test specimens were cut 2 by 2 inches for visual observation. The apparatus for this test was the same

as described for the preceding tests (page 25),

except that only Petri dishes were used as incubation chambers. In order to accommodate

liquid plasticizers, Petri dishes were modified by

fitting them with Pyrex glass rings y2 inch in length by }2 inch in diameter. The rings were ground against the central surface of the Petri

dish to assure a tight, leakproof fit. The hot

sterile agar was poured around the rings, leaving

a hollow cylinder in the center. The culture

medium was the same as described for the pre- ceding tests (page 25), except that 30 grams of sucrose per liter was added. The culture medium

was sterilized in the autoclave for 20 minutes and

was allowed to cool somewhat before pouring.

About 40 ml of the hot sterile agar medium was

poured into sterile Petri dishes under aseptic conditions. The plates were then covered and left undisturbed until the agar medium had hardened.

The hardened agar medium was inoculated by

first loading a sterile cameFs-hair brush with spores

from a Petri dish culture of Aspergillus niger

USDA 215-4247 and then brushing the surface

of the sterile agar medium uniformly under aseptic

conditions. The inoculated Petri dishes were

incubated for 42 to 48 hours at 28° to 30° C

and 90- to 95-percent relative humidity until the

white mycelium was evident over the entire sur- face of the agar medium. Cultures that did not

show a vigorous growth of mycelium were dis- carded. No other viability controls were required.

At this point leached test specimens were dipped

in a fresh aqueous suspension of A. niger and were

laid firmly on the mycelial mats and incubated

at 28° to 30° C and 90- to 95-percent relative

humidity for 14 days. At the end of the incubation period the test specimens were examined for

growth of A. niger. Any growth which could not

be seen with the unaided eye was not considered

to be significant. The average degree of growth was based on five specimens. The specimens were graded as described in the qualitative procedure of the Chaetomium-direct inoculation method (page 25). Figure 9 shows the conclusion of a mycelial mat test with A. niger.

(2) Sugar-free-medium method. —The conditions

of this test were similar to those of the Aspergillus—

mycelial mat method, except that sucrose was not

added to the medium. For the viability control,

material that is known to support a growth of A.

niger was tested in the same manner as the test sample to verify the viability of the test organisms.

Test specimens were transferred to the sterile agar

plates and were inoculated by dusting them with

the spores of a Petri dish culture of A. niger.

Dusting was effected with a dry, sterile camel'shair brush. The inoculated specimens were in- cubated at 28° to 30° C and 90 to 95-percent

26 Miscellaneous Publication 188 of the National Bureau of Standards
Figure 8. \\'ood treated with inferior fungicide <showiny growth of Chaetomium globosum when tested by the mycelial mat

method.

Figure 9. Aspergillus niger test of mildew-resistant camouflage fabric. The specimen on the right had an insufficient concentration of the fungicide. The specimen on the left was adequately treated.

; Microbiological Deterioration 27
relative humidity for 14 days. The specimens were graded as described in the qualitative

procedure of the Chaetomium-direct inoculation

method (page 25).

2. Mixed-Culture Biological Test Methods

(a) Soil-Burial Test The specimens for the soil-burial test were pre- pared as described for the quantitative procedures

of the Chaetomium tests (page 25). For this test two types of control specimens were used. Untreated cotton duck was exposed in the soil beds

throughout the periods of testing in order to

verify the microbiological activity of the soil. The soil was considered to be in a satisfactory condition when the untreated controls deteriorated in

from 7 to 10 days. The reference standard consisted of cotton fabric (similar in construction to the test fabric) treated with a solution of copper

naphthenate of such concentration that 1.0-percent

plus or minus 0.05-percent of copper was retained

by the fabric. The fabric was impregnated with

copper naphtbenate with the aid of a household

wringer. The desired roll setting was first determined by using Stoddard's solvent alone. Then

strips of cloth 6 inches long in the warp direction

and the full width of the material were immersed

in copper naphthenate solution of the determined

concentration until thoroughly wet. The strips were then passed through the rolls of the household wringer and were dried with constant motion

to prevent migration of the fungicide. Before use

the treated fabrics were analyzed for copper content. The soil used in this test was taken from

the top 6 inches of a fertile topsoil known as Chester loam. The pH was 5.7, and the moisture

equivalent was 22.6-percent, based on oven-dry

weight. The clay content was 16.9-percent, and

there was 2.0-percent of organic matter present.

Stones and roots were removed by sieving before

use. The soil was placed in wooden containers

measuring 30 by 30 by 8 inches in height. The

containers were kept in a tight, insulated cabinet

in which the air temperature was maintained at 28° to 30° C, and the relative humidity was 95 to 97 percent. After moisture equilibrium had been

established, it was rarely necessary to add water

to the soil to maintain a 23-percent water content. Test strips were inserted vertically to a depth of

4 inches by means of a metal tool. Care was

taken so that not more than % inch of the lower end

of the fabric was bent over to assist burial.

Thirty strips of each test sample were buried so that 10 specimens could be removed after each of

3 periods of time. The burial periods were 3, 6, and 9 weeks. The specimens were removed from

the soil beds at the end of each burial period,

unless they were completely degraded, and were

gently washed to remove soil particles. They

were air-dried and then conditioned for 24 hours

at 70° F and 65-percent relative humidity, after which they were broken.

(b) Soil- Suspension Test Breaking-strength specimens were used as described in the quantitative Chaetomium tests

j (page 25). Untreated material similar in all other

respects to the treated material was tested in the , same manner as the test sample to verify the via-

} bility and cellulolytic activity of the test organisms.

The apparatus and culture medium for this test was the same as described for Chaetomium tests, except that only 16-ounce screwcap bottles were

used. The inoculum consisted of a thin suspension

of composted soil containing 70 g of soil to 250 ml

of distilled water. It was freshly prepared for

each test from a stockpile of soil obtained from j Miss Margaret Furry, Bureau of Human Nutrition

and Home Economics, U. S. Department of Agriculture. Agar bottles were prepared as described

in the Chaetomium tests (page 25). The test fabric j was inoculated by immersing in the inoculum for

30 minutes with occasional agitation. Each strip was then removed from the mud and placed on the

hardened agar surface in the culture bottle. The j bottles were then incubated at 28° to 30° C and 90

to 95% relative humidity for 14 days. Data were

j obtained as described in the quantitative Chaetomium tests (page 25).

3. Accelerated Weathering Test

Breaking strength specimens (page 25), in a ;

; sufficient number for subsequent biological tests, | were exposed in an artificial weathering machine

,

for 360 hours. The apparatus consisted of a ver- I tical carbon arc mounted at the center of a vertical I metal cylinder. The arc was enclosed in a clear |

globe of No. 9200 Px Pyrex glass 0.0625 inch thick. I The globe was cleaned whenever the carbons were u i changed, which was at least once in every 36 hours : of operating time. The arc was operated at 17

j amperes, with 140-volt 60-cycle alternating current. [ i The test specimens were mounted on the inside of

j the cylinder facing the arc. The distance of the ! face of the specimen from the center of the arc was I 14% inches. The cylinder rotated about the arcjIn

at a uniform speed of approximately three revolu- 1 tions per hour. A water spray hit each specimen j in turn for about 1 minute during each revolution j of the cylinder. Ten specimens were always broken I

after a 360-hour exposure to determine whether!

there had been any deleterious photochemical effect, jl If there was no significant loss in strength, the re-jl

maining exposed specimens were then subjected toll

various mildew tests.

28 Miscellaneous Publications 188 of the National Bureau of Standards
X. Experimental Results and Discussion

1. The results in table 1 indicate that cotton osnaburg is less resistant to Cftaetomium globosum

than jute burlap, although both are cellulosie materials and have approximately the same initial breaking strength. The reason for this difference

may be found in the basic structure of the two

fibers. As the cotton fiber has a lumen, the mycelial threads can penetrate inside, and deterioration proceeds from both inside and outside. Furthermore, it is possible that there is a physical rupture of the cell wall that would tend to hasten

deterioration when it is measured by loss in breaking strength. On the other hand, the jute fiber

may have a certain amount of pectin-like material

associated with it. which may offer a slight initial resistance.

As experience has shown that every visible growth of C. globosum on cotton fabric is associated

with a loss in strength corresponding to the degree

of growth, it can readily be seen from table 1 that

the 7-day results of 45 percent and 15 percent for burlap and osnaburg. respectively, are entirely

reliable when reported on the basis of visible amount of growth in the qualitative test.

2. Table 1 shows the effect of fiber structure on

the rate of growth of C. globosum and the resultant

deterioration of the fibers, and table 2 shows how

the thickness of fabrics, of presumably the same

type of cotton fibers, affects the rate at which they

are disintegrated in the soil. Logically, it might

be reasoned that inasmuch as thicker fabrics offer a smaller relative surface per unit of fabric to the

microorganism, it should take longer to degrade

the larger quantity of fabric. This would appear

to be true if one considered only the 4-ounce sheeting, the 8-ounce cotton osnaburg. and the 20-ounce

cotton duck. These are all tightly woven fabrics

and are quite comparable hi this respect. However, the 37.5-ounce duck, which, according to its thickness, should be more resistant than the 20- ounce duck, is about half as resistant as the 20- ounce duck. Here the only difference is in tight- ness of weave. The 37.5-ounce duck is a veiy

loosely woven fabric and would be more apt to absorb moisture than the more tightly woven fabTable 1. Deterioration of untreated cellulosie fabrics by

Chaetomium globosum

Incubation

period

Residual strength of

Burlap Osnaburg

Days Percent Percent 3 84 74

4 72 47

5 49 30

45 15 8 14 7 9 12

10

Table 2. Effect of fabric thickness on rate of deterioration in soil-burial test

Burial

period

Residua] strength

Lightweight

sheeting Cotton osna- burg

20-oz cotton duck37.5-oz cotton duck

Days

610

14

2128

Percent

17 Percent

1910

Percent

100 10097

27

Percent 7046

38

15

rics. It may well be that the moisture holding

capacity of cotton fabrics is of prime importance

in their microbiological degradation.

3. Table 3 shows the results of vertical and of

horizontal burial of cotton osnaburg strips. By

the seventh day the vertical strips were completely

deteriorated, whereas, by the ninth day, the

horizontal strips retained 13 percent of then- strength. Many workers concur in the opinion

that most of the damage in soil burial occurs at the soil-air line. "When a specimen is buried

vertically to a depth of 4 inches, 2 inches of the

specimen remain on the surface. Xot only does

the specimen have greater aeration at the soil-air

line, but it also receives the attention of the

different types of organisms that exist at different

levels of the sod. According to Waksman, the

top 4 inches of a soil are most active microbiologically. As to horizontal burial, the aeration of

the specimens is especially poor. Restricting a specimen to one level of the soil may be restricting

it to one type of organism. For these reasons it woidd appear to be advisable to employ vertical

burial of specimens in the soil-burial test for maximum degradation.

Table 3. Comparison of vertical and horizontal soil- burial tests

Residua] strength

Burial period

Vertical Horizontal

burial burial

Days Percent Percent

2 82 99

4 34 91 5 8 51 7 34

8 24 9 13

4. Table 4 shows the resistance to leaching of three copper compounds used for treating jute

sandbags. In order to be able to recommend

concentrations of these fungicides, which would

permit a margin of safety, this rather rigorous

test was carried out. The excellence of copper

naphthenate stands out. After 96 horns of leachMicrobiological Deterioration 29
Table 4. Effect of prolonged leaching at 70°C on mildew

resistance of treated jute fabrics

Treatment

Cuprammonium _

Copper ammonium fluoride.

Copper naphthenate _

Hours

of extrac- tion

2448

72

2448

72

2448

72

Growth of Chaetomium

globosum

None

r>o. Do.

Do.

None. Do.

Do. Moderate None

Do.

Do.

Do.

ing at 70° C, the treated jute fabric is still resistant to C. globosum. Whereas 0.8 to 1.0 percent of

copper naphthenate would be entirely satisfactory as a cellulosic fungicide, higher concentrations of

cuprammonium and copper ammonium fluoride

would be required to give the same protection.

5. Bartlett and Goll [15] investigated the possible photochemical oxidation of cellulose by

copper compounds, acting as catalysts. Experiments with fabrics impregnated with 0.6 per- cent of copper naphthenate (based on metallic

copper) gave no evidence of fabric tendering.

On the other hand, copper sulfate in a similar

concentration produced a great loss in tensile

strength. They concluded that copper naphthenate does not hasten oxidative degeneration of

cellulosic fabrics, although copper sulfate seems

to have such an effect and might be the origin

of reports about this phenomenon. Bayley and

Weatherburn [20] exposed cotton, hemp, manila,

and sisal cordage treated with 0.1 and 0.5 percent

of copper as copper naphthenate for 7 summer

months and found no increase in the rate of deterioration due to the presence of copper. In

another paper [21] they reported that untreated

No. 8 cotton duck showed substantial loss in breaking strength after outdoor exposure for 3

months. Similar fabric treated with 0.1 to 1.0 percent of copper naphthenate, copper hydroxy

naphthenate, and copper oleate showed losses of

breaking strength similar to that of the untreated

fabric. However, when measured by the cuprammonium fluidity technique there was some evidence of increased actinic degradation.

In order to gather as much information as

possible regarding the supposed photochemical

effect of copper compounds, a wide range of concentrations of copper naphthenate was used in experiment 5. In table 5, a, it can be seen that the untreated

fabric is the only one that loses all its strength

during the test. It would appear that the copper

naphthenate has served as a protective coating

rather than as an oxidative catalyst. With in- crease in copper concentration there is increased

protection. Above 1.0 percent of copper con- Table 5. Effect of copper naphthenate on cotton osnaburg in presence of light

(a) EXPOSURE TO ULTRAVIOLET LIGHT

Treatment of sample

Residual strength after

Residual copper after

50 hr 100 hr 150 hr 50 hr 100 hr 150 hr

Percent Percent Percent Percent Percent Percent Untreated - - - - 3138

10 Copper naphthenate, 0.5%. _ ._ _._ 11 5 0.31 0. 17 0.04 Copper naphthenate. 1.0% _ 42 23 7 .74 .53 .41 Copper naphthenate, 2.0% -- -- - 44 24 11 2.0 1.9 1.7 Copper naphthenate. 3.0% . -.. - 55 37 19 3.0 2.9 2.8 Copper naphthenate. 4.0%

.

_-- - --- - 72 45 29 4.0 3.9 3.7

(b) EXPOSURE ON NBS ROOF

Untreated Copper naphthenate, 0.5%___ _.. Copper naphthenate, 1.0% ,_ Copper naphthenate, 2.0% _ Copper naphthenate, 3.0% Copper naphthenate, 4.0%-_-

Residual strength after—

1 mo 2 mo 3 mo 4 mo 5 mo 6 mo 7 mo 8 mo 9 mo 10 mo

Percent

93

8184

8282

95

Percent

64

49

4849

50

61

Percent

62

38

34

30

36

44

Percent

5132

28

2532

36

Percent

49

3729

23

3037

Percent

47

34

25

2126

32

Percent

46

30

21

1724

27

Percent

38

2720

1420

26

Percent

34

22

17

12

1925

Percent

33

16

15

1016

22

Copper naphthenate, 0.5% _ Copper naphthenate, 1.0%. Copper naphthenate, 2.0% Copper naphthenate, 3.0% Copper naphthenate, 4.0%

Residual copper

0.4

.8

1.9 3.0

4.0

0.1.8

1.9 2.9 4.0

0. 06

.4

1.5 2.6

3.9

0.02

.14 1.2

2. 5 3.6

0.008

.01

.8 2.0 3.3

0.005

.01 .7 2.0 3.3

0.004

.01 .3 1.9 2.9

0. 003

.008

.3

1.5 2.8

0.003

.008

.2

1.1 2.4

0.003

.003

.098

.96

2.4

30 Miscellaneous Publication 188 of the National Bureau of Standards
' centration the loss in copper is negligible, whereas,

: at 0.5 and 1.0 percent the loss is considerable,

i However, if 0.3 percent of copper is considered

| to be the lower limit of fungicidal effectiveness,

then even after 150 hours of exposure the 1.0-

\ ^percent sample still retains 0.41 percent of copper.

The results of the roof exposures, listed in

. table 5, b, are not as clear-cut as are those for

i the ultraviolet-light-exposure samples. In the

| first month of exposure there is a loss in strength

i for all samples. However, the untreated fabric

I at 93 percent has not lost as much strength as

I the treated samples, up to the 3-percent concentration. It would seem that copper does have a

| deteriorative effect on cotton fabric. Yet, the

| 4.0-percent sample, which has more copper than

. any of the other samples, has a greater residual

. strength (95%) than the untreated fabric.

After 2 months the original fabric has dropped

; to 64-percent residual strength and the 4-percent

| copper fabric has dropped to 61 percent. The

. residual strength of the remaining copper-treated

i fabrics has dropped to 48 to 50 percent. After

3 months the untreated fabric has lost an addiI tional 2 percent in strength, whereas the 4.0-

( percent-copper sample has dropped 17 percent,

, and now is not very much better than the other

, treated fabrics. It may be, assuming that there

is a photochemical effect, that it takes longer for

I the ultraviolet rays to penetrate the 4-percentcopper sample and thus effect its degradation.

In the ensuing months the untreated fabric continues to lose strength slowly until the tenth

month, when only 33 percent of its original

strength remains. The 4-percent sample also continues to lose strength slowly and has only

22 percent of strength retained at the end of 10

months. Of the remaining concentrations, the

. 2-percent-copper sample seems to show the

greatest deterioration during each month after

I the second month. On the other hand, the 0.5-

i percent-concentration sample loses less strength

as time goes on, and by the fifth month is as strong as the 4-percent sample. From there on

until the ninth month it loses less strength than

the 4-percent sample. Again, the copper loss is proportionately less for high concentrations than

for low. The 0.5- and 1.0-percent concentrations would probably be ineffective as fungicides

after the fourth month.

From the data in table 5, a, it appears that

copper naphthenate does not increase the tendency

j to photochemical oxidation of cellulose under the

conditions of the experiment. In fact, there

appears to be a protective action with increasing

concentration. However, table 5, b, indicates

that although untreated fabric loses considerable

strength during outdoor exposure, fabric treated

| with copper napthenate loses strength to a greater

extent. Whether this increased loss is due to a photochemical effect in which the copper ion

takes part is difficult to say. It would appear

that the ultraviolet exposure, which completely

deteriorated cotton osnaburg in 150 hours, was much more severe than the 10-month roof

exposure. One possibility presents itself. The

difference in deteiioration may be due to differ- ences in moisture. Although the samples were

sprayed in the accelerated weathering test, the

spray was of short duration, and, furthermore,

copper naphthenate is known to render fabrics

slightly water repellent. This combination might

be sufficient to keep the treated samples relatively

dry, whereas the untreated samples would tend

to become wet enough to be affected photochemically. In this respect, the high degree of

copper retention, above the 0.5-percent concentration, may be considered as circumstantial

evidence pointing to the lack of leaching. On

the other hand, the outdoor samples were subjected

to alternate periods of sunshine and rain and

morning dew. This is reflected in the relatively

greater loss of copper. Furthermore, Bartlett

and Goll [15] exposed their treated fabrics to

ultraviolet light without the addition of moisture. In a 48-hour exposure test they found that copper

naphthenate (0.6% Cu) treated cotton duck

retained 75 percent of its strength, whereas the

untreated fabric retained only 69 percent of its strength. The results of the 50-hour exposures

in table 5, a, are approximately twice as severe but are in the same proportion.

6. Although most of the microbiological laboratories in the country have standardized on the

use of C. globosum, several laboratories prefer

other organisms or test methods with many

claims for superiority. For example, Great-house

and Marsh have extolled the merits of Myrothecium

verrucaria {Metarrhizium glutinosum), although

they also find merit in C. globosum as well as a group of cellulolytic Penicillia. Furry is especially partial to the soil-suspension test, for

which she makes great claims. Bertolet has

discarded all pure-culture tests in favor of a soil- burial test with horizontal burial of specimens.

Several laboratories favor the use of mixtures of

organisms either in pure-culture technique or in

mold chambers. Outstanding among these is the

"Tropical Room" at the Engineer Board, Fort

Belvoir, Va.

Table 6 shows that no single test can be used

to determine the usefulness of a fungicide under

all service conditions. Although the Aspergillus

niger test (column a) is not normally used for testing fungicidal resistance of cellulosic materials,

it was included in this series of tests to show the

effect of A. niger on copper compounds. There

is a slight growth of mildew on the 0.5-percent

concentrations of copper napthenate and copper

hydroxy naphthenate, but the higher concentrations of both compounds show no growth of

mildew, although all specimens originally green

Microbiological Deterioration 31
Table 6.

Relative effectiveness of various mildew-proofing agents on 10-oz cotton dvck as evaluated by a variety of test methods

Fungicide Growth of— Residual strength

Kind i~ oncGii- tr&tion

(a)

Aspergil-

. lus mger

(b)

Chneto- mium

d lobOS U771

(0

l nueioffllUW.- myeelial mat

(d)

Accel- erated

artificial weathering

(e)

Accelerated

artificial weathering plus

L ntie.lv- mium—

mycelial mat

(0

Pen icil Hum 66

(?)

boil sus- pension So

3 wk

(h)

il burial

6 wk

for—

y \\ K

Percent Percent Percent Percent Percent Percent Percent Percent Percent.

| 1.0 NM NM 14 98 2fi 55

< 2. XT A/T IN XVI XTA/T IN XVL 18 92 39 79

{ 3.0 NM NM 22 91 51 100

[ 0.

5

HM MM 98 10 54

•J 1.0 OlVl OlVI 9 96 1 1 63

I 1.

5

NM NM 45 90 45 79

| 1.0 NM NM 16 93 70 66 97

J 9 o NM NM iy ooy/ 71 GO 1 nn xuu /ft 1 z.o NM NM 26 90 90 94 100 61

1 0.

5

SM NM 100 43 46

J I Q NM \TAT IN 1VX 9 100 Au 4/ 51 Q Q A

1 L5 NM NM 19 100 58 64

( 1.0 NM NM 14 90 55 82 76

J o o NM NM^ 36 QQOO U

77 Q

1 3>, NM NM 96 82 68 86 100

( 0.5 MM NM 96 52 81

) 1 (1 \ 1. u SM XTA/T IN XVI 98 66 85 Q Q

I 1.5 NM NM 53 100 71 89

1 0.5 NM NM 100 100 29 100

\ 1.0 NM NM 100 93 80 100

I 1.5 NM NM 100 87 100 100

[ 0.5 NM NM 12 100 22 49 21

\ 1.0 XN XVI NM 33 100 35 78 OO

I 1.5 NM NM 83 100 69 83 39

| 0.5 NM NM 33 100 38 69 C

s 1.0 MAT IN XVi NM 65 97 67 • /jO i u Q

I 1.5 NM NM 97 96 83 94 39

1 0.5 NM NM 24 97 25 48 15

I 1.0 IN XVL NM 52 100 29 57 e;7 40 u

I 1-5 NM NM 89 100 48 85 91 37

1 1.0 NM NM 30 96 39 87 96 17

\ 2. NM NM 36 94 c 45 98 100 44

[ 3.0 NM NM 45 88 57 98 100 91 12

I 0.5 NM NM 24 100 35 82 55 15

< 1.0 NM NM 29 100 36 csy 40 A

I 1.5 NM NM 33 100 41 100 91 37 41

1 0.5 NM NM 100 96 56 100 36 9

\ 1. NM NM 100 85 100 83 46 38

I 1.5 NM NM 100 78 100 100 96 63 46

| 0.5 MM NM 100 100 87 97 71 19

< 1.0 SM NM 100 100 100 100 OO 47

I 1.5 NM NM 100 100 100 100 98 63

I 1.0 HM HM 93 8 58

< 2. HM HM 90 15 64 Q

I 3.0 HM HM 32 84 27 73

1 0.5 NM NM 23 79 53 76 100 55 10

\ 1.0 NM NM 57 78 59 88 100 93 59

1 1.5 NM NM 76 72 61 100 100 100 97

1 0.5 NM NM 48 94 23 72 86 22

\ 1.0 ,vT A/T iN XVI IN XVI 51 95 28 76 86 40

I 1.5 NM NM 69 95 30 85 87 37

I 0.5 NM HM 98 100 63

\ 1.0 NM HM 94 100 74 28

{ 1.5 NM HM 98 100 78 36

| 0.5 NM NM 58 98 50 79 21

\ 1.0 NM NM 68 96 9 59 80 49

I 1.5 NM NM 90 100 24 63 89 97

I 0.5 NM NM 48 100 67 69 8

< 1.0 NM NM 76 100 74 81 44

I 1.5 NM NM 99 100 23 92 96 76

o-Phenylphenol

(/-Phenylphenol. sodium salt,

2. 4, 5-Trichlorophenol

2,4, 5-Trichlorophenol, sodium salt

Chloro-2-phenylphenol

Chloro-2-phenylphenol, sodium salt-. --

2-Chloro-4-phenylphenol

2-Chloro-4-phenylphenol, sodium salt

2-Bromo-4-pheny]phenol

2-Bromo-4-pheiiylphenol, sodium salt

2, 3, 4, 6-Tetraehlorophenol

2, 3, 4, 6-Tetrachlorophenol, sodium salt

Pentachlorophenol

Pentachlorophenol, sodium salt

2, 4, 6-Trichlorophenol

2,2' Dihydroxy, 5,5' Dichloiodiphenylmethane

Salicylanilide

Paranitrophcnol.

Phenyl mercuric acetate.

Phenyl mercuric salicylate.

32 Miscellaneous Publication 188 of the National Bureau of Standards
Table 6. Relative effectiveness of various ?nildew-proofing agents on 10-oz cotton duck as evaluated by a variety of test methods—

Continued

Fungicide Growth of

Residual strength

Kind

Phenyl mercuric hydroxide.

Phcnyl mercuric oleate

.

Phenyl mercuric naphthenate.

Phenyl mercuric acetoxv octadecanoic acid.

Phenyl mercuric triethanolamine lactate.

Pyridyl mercuric chloride.

Pyrid;. 1 mercuric stearate.

Pyridyl mercuric acetate

.

Mercaptobenzothiazole

.

2.3-Dichloronaphthoquinone.

Ziuc naphthenate.

Zine dimethyldithiocarbamate..

Copper naphthenate.

Copper hydroxynaphthenate.

Copper oleate

Untreated

Concentration

(a)

Aspergillus

Percent

0.5

1.0 1.5

0.5

1.0 1.5

0.5

1.0

1.5

1.0 2.0

3.0

1.0 2.0

3.0

n. 5 1.0 1.5

0. 5 1.0

1. 5

1.5 1.0 1.5

0.5

1.0 1.5

0. 5 1.0

1.5

1.0

1. 5 2.0

0. 5

1. 5 2.0

0.5

1.0 1.5

0.5

1.0

1.5

0.5

1.0

1.5

NMNM

NM

NM

NMNM

NM

NM

NM

NM

NM

NMNM

NM

NM

NM

NMNM

NM

NM

NM

NMNMNM

MMMM

NM

NMNM

NM

NM

SMNM

NM

SMNM

HMMMMM

VHM

(b)

Chaeto- mium

jlobosum

(0

Chaeto- miummycelial mat

NM

NM

NM

NMNM

NM

NM

NM

NM

NMNM

NM

NM

NM

NM

NM

NM

NM

NM

NMNM

NM

NM

NM

NM

NM

NM

NMNM

NM

NM

NM

NM

NM

NM

NM

NM

NM

VHM

Percent

64

66

9

22

100

100

100

95

100

(d)

Accelerated

artificial weathering

Percent

100 100

1C0

100

100 100

10094

95

100

10097

97

100

100

100

100

100

100

10097

97

100

100

10099

100

100 100

100

100

100

100

100

(e)

Accel- erated

artificial weathering plus

Choeto- miummycelial mat

Percent

30

1018

30

20

4342

(f)

Penicil- liun 66

Percent

38

5366

52

70

(g)

Soil sus- pension

Percent

7282

86

89

94

6267

76

96

96

100

98

9392

100

9194

99

Soil burial for3 wk 6 wk 9 wk

Percent

23

62

7075

100

88

90

29

3436

82

100

100

79

91 100

40

100

100

13

41

98

97

100

78

9499

5170

Percent

were decolorized, indicating a loss of copper.

The presence of the naphthenic acid conferred

a degree of mildew resistance upon all the specimens, including the 0.5-percent concentration.

The copper oleate specimens, which were also decolorized, showed little resistance to A. niger.

The loss of copper left only the oleic acid radical,

which has no mildew-resistant power. Other

compounds that showed little resistance to

A. niger were 2,3-dichloronaphthoquinone,

2,4,6-trichlorophenol, lower concentrations of pentachlorophenol, and the sodium salts of chloro-2-phenylphenol, 2,4,5-trichlorophenol, and

orthophenylphenol.

The Chaetomium-dhcct inoculation-sterilized

specimen-qualitative test (column b) was first used in studying the mechanism of the microbiological deterioration of cotton. Later it was

Microbiological Deterioration 33
used as a relatively rapid means for determining

the thoroughness with which acceptable fungicides

had been applied to cellulosic fabrics. With this

method the National Bureau of Standards was

able to test the fabric for 500 million sandbags

during the first year of the recent war. This test was also useful in screening unknown fungicides.

If a fabric, treated with an unknown compound,

failed to pass this test, no further tests were

necessary. In this investigation it is not surprising that C. globosum failed to grow on so many of

the compounds. In the first place, the compounds

were selected because of their excellence for some

particular purpose, and, secondly, the concentrations are close to the upper limit of usefulness.

Treatments that do not resist the growth of C. globosum in this test are: sodium salts of

orthophenylphenol, 2,4,6-trichlorophenol, and

paranitrophenol. Inasmuch as these compounds

are rather unstable at high temperatures, it is quite likely that the use of steam sterilization is objectionable.

The Chaetomium-mjcelial mat test represents

the work of several laboratories. It was pioneered

by Greathouse and Marsh and was further

developed in the laboratories at the National

Bureau of Standards. The results in column (c) show that very few compounds can resist the

growth of Chaetomium globosum under the conditions of this test. A decided advantage of this

test is that treated samples need not be subjected

to steam sterilization—a condition not encountered

in service. Treatments that resist C. globosum

in this test are 3.0 percent of chloro-2-phenylphenol, 0.5 percent of 2-chloro-4-phenylphenol

and its sodium salt, 1.5 percent of 2-bromo-4-

phenyl-phenol and its sodium salt, 0.5 percent of pentachlorophenol and its sodium salt, 1.5 percent

of phenyl mercuric acetate, 1.5 percent of phenyl

mercuric salicylate, 1.5 percent of phenyl mercuric

hydroxide, 1.0 percent of phenyl mercuric oleate,

1.0 percent of phenyl mercuric naphthenate,

0.5 percent of pyridyl mercuric chloride, 0.5 percent of pyridyl mercuric stearate, 1.0 percent

of pyridyl mercuric acetate, 2 percent of zinc

naphthenate, 1.5 percent of zinc dimethyl-dithio—

carbamate, 0.5 percent of copper naphthenate,

0.5 percent of copper hydroxy naphthenate, , and

0.5 percent of copper oleate. With the exception

of the copper and mercury compounds, the concentrations of these compounds are somewhat

higher than those customarily used.

Experience has shown that certain compounds

promote deterioration of cellulose. Regardless of

how they achieve their destructive ends, it is desirable to know whether a compound will affect a cellulosic fabric adversely even before it has the

opportunity to protect the fabric from the

ravages of the microorganisms. Such information

could be obtained by lengthy outdoor exposures,

but the accelerated weathering test is much

faster. It must be emphasized that no claim is made as to the correlation of accelerated laboratory exposure with actual field exposure. There

are many factors involved in outdoor exposure

that are not yet fully understood. For example,

copper naphthenate-treated fabrics actually show

no loss of strength in the usual 360-hour exposure

in the accelerated weathering test, whereas

untreated fabrics do show a loss in strength. Yet

the reverse is true in outdoor exposure, although

here the untreated fabric also shows a loss in

strength. At any rate, the accelerated weathering test has been useful in eliminating fungicides

that break down in the presence of light and either

liberate harmful products or are inactivated. In

either case, the fabric is no longer protected.

Column (d) shows that the chlorinated phenols

tend to weaken the fabric as the concentration is increased. This effect is serious only in the case

of chloro-2-phenylphenol, pentachlorophenol, 2,4,-

6-trichlorophenol and dihydroxydichlorodiphenylmethane. However, a breaking-strength test alone does not indicate whether any of these

compounds has retained its fungicidal potency, even though the residual strength may be 100

percent.

In column (e) are listed the results of a Chaeifommm-mycelial mat test, following 360 hours of

accelerated weathering. The results are quite

striking. Very few compounds are shown to possess the permanence necessary for the most

rigorous service conditions. These superior compounds include pyridyl mercuric chloride, pyridyl

mercuric stearate, zinc dimethyl dithiocarbamate,

copper naphthenate, and copper oleate. As a comparison of the effectiveness of C. globosum in the mat method another cellulolytic

organism was chosen. Penicillium 66 was recom- mended by Dr. Paul Marsh, United States Department of Agriculture, Beltsville, Md. The

results of tests with this organism are listed in

column (f) Although it is a competent cellulolytic

organism, it does not appear to be as tolerant of

fungicides in general as C. globosum, although there

are exceptions. Some of these are: 2-chloro-2-

phenyl phenol and pentachlorophenol which are

tolerated by Penicillium 66 at 0.5-percent concentration, whereas C. globosum is completely in- hibited. Salicylanilide is better tolerated, to a

slight extent, by Penicillium 66 than C. globosum.

On the other hand, paranitrophenol completely

inhibits Penicillium 66, whereas C. globosum is not

affected by it at all. Phenyl mercuric acetoxy

octadecanoic acid is vulnerable to C. globosum,

although Penicillium 66 is fairly well inhibited by

it. All pyridyl mercuric compounds are fairly

easily attacked by Penicillium 66, although they

are quite resistant to G. globosum.

It is obvious, then, that fungicides are effective

only with respect to the test organisms used.

Unfortunately, one cannot specify the type of or34 Miscellaneous Publication 188 of the National Bureau of Standards
ganisms that shall attack a particular treated

fabric in the field. Furthermore, it would be impossible to foresee all contingencies by attempting

to use a large variety of test organisms. In this dilemma, the solution has been the soil-burial test, which is the most potent microbiological test in use at the present time. In column (h) are listed

the results of the 3-, 6-, and 9-week exposures in

the soil beds. The 6-week results show that great

reliance may be placed on the Chaetomiummycelial mat test, particularly after accelerated

weathering. Two exceptions to this are pentachlorophenol and dihydroxydichlorodiphenylmethane,

which are excellent in the soil-burial test. In an effort to secure the obvious advantages of

the many different types of organisms existing in

composted soil, Furry developed the soil inoculum

test. Unfortunately, the standardization of a

test of tins type entails the control of a great

number of variables. For example, the soil used

in this test was obtained from Miss Furry and had

been stored for several months before use, with

occasional watering to keep it from drying out.

Strictly speaking, the soil should have been kept

at standai'd conditions until needed for use in the

test. However, it was not practical to do so. This lack of standardization in handling of the soil bank may account for the variation in test results

encountered from laboratory to laboratory. The

results of the soil-suspension test in this investigation appear in column (g).

Although this investigation was primarily con- cerned with the evaluation of the most widely used

test methods, the use of such a large number and

variety of fungicides provides an opportunity for

evaluating these fungicides. For want of a better

term, "fungicide," as used here, refers to all mildew-resistant compounds, regardless of their

mode of action.

It is quite obvious that, in regard to any particular fungicide, resistance toward any particular

test organism does not necessarily imply resistance,

to all organisms. Furthermore, a compound may

be an effective fungicide under certain conditions

but may lose its potency when subjected to the

physical agents of weathering. Thus the chlorinated phenols, which are powerful fungicides, are

limited in their use because of their high degree of

solubility and because of their photochemical instability. With increasing concentration there is a tendency for these compounds to cause deterioration of the fabric during accelerated weathering.

This effect is most pronouuced in the case of

pentachlorophenol, which shows a strength loss of

22 percent for the 1.5 percent concentration.

Dihydroxydicliloi'odiphenylmethane, although

not a member of the Dowicide series of chlorinated

phenols, is also a chlorinated phenol. Its photochemical effect on the fabric is even more severe than that of petachlorophenol. At 1.5-percent

concentration, 28 percent of the fabric strength

Microbiological Deterioration

was lost. However, unlike the other chlorinated

phenols, this compound appears to be exceptionally resistant to soil burial, particularly at the

1.5-percent concentration.

Salicylanilide, which has been widely used under

the trade name of Shirlan, is shown to be readily

inactivated by accelerated weathering. Furthermore, its performance in the soil-burial test in- dicates either excessive solubility or the presence

of organisms that hydrolyze the compound to

salicylic acid and aniline. Although paranitrophenol appears to be unusually resistant to PeniciUium 66 at all concentrations, it is seen to be ineffective in all the other

biological tests except the A. niger test. Although

this compound appears to be of little value as a

cotton-fabric fungicide, we shall see later (table 8)

how it is effective as a leather fungicide.

The mercury derivatives have been demonstrated to be rather powerful fungicides. Although

PeniciUium 66 appears to tolerate these fungicides

to a slightly greater degree than C. globosum, the

difference does not seem to be significant. Furthermore, phenyl mercuric acetoxyoctadecanoic

acid and phenyl mercuric triethanolamine lactate are tolerated to a greater extent by C. globosum

than by PeniciUium 66. On the other hand, the

excellent pyridyl mercuric compounds show only

moderate resistance to PeniciUium 66, whereas C. globosum is almost completely inhibited by them.

Pyridyl mercuric acetate appears to be the most

soluble of the pyridyl mercuric group.

Mercaptobenzothiazole and 2,3-dichlonmapthoquinone are seen to be more or less ineffective

against all organisms used. Inasmuch as these

two compounds are known to be powerful fungicides, their ineffectiveness on fabrics is probably

due to excessive solubility.

The two zinc compounds tested are seen to be

moderately good fungicides. The protection, at higher concentrations, approaches the ideal of 90-

percent residual strength. A tendency toward sol- ubility is indicated by the results of the mildew

test after accelerated weathering. These compounds, although not as effective as the copper

compounds, can be recommended where the use of copper is objectionable, as, for instance, in rubber.

Of the three copper compounds tested, copper

naphthenate is by far the superior fungicide. It

is interesting to note that all of these compounds

permitted a growth of A. niger to some extent. Where there was no growth, as in the higher concentrations of copper naphthenate and copper hydroxy naphthenate, the specimens were decolonized by the growth products in the mycelial mats.

This phenomenon offers a clue as to the superiority

of copper naphthenate and copper hydroxy naphthenate over copper oleate. The loss of color due

to the action of the end products of A. niger may

be simulated by immersing a test specimen in

0.4-percent solution of oxalic acid. Obviously,

35
the copper is removed. In the case of the naphthenate, the naphthenic acid that remains in the

fabric has a degree of fungicidal potency and resists the growth of the microorganisms. However,

the oleic acid residue of copper oleate is a wellrecognized source of food for microorganisms.

7. Inasmuch as vegetable-tanned leather was

found to support a vigorous growth of A. niger

in the absence of the sucrose and minerals of a

culture medium, it became desirable to determine

the constituent of the leather that was promoting

this growth. Table 7 shows that when degreased

leather is subjected to the A. niger test, the growth

is slight before leaching and very slight after leaching. This would indicate that the slight degree

of growth is stimulated by the soluble materials

in the leather, such as minerals and glucosides.

When fatty oil is added to the leather, the growth

both before and alter leaching is heavy. When

fatty oil and 1 percent of salicylanilide are added

to the leather, there is no growth of A. niger,

indicating that this fungicide at high concentration can inhibit the growth of A. niger when it obtains

its nourishment from fatty oil. When leather

grease was added to degreased leather, the growth

of A. niger was very heavy both before and after

leaching. Finally, when fatty oil, grease and 1 percent of salicylanilide were added to degreased

leather, the salicylanilide was no longer able to completely inhibit the growth of A. niger. Apparently, the leather grease is the material that

promotes the vigorous growth of A. niger. This

corroborates previous work by Kanagy, et al. [90], in which it was shown that when vegetabletanned leather was exposed to various microbiological tests the greatest damage to the leather was caused by stiffness due to a loss of grease.

Table 7. Effect of constituents of vegetable-tanned leather on growth of Aspergillus niger

Materials added to degreased vegetable-tanned leather

Growth of Aspergillus niger

Before leaching Leached 48 hr

None. SIight_ Very slight. Heavy. Very heavy.

Slight. None.

Fatty oil .._ Heavy

Leather grease Fatty oil plus grease plus Shirlan Fatty oil plus Shirlan

Very heavy. .

.

Slight None

8. As vegetable-tanned leathers are so susceptible to attack by molds, only the most powerful

fungicides will give protection. However, what

may be an effective fungicide for fabric may not be

at all effective for leather. Furthermore, many

fungicides cannot be used on leather because of

their undesirable effects. For example, copper

naphthenate cannot be used because it darkens

leather. Mercury compounds cannot be used on

account of their toxicity. Table 8 shows the re- sults of the A. niger test on the more effective

fungicides. An interesting compound is trimethyl

cetyl ammonium pentachlorophenate (Hyamine

3258), which is said to be substantive to cellulose. However, it offers no protection to leather. Similarly, dihydroxydichlorodiphenylmethane is an

excellent fabric fungicide, except in the presence of

light, yet it has little effect as a leather preservative. Paranitrophenol and pentachlorophenol are

of especial interest. Paranitrophenol was found to be effective at 0.35 percent and pentachlorophenol

at 0.25 percent. However, both of these concentrations are too high from the point of view of

toxicity to human beings. By combining these two

fungicides in equal parts it is found that as low a combined concentration as 0.20 percent will effec- tively control the growth of A. niger. At this concentration the compound may be used for leather

Table 8.Relative effectiveness of various mildew-proofing

agents on vegetable-tanned leather as evaluated by the Aspergillus niger test

Fungicide

8-H yd rosyquinoline

Mercaptobenzothiazole

Tetrabrom-ortho-cresol.

Beta-naphthoL

Trimethyl cetyl ammonium penta- chlorophenate.

Anilino methyl mercaptobenzothiazole.

2 , 2' Dihydroxy -5,5' dichlorodiphenyl methane.

Paranitrophenol.

Pentachlorophenol.

Paranitrophenol and pentachloro- phenol.

Salicylanilide.

Concentration on

leather

Percent

0. 22

.48

.72

1. 06

0.34

.52 .58

1.07

0. 21 .26

.51 .83

.24

.55

.82 1.38

0. 29 .45 .57

. 77

.28

.45

.76

1.01

0. 23

.39

.62

1.03

0.30

.57

.65

1. 01

0. 25

.59

.89

1. 16

0.11 .20

.26 .37

.26

.41 .73

1.24

Growth of Aspergillus niger

Before leaching

Slight Moderate. None. Slight. do None. do Do.

Moderate... Very heavy. do Heavy.

do Do. do Moderate. do Very heavy. do Do.

do Heavy. do Moderate. do Heavy.

None.. _ ... Moderate.

do. _ None. do Do. Moderate... Heavy.

do Do. do Moderate.

Slight Slight. Moderate... Very heavy.

Slight Heavy.

do Moderate. do Do.

Moderate. __ Very heavy. do Heavy.

do Do.

do.. Moderate. None Do. do Do. do None. do Do. do

do

do.. do

Slight. None

dodo Very heavy. Heavy

do

Moderate. ..

Leached 48

hr.

Slight. None.Do. Do.

Moderate. None.Do.

Do. Very heavy

Do.

.Do. Do.

30 Miscellaneous Publication 188 of the National Bureau of Standards
articles that come in contact with the human body.

This fungicidal combination, developed at the

National Bureau of Standards, is the basis of a mixture for use on leathers and was supplied to the

soldiers in the tropical regions during the latter part of the recent war. It was found to be highly

satisfactory.

9. Table 9, a, shows the susceptibility of 15 unplasticized polymers to Aspergillus niger. The

results of the test with the complete medium

(mycelial mat method) indicate that only urea- formaldehyde has any fungicidal power. The

remaining plastics all permit a growth of ^4. nigt r

. over them, varying from slight to heavy. This

. test alone would not suffice to tell whether these

materials actually supported a growth of the

organism. To be sure, a material, such as urea- formaldehyde, which gives off formaldehyde,

. manifests its fungicidal character in this test. The results of the tests with sugar-free medium,

however, show that only cellulose nitrate, polyvinyl acetate, phenol-formaldehyde, and melamine-formaldehyde are sources of food for A. n iger.

Although none of the remaining polymers sup-

. ports a growth of mildew, it does not prevent the

growth of mildew. These tests indicate that

when these materials are accompanied by substances that promote the growth of micro-organisms, no inhibition will take place because of the

polymeric material itself, except with urea—for- maldehyde. Presumably when commercial plastic

materials support a growth of mildew, components

other than the pure polymer are responsible for it. Table 9, b, which lists the susceptibility of 83

plasticizers to ^1. niger, shows the prime source of mold susceptibility of plastic materials. Although

many of the plasticizers show no growth of mildew

in the sugar-free test, indicating that they are

l not sources of mold nutrition, all of them show

some degree of permissive growth when sugar is added to the medium. Of the plasticizers that

support a growth of mildew on sugar-free agar, the worst offenders include: glycol sebacate resin,

sebacic acid alkyl resins, castor oil and ricinoleate

derivatives, tung oil, glyceryl triacetate, and

pentaerythritol triacetate monopropionate. It is interesting to note that of the 10 pentaerythritol

derivatives tested, three showed no growth, five showed slight growth, one showed moderate

growth, and one showed heavy growth. In like manner it is difficult to correlate the results of the

remaining compounds on the basis of chemical

structure, particularly since, in general, there is less similarity in structure among the other compounds than among the pentaerythritols. However, certain trends are evident. As a group the

phthalates may be said to offer no source of nutriment for the development of A. niger with several

exceptions. Ricinoleates invariably support copi- ous growths of this organism as do the sebacates

and stearates.

Table 9. Mold susceptibility of pure plastics and

plasticizers

a. PURE PLASTICS

Pure polymer

Cellulose acetate.

Cellulose acetate butyrate.. - Cellulose acetate propionate.

Cellulose nitrate Ethyl cellulose Polyethylene.—

Polymethylmethacrylate.

Polystyrene Polyvinyl acetate Polyvinyl chloride Polyvinyl acetate-chloride VYNW.

Polyvinyl acetate-chloride VYHH.

Phenol-formaldehyde Melamine-formaldehyde

Urea-formaldehyde

Growth of Aspergillus niger

Complete

medium

Slieht. do. Moderate. do

do

Slight ....do Moderate. Heavy

Moderate .

Slight Moderate.

do Heavy None

b. PLASTICIZERS

Hydrogenated methyl abietate. 7>/-n-butyl aconitate Triethyl aconitate Ethyl-o-benzoyl benzoate Benzyl benzoate

Tri-n butyl citrate Triethyl citrate Glyceryl triacetate Diethylene glycol ethyl ether acetate Diacetate of 2-nitro-2-methyl-l,3-propanediol.

Dipropionate of 2-nitro-2-methvl-l,3-propanediol. Diethylene glycol dipropionate Triethylene glycol di-(2-ethylhexoate) Triethylene glycol di-(2-ethyIbutyrate) Polyethylene glycol di-(2-ethylhexoate) Ethyl phthalyl ethyl glycolate Methyl phthalyl ethyl glycolate Butyl phthalyl butyl glycolate Ethylene slycol methyl ether oleate. Dipentaerythritol hexaacetate

Dipentaerythritol hexapropionate

Dipentaerythritol hexabutyrate

Pentaerythritol diaeetate-dibutyrate Pentaerythritol diacetate-dipropionate Pentaerythritol monoacetate-tripropionate.

Pentaerythritol triacetate monopropionate

Pentaerythritol tripropionate monomyristate.

Pentaerythritol tetrabutyrate

Pentaerythritol tetrapropionate Triethyl phosphate

Tributyl phosphate

Triphenyl phosphate.

Tricresyl phosphate . Tri-(2-nitro-2-methylpropyl) phosphate Monophenyl rf/-(p-tert.-butylphenyl) phos- phate .-- Diphenyl mono-o-xenyl phosphate...

-Df'-o-xenyl monophenyl phosphate. .. Tn'-(p-tert. butylphenyl) phosphate.

Tri-(o-xenyl) phosphate Dimethyl phthalate

Diethyl phthalate

Z>i- n-propyl phthalate. _ -Di-isopropvl phthalate . Dibutyl phthalate

.Di-isobutyl phthalate.

Diamyl phthalate. Dicapryl phthalate

Dioctvl phthalate

Z>;'-(2-ethylhexyl) phthalate. Dicyclohexyl phthalate

Moderate. do

do.,

do.. HeavyModerate. . Heavy

do

Moderate.

do Heavy

Moderate. Heavv

do —

_

do.--_

Slight Moderate. do

.do- Heavy. ... Moderate. do

do

do Heavy

do

do Moderate . Heavy

Moderate .

Slight. Moderate- do

.do.

-do- Heavy

Moderate. Heavy

Moderate

do

do Heavy

Moderate . do

Slight. Moderate. do

do Heavy

do

Sugar-free medium

None.

Do. Do.

Moderate. None.

Do.

Do.

Do.

Moderate, None.

Do.

Do.

Slight. Moderate. None.

None.

Slight. Do.

None.

Moderate. None.

Do. Heavy.

Moderate. None.

Do.

Do.

Do. Do. Do.

Slight. None.

Moderate. Do. None.

Do.

Do.

Slight. Do. Do.

Heavy. Moderate

Slight. Do. None.

Do.

Slight. None.

Moderate.

Slight. Do.

None.

Do.

Do. Do.

Do.

Do.

Do.

Do.

Do. Do.

slight. None.

Do. Do.

.1/ icrobiological Deterioration 37
Table 9. Mold susceptibility of pure plastics and

plasticizers—Continued

b. PLASTICIZERS—Continued

Dibenzyl phthalate Dimetho.xy ethyl phthalate Diethoxy ethyl phthalate Dibutoxy ethyl phthalate Methyl-2-methyl-2-nitropropyl phthalate..

Ethyl-2-methyl-2-nitropropyl phthalate Butyl-2-methyl-2-nitropropyl phthalate

Bis-(diethylene glycol ethyl ether) phthalate. Glyool sebacate resin Sebacic acid alkyd resin-G25 _.

Sebacic acid alkyd resin-RG-2___

Sebacic acid alkyd resin RG-20 ,_. Butyl acetyl ricinoleate ... Ethylene glycol methyl ether acetyl ricinoleate. Dimethyl sebacate _ Dibutyl sebacate «-Butyl stearate Cyclohexyl stearate Butoxy ethyl stearate. Z>!-«-butyl tartrate

Ethyl-p-toluenesulfonate

o-Cresyl-p-toluenesulfonate...

o- and p-Toluene ethylsulfonamide.

Triethyl tricarballylate Tri-7i-butyl tricarballylate.-.

Castor oil Tung oil Diphenyl

Diamylnaphthalene.

Cyclohexyl lactate. _. Methylcyclohexyl oxalate. Diphenylsulfone Triphenylguanidine

Growth of Aspergillus niger

Complete

medium

Moderate. _. do

Slight Moderate... Heavy

do

Moderate... do Very heavy

.

do _. do

do

do

do.... Moderate . Heavy

do

do

Moderate.

dodo

Slight Moderate . Slight Heavy

Very heavy. Heavy

Slightdo

Moderate...

Slight Moderate.

Slight

Sugar-free medium

Do. Do.

Do.

Do.

Do.

Slight. None.Do.

Very heavy. Do.

Do. Do.

Do. Heavy.

Moderate. Do. Do.

Do. Do.

None.Do.

Do.

Do.

Slight. Do. Heavy.

Do.

None.

Do. Do.

Do. Do.

Do.

10. As a result of the investigation of the mold

susceptibility of plastics and plasticizers, other

materials that had formerly been considered to be

inert, as far as mold nutrition was concerned, were

investigated in the same manner. The results are recorded in table 10. It is not at all surprising to find that natural crepe rubber supports mold

growth. The literature on mildewed crepe rubber

is extensive. However, of the synthetic rubbers

both Buna S and butyl rubber support a growth of A. niger. Neoprene, on the other hand, does not; support a growth of mildew, although it does not

inhibit growth when sugar is added to the culture! medium.

Table 10. Mold susceptibility of various materials

Material

Natural crepe rubber. Buna S_ ._. Butyl (isoprene) Neoprene -- Nylon fabric Glass fabric _ Mineral wool

Growth of Aspergillus Niger

Complete

medium

Very heavy- Heavy

do - do.

do.

do. None..

Sugar-free medium

Heavy.

Moderate. Do.

None.

Do. Do.

Do.

By making use of the information in table 9, a and b, it has been possible to compound a plastic

material that does not support a growth of mildew.

For example, polyvinyl acetate-chloride copolymer

has been successfully plasticized with tricresyl

phosphate as a result of this investigation. Previously, the most common plasticizers had been the

ricinoleates.

Nylon fabric, glass fabric, and mineral wool all offer no nourishment to A. niger. However,

mineral wool is the only one that resists the growth

of mildew even when sugar is added.

As a result of this investigation it became

possible to recommend a material for use in fabricating mildew-resistant portable water tanks.

Canvas tanks had formerly been used, but because

of their susceptibility to rot it became necessary

to develop mildew-resistant treatments for them.

Unfortunately, none of the superior fungicides

could be used for this purpose because of the

possible contamination of drinking water. Consequently, the value of a material such as neo- prene-coated nylon can be readily seen.

XI. Summary and Conclusions

A series of 10 investigations is reported in which

the basic problems of the testing of mildewresistant treatments for organic materials are

covered.

1 . It has been shown that cellulosic fibers of

different origins have varying resistance-to microorganisms. Cotton fibers are more easily deteriorated than jute fibers.

2. Fabrics of the same cellulosic fibers, which

differ as to thickness and tightness of weave, also

vary in their resistance to microbiological attack.

In general, thicker fabrics tend to resist deterioration for longer periods, unless they are loosely woven.

3. It has been shown that the results in the

soil-burial test can be affected by the manner in which specimens are buried. Vertically buried

specimens appear to offer greater opportunities

for microbiological attack than horizontally buried

specimens.

4. The excellence of copper naphthenate as compared to two other copper compounds is demonstrated by its resistance to leaching for 96-hour at 70° C.

5. Copper naphthenate appears to protect

cellulosic fabrics from photochemical degradation

to a slight extent when exposed to strong ultra- violet rays, but the situation is reversed when the

38 Miscellaneous Publication 188 of the National Bureau of Standards
same samples are exposed outdoors for as long

as 10 months. It appears that in the presence

of moisture and light, as in the outdoor exposure,

there is a slight tendency for copper naphthenate

to reduce the breaking strength of cellulosic

j fabrics. In the absence of large quantities of moisture, as in the ultraviolet exposure, copper

naphthenate tends to retard the degradation of

| cellulosic fabrics.

6. For the purpose of evaluating cotton fabrics

treated with mildew-resistant compounds, eight

test methods have been studied. No single test

offers a complete picture of the performance of a

particular treatment under all service conditions.

The tests that stand out as most useful are Chaetomium-Mjcelial mat test; Accelerated weathering test, followed by a subsequent Chaetomium

test; and the soil-burial test. Where the particular conditions of service are known, it may be

sufficient to select a single test. But where

a mildew-resistant treatment is required to protect

under a wide variety of conditions, all three of

the above tests should be used.

In evaluating these test methods, 35 of the

most commonly used fungicides were used.

The superiority of the copper compounds, especially copper naphthenate, has been demonstrated.

Although not as effective, zinc naphthenate and

zinc dimethyldithiocarbamate have been shown to be acceptable substitutes where copper compounds

cannot be used. The chlorinated phenols have

been shown to be excessively soluble and rather

photochemically unstable. However, in the absence of light, pentachlorophenol and dihydroxydichlorodiphenylmethane would be excellent fabric

fungicides. The pyridyl mercuric compounds

appear to be the best of the mercury fungicides

although the acetate is probably too soluble for

general use. Finally, it is obvious that no single

compound may be considered the universal fungicide. Even copper naphthenate, which withstands extemely rigorous exposure, is not the

ideal treatment. However, with a knowledge of the end use of the treated fabric, it is possible to

select an effective fungicide from these 35

compounds.

7. The material that imparts to vegetabletanned leather its great susceptibility to mold

growth appears to be the grease commonly used

in leather manufacture.

8. Paranitrophenol and pentachlorophenol have

been demonstrated to be effective fungicides for

vegetable-tanned leather. Inasmuch as the effec- tive concentrations of these compounds are too

high, for practical reasons, a compound containing

equal parts of these two fungicides has been

developed in which the combination is effective at as low a concentration as 0.20 percent.

9. Fifteen unplasticized polymers and 83 plasticizers have been investigated for resistance to Aspergillus niger. Of all the materials tested only

ureaformaldehyde showed a resistance to the

growth of A. niger on a complete medium. On

sugar-free medium, 4 polymers and 37 plasticizers were shown to support a growth of A. niger.

The relatively large proportion of mold-susceptible

plasticizers indicates the need for careful selection

of plasticizers in compounding a plastic material.

This data can be used in the selection of resistant

materials for various plastic formulations.

10. A group of seven miscellaneous materials

has been investigated for resistance to A. niger.

Of all the materials tested only mineral wool

resisted the growth of A. niger on a complete

medium. On sugar-free medium, natural crepe

rubber, Buna S and butyl rubber manifested the

ability to support a growth of A. niger. Neoprene,

nylon fabric, glass fabric, and mineral wool failed to support the growth of the organism.

XII. References

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[2] Armand, L., Tiba 12, 675 (1934).

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[5] —, —, Color Trade J. 13, 72 (1923).

[6] Armstrong, E. F., Dver & Textile Printer 86, 207

(1941).

[7] —, Chemistrv & Industry 60, 668 (1941).

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[9] Artari, A., Jahrb. wiss Bot, 46, 443 (1910).

[10] Ayres, G. B., and Tobie, W. C, J. Bact. 45, 18 (1943).

[11] Azevado, P. da R., and Maffei, F. J., Anais assoc. quim. Brasil 3, 120 (1944).

[12] Barker, S. C, Chemistrv & Industry 57, 1222 (1938).

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[16] Batson, D. M., Teunisson, D. J., and Porges, N., Am. Dyestuff Reptr. 33, 449 (1944).

[17] Bayley, C. H., and Weatherburn, M. W., Cua

Textile J. 62, 34 (1945).

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[21] —, —, Can. J. Research 24F 193 (1946).

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[23] Bertolet, E. C, Am. Dyestuff Reptr. 32, 214 (1943).

[24] Binns, C., Proc. Tech. Sect. Paper Makers' Assn.

Gt. Brit. & Ireland 25, 280 (1944).

[25] Borghetty, H. C, Rayon Textile Monthly 26, 479

(1945) . [26] Borlaug, N. E., Ravon Textile Monthly 24, 416

(1943).

[27] Bright, T. B., Morris, L. E., and Summers, F., J. Textile Inst. 15, T547 (1924).

[28] —, J. Textile Inst, 17, T396 (1926).

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Microbiological Deterioration 39
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40

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