September 1964

SOME METHODS

USED IN STUDYING

MICROBIOLOGICAL DETERIORATION

OF WOOD

U.S. FOREST SERVICE RESEARCH NOTE

FPL-063

Table of Contents

Page

Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Microscopical Examination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Surface Observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Maceration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Sectioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Freehand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Sliding Microtome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Rotary Microtome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Microscopical Appraisal of Decay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Decay Rating System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Measurement of Area on Cross Sections . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Staining of Wood Sections for the Observation of Hyphae . . . . . . . . . . . . . . . . . . . 7 Picro Aniline Blue and Safranin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Pianeze IIIb . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Safranin and Fast Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Fast Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Additional Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

8 Isolation from Sporophores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Isolation from Wood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Culturing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Control of Mites in Fungus Culture Work . . . . . . . . . . . . . . . . . . . . . . . . . 9 Purification of Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Acidification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Van Tiegham Ring Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Addition of Inhibitory Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Culture Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

General Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Cultural Keys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Oxidase Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

12 General Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Osmium Tetroxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Alizarine Red S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

13 Test for Iron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Isolation, Culturing and Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Additional Methods for the Diagnosis of Decay . . . . . . . . . . . . . . . . . . . . . . . .

Diagnosis of Stains and Discolorations . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Test to Differentiate Between Biological and Nonbiological Stains . . . . . . . . . . . . 14 Preservative Testing Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Agar-Plate Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Agar-Block Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Soil-Block Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Testing of Natural Decay Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Miscellaneous Methods and Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Differentiation of Heartwood and Sapwood . . . . . . . . . . . . . . . . . . . . . . . . . 16 Wide-Range pH Indicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Detection of Oil-Borne Preservatives . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Gaseous Sterilization at Room Temperature . . . . . . . . . . . . . . . . . . . . . . . . 17 Inoculating Punch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Petri Plate Shield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Fungus Growth Tube . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Growth-Tube Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Inoculation Tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Chisel Forceps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Literature Cited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

FPL-063

SOME METHODS USED IN STUDYING

MICROBIOLOGICAL DETERIORATION OF WOOD

W. WAYNE WILCOX, Pathologist

1 Forest Products Laboratory, Forest Service

U. S. Department of Agriculture

Summary

Methods useful in detecting and studying microbiological deterioration of wood are discussed. They concern aspects of wood microtechnique fundamental to the execution of microscopical examinations of decayed wood, isolation and culture of wood-destroying microorganisms, diagnosis of decay and discolorations, and testing of preservatives and natural decay resistance. Special equipment devel- oped for the study of wood-destroying fungi is also discussed.

Introduction

This report is a brief summary of methods which have proven useful, or could prove useful, in the detection and study of microbiological deterioration of wood and wood products. Wherever possible, the source of more detailed information on each method is given. Cartwright and Findlay ( 9 ), 2 Hartley ( 23 ) and Hubert ( 26 ) have provided summary discussions of many methods applicable particularly to the detection of decay in wood.

Toughness and weight loss have been considered the most sensitive indicators of the degree of wood deterioration caused by decay ( 23 ). However, since both of these methods require nondecayed controls for determination of the magnitude

1 Maintained at Madison, Wis., in cooperation with the University of Wisconsin. 2 Underlined numbers in parentheses refer to Literature Cited at the end of this report.

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of their alteration, they are seldom applicable to the detection of deterioration of wood in service. Often it is sufficient to know whether or not decay exists, regardless of its severity.

Microscopical examination and culturing probably are the most reliable means of determining the presence of decay in wood ( 26 ). However, other methods have been devised for this purpose, which are more rapid and which may be fairly reliable with respect to certain applications. Several of these methods are described. In addition, certain procedures which have proven useful in the study of other wood-inhabiting microorganisms, and of the effects of some nonbiological types of deterioration, are presented.

Microscopical Examination

Surface Observation

Often a great deal of information may be obtained simply by critical study of the wood surface. The type of equipment used depends upon the required magni- fication and resolution. Magnifications of approximately 10X may be obtained with a hand lens, while magnifications of up to 40X or 50X may be obtained with a dissecting microscope. Surface examinations may also be made at higher magnifications by using a compound microscope equipped with a device for obtaining incident illumination. With such equipment it is also possible to study fully hydrated, living microorganisms by means of high-power, water-immersion lenses. The best type of surface for such examination will depend upon the features under consider at ion.

Rough Surface.--Much information may be obtained by studying fungal hyphae directly upon the wood surface which has received no preparation prior to observation. Such observation is often sufficient to partially identify many of the mold or stain fungi.

Smoothed Surface. --A jointer- or razor-smoothed surface can be prepared rapidly to permit the observation of hyphae and other features in the surface layers of the wood.

Maceration

Some deterioration of secondary walls, such as bore holes and soft-rot cavities, may be viewed most easily by examining the individual wood elements.

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Wood is separated into its individual elements by maceration. Many methods are available for the maceration of wood ( 55 ). One which has proven satisfactory for application to decayed wood is Jeffrey’s method ( 28 , 45 , 54 ).

Jeffrey’s macerating fluid consists of equal parts of 10 percent aqueous nitric acid and 10 percent aqueous chromic acid. Thin slivers of the wood to be macerated should be placed in the macerating fluid in an oven at 40° to 50° C. overnight. Additional treatment with fresh liquid may be necessary to completely macerate some woods. When separation is complete, the spent macerating fluid should be completely washed out of the macerated sample with water. The sample may be stored in 50 to 70 percent alcohol and observed as a wet mount directly in this storage fluid.

Sectioning

General information on sectioning may be found in Sass ( 45 ) and Johansen ( 28 ). A bibliography on the subject of the preparation of wood for microscopic study was prepared by Kryn ( 32 ), and detailed informationon the sectioning of decayed wood has been provided by Wilcox ( 54 ).

Freehand.--Considerable information can be obtained from the relatively thick sections produced by freehand sectioning. Often hyphae are observed more easily in such material than in thin sections. A razor blade is usually sufficient for such sectioning, but a microtome knife may also be used. Sections of fairly uniform thickness may be obtained by guiding the blade with a fingernail, while observing the procedure under a dissecting microscope. In addition to true ‘‘freehand” sectioning, devices are available for holding and carefully advancing material to be sectioned by hand. Several such instruments are available commercially.

Sliding Microtome.--Sound or slightly decayed wood often can be sectioned on the sliding microtome without embedding. However, very soft or heavily decayed wood may require the support of an embedding matrix before sectioning can be performed satisfactorily.

Softening.--If wood is to be sectioned without embedding, it is usually necessary to soften or at least thoroughly hydrate the material prior to sectioning. This may be accomplished by boiling small blocks until they sink, or by thoroughly infiltrating them with cold water under vacuum. For decayed wood, especially if hyphae are to be observed, submersion in water under vacuum at room tem- perature is recommended. Very hard wood can be softened by storage in concentrated hydrofluoric acid.

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Steaming.--A rapid and very effective method for sectioning hard wood involves the application of a small jet of steam to the surface of the specimen to be sectioned. No other treatment is necessary, and excellent sections may be obtained from very hard material in a short time.

Freezing.--Adequate support for the sectioning of many soft or decayed wood samples may be provided by freezing these specimens in the water-soaked condition. For sectioning by this method, a small, water-saturated wood block is surrounded with water on top of a special freezing attachment, through which pressurized carbon dioxide is allowed to flow. The rapid release of gas pressure cools the attachment and freezes the water in and surrounding the wood block. Greater support of the section is obtained if the microtome knife is also cooled. This can be done by deflecting the escaping carbon dioxide onto the knife. Since the ice melts immediately after sectioning, the resulting sections are handled in the same manner as sections from other nonembedded specimens.

Celloidin.--One of the most satisfactory methods for the preparation of sections of wood in all stages of decay is that of embedding with celloidin. Sections as thin as several microns may be obtained from celloidin-embedded wood, and all structural features of the decayed wood are held carefully in place by the embedding matrix.

The celloidin method involves the infiltration of the wood block to be sectioned with a purified cellulose nitrate product. The use of ethylene glycol monomethyl ether as the celloidin solvent, when this method is applied to decayed wood, was recommended by Wilcox ( 54 ). The wood blocks are first air-dried, or dehydrated by means of an ethyl alcohol series. They are transferred through several changes of absolute alcohol under vacuum and then to ethylene glycol monomethyl ether. The blocks are then transferred successively through 2, 4, 6, 8, and 10 percent solutions of celloidin in ethylene glycol monomethyl ether, and main- tained at 52° to 54° C. for a minimum of 2 days in each grade. Next they are transferred to 12 percent celloidin and placed in hardening chambers, in which the celloidin concentrates by evaporation at room temperature ( 54 ). Twenty percent and later 40 percent celloidin solutions are added to the hardening chambers as the celloidin concentrates. When the celloidin has become sufficiently hard, embedded specimens are removed from the hardening chambers, placed upon wooden mounting blocks, and surrounded with additional 40 percent celloidin. The celloidin then is thoroughly hardened by submersion in chloroform overnight. The embedded specimen is removed from the chloroform, trimmed, and stored in a 50/50 mixture of glycerin and 95 percent alcohol.

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Cellulose Tape. --Another m i c r o t o m e employs cellulose material ( 5 ). This method may be

method available for use with the sliding tape to hold together difficult-to-section applied to either embedded or nonembedded

material and is useful when the material to be sectioned is either too hard or too weak to maintain its integrity during sectioning. The procedure consists of allowing a loop of cellulose tape to hang down over the specimen mounted in the sliding microtome. The tape is firmly affixed to the thoroughly dried surface of the specimen. The section is cut with a slight tension applied through the tape to its leading edge, to keep the tape from sticking to the knife. Difficulty has been encountered, however, in removing the tape from the sections following cutting. Soaking in xylene, as recommended by Bonga, proved unsatisfactory. Removal of the tape and adhesive with acetone priortoaffixing the sections to the slides was successful, but some disintegration of the sections usually accompanied this process ( 54 ).

Rotary Microtome

Paraffin. --The most common method of preparing specimens for sectioning on the rotary microtome is paraffin embedding. In this method the blocks to be embedded may be dehydrated in a tertiary butyl alcohol (TBA) series, according to the methods of Sass ( 45 ). Fromanhydrous TBA the blocks are trans- ferred to a 50/50 mixture of anhydrous TBA and paraffin oil. They are then gently poured onto the solidified layer on the surface of molten paraffin which has cooled slightly. The assembly is placed in an oven and the blocks drop into the pure paraffin when the surface film melts. The embedded blocks in paraffin are then poured into a paper boat, and the paraffin is rapidly solidified in a freezing compartment. Individual embedded blocks may be removed with a hacksaw blade and placed upon mounting blocks for sectioning.

Polyethylene Glycol.--An alternative embedding method for use with the rotary microtome employs polyethylene glycol (PEG) in place of paraffin. Several advantages of the use of PEG instead of paraffin are that green or moist samples may be used directly, reducing the problem of distortion due to dehydration; the method is rapid; and it requires a minimum of specimen handling. Commercial PEG with a molecular weight of 1450 was found by Gjovik to be most suitable for the embedding of woody tissues. By this method green or moist blocks are placed in a 50/50 mixture of PEG-1450 and distilled water at 50° C. for 2 hours. Next the 50/50 mixture is decanted, replaced with melted, pure PEG-1450, and stored at 50° C. for 2 hours, The old PEG-1450 is exchanged for fresh liquid and allowed to remain at 50° C. for 4 to 6 hours. The blocks can then be cast in paper boats and handled as in the paraffin method. Acceptable

3

3 Gjovik, L. R. The application of fluorescence microscopy to the study of the permeability of aspen ( Populus tremuloides Michx.). M.S. thesis, University of Minnesota. 1961.

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sections may be obtained in less than 1 hour by using an abbreviated schedule, consisting of the same steps as outlined above, but allowing only 15 to 20 minutes of storage at each stage.

Microscopical Appraisal of Decay

Decay Rating System

It was mentioned previously that the major application of microscopical examination in the study of wood deterioration is the qualitative determination of whether or not decay or other microbiological damage is present. However, an attempt has been made to quantitatively determine the extent of decay, on the basis of microscopical observations, by correlating such observations with changes in toughness of matched samples ( 53 ). The woods examined were rated on the basis of the size and quantity of bore holes, the occurrence of cell wall separation, the degree of cell wall thinning as observed in cross section, and the presence of hyphae. Although trends were apparent, the correlation between the microscopical rating and the toughness of a wood specimen was not great. However, the features observed give a good indication of the type of information that may be obtained from microscopical examination of decayed wood.

One of the criteria used in the decay rating system of Waterman and Hansbrough ( 53 ) was the microscopically visible quantity of cell wall material removed. For some purposes it may be necessary to obtain a quantitative estimate of the amount of cell wall material removed, as measurable in cross section. Since such a feature may vary considerably, it is necessary to measure a large number of cells to arrive at a suitable estimate. The laborious methods of optical micrometry and photographic weighing, or planimetry, are not satisfactory where such a large number of measurements is desired. A method was developed by Ladell ( 34 ) for rapidly measuring cross-sectional areas of large numbers of wood cells. This method has been applied to decayed wood by Wilcox ( 54 ).

The method consists of projecting the image of a section from the microscope onto a white card containing 100 pinholes punched on grid coordinates determined from a table of random numbers. The card containing the randomly spaced pinholes is illuminated from below so that the holes appear as bright spots of light on the projected image of the specimen. In the area occupied by the 100 spots, the number of spots falling upon a given structure provides a measure of the percentage of the area occupied by the structure.

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Measurement of Area on Cross Sections

Staining of Wood Sections for the Observation of Hyphae

Picro Aniline Blue and Safranin

The most satisfactory results in the differential staining of microorganisms and wood at the Forest Products Laboratory have been obtained with the picro aniline blue-safranin staining procedure. By this method wood cell walls appear pink, and hyphae or bacterial cells appear blue. The procedure in use at the Laboratory is a modification of that originally developed by Cartwright ( 8 ), in which the staining solutions have been diluted as suggested by Proctor ( 40 ). The staining schedule as applied at the Laboratory is presented by Wilcox ( 54 ). The stain stock solutions consist of 1 percent aqueous safranin-0 and 25 milliliters of a saturated, aqueous solution of aniline blue, to which 100 milliliters of saturated, aqueous picric acid has been added. For use, the safranin is diluted at the rate of 3 drops of stock solution in 10 milliliters of distilled water, and the picro aniline blue stock solution is diluted by adding 5 drops to 10 milliliters of distilled water. The safranin is applied first, followed by the picro aniline blue which is gently warmed until the stain begins to steam slightly. The sections are washed in water after the application of each stain.

Pianeze IIIb

This is an alternative method of differentially staining fungal hyphae in wood and has the advantage of requiring only a single stain application, therefore making it desirable for routine work. This method colors the hyphae red and the wood green. The procedure followed at the Forest Products Laboratory is an adaptation of the method presented by Vaughan ( 52 ). The stain stock solution consists of 1 gram malachite green: 0.5 grams acid fuchsin; 0.05 grams martius yellow: 150 milliliters distilled water; and 50 milliliters 95 percent ethanol. With this method hydrated sections are stained in the stock solution for 10 to 45 minutes. After washing, the stain is decolorized in acid alcohol, and clearing is performed with carbol-turpentine made by adding 400 milliliters melted phenol to 600 milliliters oil of turpentine. The sections are washed in xylene and mounted.

Safranin and Fast Green

A number of modifications of this method have appeared ( 28, 45 , 54 ). By this method hyphae are stained very lightly by the fast green and sometimes take on a faint pink tinge. Although this method is very satisfactory for application to the observation of wood structure, hyphae of decay fungi stained by this method

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remain indistinct. The sections are first stained in a 1 percent solution of safranin in 50 percent alcohol for 1 hour or more. They are washed in 50 percent alcohol and stained for 1 minute in a 1 percent solution of fast green in a mixture of 9 parts 95 percent alcohol and 1 part clove oil. They are then washed in 95 and 100 percent alcohol, cleared in clove oil, washed in xylene, and mounted.

Fast Green

The use of a 1 percent alcoholic fast green solution for the staining of decayed wood, to facilitate the observation of hyphae, has been suggested by Dr. T. C. Scheffer of the Forest Products Laboratory. The time of staining is not critical, provided that the excess stain is washed out with 95 percent alcohol. By this method, both the hyphae and the wood are stained green, but such addition of color to the hyphae facilitates their observation.

Additional Methods

Additional methods have been suggested for the staining of hyphae in wood, although they are not presently in use at this Laboratory. The stains suggested include methyl violet ( 49 ); bismark brown and methyl violet ( 25 ); gold chloride ( 21 ); and picro-pontamine pink ( 40 ).

Isolation, Culturing and Identification

Since the cultural identification of many wood-rotting fungi is laborious and uncertain, isolation from an identifiable sporophore is preferable to isolation from a nonfruiting mycelium. As the spores of many wood-rotting fungi germinate erratically, if at all, it is best to prepare cultures from the internal tissue of the sporophore itself ( 9 ). This may be performed by aseptically stripping off the external sporophore tissue and transferring a small portion of the internal tissue to a nutrient medium.

Isolation from Wood

Methods for the isolation of microorganisms from plant tissues were discussed by Riker and Riker ( 42 ), and the procedure for isolation of fungi from wood was described by Cartwright and Findlay ( 9 ). Since the surfaces of wood specimens may be contaminated with spores of fungi capable of growing more rapidly than the wood-destroying Basidiomycetes, it is necessary to aseptically remove the

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Isolation from Sporophores

surface layers before transferring tissue to a nutrient medium. Very small pieces of wood from the interior of the specimen should be transferred to minimize the chances of obtaining more than one organism Since wood-inhabiting organisms are sometimes irregularly distributed within wood, it is necessary to transfer from several areas within a given sample. A chisel forceps and other special equipment designed to facilitate the isolation of fungi from wood were described by Hubert ( 24 , 27 ), but a flamed scalpel is also satisfactory. An isolating tool also has been designed by Dr. W. E. Eslyn of the Forest Products Laboratory. It consists of a small-bore leather punchmounted on a wood handle. The punch is fitted with a rod which may be pushed through the bore of the instrument to extract the small cylindrical plug which is formed. A rapid isolation method was suggested by Scheffer for isolation from wood which is not appreciably contaminated with molds or other rapidly growing organisms. By this method a wood sample is sawn into small blocks. Each block is speared with a dissecting needle at one corner and rotated quickly in a flame to surface- sterilize it. The block is then placed on a nutrient medium with the needle.

Methods designed specifically for isolation of bacteria from wood have also been developed by Knuth and McCoy ( 31 ) and Knuth. A general review of isolation techniques has been presented by Durbin ( 17 ).

4

Culturing

Wood decay fungi are commonly cultured on an agar medium containing 2 percent malt extract. Although this medium appears to be quite satisfactory, other possible culture media have been discussed by Cartwright and Findlay ( 9 ) and by Riker and Riker ( 42 ).

Control of Mites in Fungus Culture Work

The best method for the exclusion of mites from fungus-culturing facilities is strict sanitation ( 42 ). Freshplant material or organic debris should not be brought into the laboratory or should first be disinfested. A chamber filled with fumes of p-dichlorobenzene has proven effective for disinfestation of wood specimens, and a thorough steaming has been adequate for implements.

To prevent the entrance of mites, painting the entrances to cabinets (used for storage of fungus cultures) with a suspension of 7.5 percent chlordane has been tried. This method appears to be effective, although no controlled experiments

4 Knuth, D. T. Bacteria associated with wood products and their effects on certain chemical and physical properties of wood. Ph. D. thesis, University of Wisconsin, 1964.

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have been made. A combination of shelf painting and room spraying was rec- commended by Rose1 ( 43 ) for scrupulous control of mites, Annually, shelves are smeared with a 3 percent solution of beta-napthol in medicinal paraffin oil. Every 2 months the incubation room is sprayed with a 0.25 percent water emulsion of a commercial miticide containing di (p-chlorophenyl) methyl carbinol. A method of excluding mites from individual culture tubes by means of cigarette-paper caps was described by Hansen and Snyder ( 22 ). Simply wiping culture shelves with fuel oil also reportedly decreases the chances of mite infestation without harming the fungi ( 9 ). Curl ( 13 ) recommended the use of a 3 percent solution of Kelthane as a shelf spray for preventing mite infes- tation of fungal cultures.

The use of 1 milliliter of pyridine or carbon tetrachloride for each 4 liters of space has been recommended by Cartwright and Findlay ( 9 ) for eliminating mites from already infested fungal cultures. The fumigation should be applied overnight and should be reapplied after 4 days to kill mites hatched from eggs not killed by the first treatment. Caldwell ( 7 ) reported that exposure to 2 to 3 atmospheres pressure of pure oxygen for 24 hours killed mites and mite eggs but had no detectable effect upon fungi or bacteria. He suggested that this might prove to be an effective method of disinfesting mite-contaminated fungal cultures.

Purification of Cultures

Acidification.--Since most fungi can tolerate acid conditions of less than pH 5 and most bacteria cannot, the acidification of the culture medium by the addition of a sterile organic acid has been suggested to free fungal cultures from bacterial contaminates ( 9 , 42 ).

Van Tiegham Ring Method.--A method of freeing cultures of fungi which produce no aerial mycelium from bacterial contamination was suggested by Raper ( 41 ). This methodutilizes a glass ring with three small glass beads fused to its lower side. Agar is poured in a petri dish containing the ring at its center, and a portion of the contaminated culture is transferred to the agar inside the ring. Fungal hyphae grow under the ring and out into the surrounding medium, leaving the bacteria behind.

Addition of Inhibitory Chemicals. --A selective medium was reported by Russell ( 44 ) for the isolation of Basidiomycetes containing 0.006 percent o-phenylphenol which was added after the medium had been autoclaved. Such a medium should selectively allow the growth of white-rot fungi, which are capable of breaking down the toxic phenolic substance. However, it has been reported ( 37 ) that this substance suppresses as many white rots as brown rots. A medium which reportedly eliminates bacteria and greatly restricts the growth

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of molds was developed by Martin ( 36 ). This medium is a peptone-dextrose agar, containing rose bengal in a dilution of 1:30,000, and 30 µg. per milliliter of streptomycin. Since streptomycin reportedly inhibits the growth of some fungi, Johnson ( 29 ) recommended replacing the streptomycin with aureomycin. The use of antibiotics and other chemical additives in culture media was reviewed by Durbin ( 17 ).

Culture Storage

Probably the most common method of storing cultures of wood-inhabiting fungi is simply by means of agar slants which require periodic transfer. By storing the cultures at approximately 6° C., the period between transfers can be lengthened to nearly 1 year. Sealing the culture tubes with a rubber stopper and wax has been suggested for some fungi, but the method is considered by Fennell to be too drastic for most fungi ( 19 ). Mineral oil may also be used for the storage of cultures of wood-inhabiting fungi.

A promising method for the storage of wood-decay fungi has been investigated by Dr. Catherine G. Duncan of the Forest Products Laboratory. By this method, small, autoclaved wood blocks, or corn kernels, are inoculated with the decay fungus and stored in a sub-zero freezingcompartment. In order to transfer from such a stock culture, it is necessary only to remove one small block or corn kernel and place it on a suitable nutrient medium.

Recently a successful procedure for lyophilization of minute cultures of wood- rotting fungi, growing upon 1/10 .to 1/20 milliliter of malt agar in small tubes, was reported by Bazzigher ( 4 ). The subject of storage of fungus cultures has been reviewed by Fennell ( 19 ).

Identification

General Characteristics.--A characteristic feature of wood-rotting Basid- iomycetes is the presence of clamp connections. However, since monokaryotic mycelia possess no clamp connections, and since even in the dikaryotic mycelia of some fungi abundant clamp connections are lacking, the absence of such structures on hyphae in wood does not rule out the possibility of their being wood-rotting fungi.

By far the most reliable form of identification of a wood-rotting fungus is that made from the sporophore itself. If the culture was not originally obtained from an identifiable sporophore, it may be desirable to attempt to induce sporophore formation in culture. Some methods for this often difficult process are discussed by Cartwright and Findlay ( 9 ).

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Cultural Keys.--The first comprehensive attempt at the development of a system for cultural identification of wood decay fungi was made by Fritz ( 20 ). More recently, Davidson, Campbell, and Vaughn ( 15 ) developed a key for the cultural identification of a number of fungi found in living oak trees. Nobles ( 38 ) developed an extensive key to the identification of cultures of wood-rotting fungi.

Oxidase Tests.--In 1928 Bavendamm discovered that white rots formed a dark zone under the fungus mat when grown on a medium containing gallic or tannic acid, while the brown rots gave no such reaction. In 1938 Davidson, Campbell and Blaisdell ( 14 ) determined the reactions of a number of white-rot and brown- rot fungi on such media. The media consisted of 2 percent agar and 1-1/2 percent malt extract, to which 1/2 percent of gallic acid or tannic acid was added after sterilization.

A test for polyphenol oxidase, which utilized an extract from red cabbage leaves, was developed by Jorgensen and Vejlby ( 30 ). White rots turned the purple of the medium to yellow, while brown rots produced a red color. Nobles ( 38 ) developed a rapid oxidase test, which employed a solution of 0.5 grams gum guaiac in 30 milliliters of 95 percent alcohol. By this test, most white rots produce a bright blue color. Etheridge ( 18 ) has developed an oxidase test which utilizes finely ground, extracted wood meal in a liquid medium. When this medium is inoculated with a white-rot fungus, a brown zone occurs in the wood meal within 10 days.

Additional Methods for the Diagnosis of Decay

General Methods

Several rapid methods are commonly applied when surveying wood or wood products for the presence of decay. Picking or probing with a knife or other sharp instrument may reveal the presence of decay. The pick test consists of prying up splinters of wood and observing the type of splintering which occurs. This is essentially a test for toughness, since sound wood generally produces a long, fibrous splinter, while decayed wood, which is characteristically brash, produces a short splinter which breaks easily across the grain. Probing wood with a sharp instrument may reveal the significant loss of hardness which accompanies advanced stages of decay. Both of these survey methods were discussed by Boyce ( 6 ) and by Hubert ( 26 ). Collapse of the surface layers of wood, or of its finish, may indicate localized shrinkage which might result from pockets of advanced decay.

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Osmium Tetroxide

A rapid method for the detection of brown rot in either incipient or advanced stages was suggested by Cowling and Sachs ( 12 ). The reagent for this test is prepared by adding 100 milliliters of water to 1 gram of melted osmium tetroxide. Upon application of this reagent, brown-rotted wood becomes blackened within 5 minutes, whereas the blackening is delayed for 20 minutes or more in sound or white-rotted wood.

Alizarine Red S

A rapid color test has been proposed for the detection of decay caused by Peniophora gigantea , which is the most common storage decay in southern pine poles, posts, and pulpwood ( 35 ). The color reaction is dependent upon the increase in acidity which accompanies the presence of Peniophora in wood. The reagent employed is a 0.75 percent solution of sodium alizarine sulfate (alizarine red S) in distilled water. A small amount of this reagent is sprayed upon the freshly exposed, end-grain surface. Sapwood infected with Peniphora gigantea is stained yellow following the application of the reagent, while uninfected sapwood stains pink to red.

Diagnosis of Stains and Discolorations

Moist wood is subject to a number of discolorations other than those caused by the presence of mold or stain fungi. The exposure of moist wood to oxygen may cause color changes, due to the oxidation of constituents of the wood ( 9 ). A dark blue-black stain is formed when wood containing tannin comes in contact with iron or iron salts. It is often necessary to distinguish between such so-called chemical stains and discolorations caused by the presence of microorganisms. Several tests have been developed for this purpose.

Test for Iron

The presence of iron, which could c o n t r i b u t e to the f o r m a t i o n of dark blue-black iron tannate in wood, may be detected by applying a 19 percent solution of hydrochloric acid, followed by a 12 percent aqueous solution of potassium ferrocyanide. The formation of a blue color upon application of these reagents indicates the presence of iron.

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Test to Differentiate Between Biological and Nonbiological Stains

A test was developed by Scheffer at the Forest Products Laboratory for clearly differentiating between the so-called chemical stains and discolorations caused by the presence of microorganisms. This test involves the application of a saturated, aqueous solution of oxalic acid to the discoloration in question. This reagent does not affect stains caused by fungi, but removes chemical stains.

Preservative Testing Methods

A great deal of methodology in the study of fungus deterioration of wood products and its prevention has necessarily been directed toward the qualitative and quantitative measurement of the effectiveness of preservatives and of the degree of natural decay resistance.

Agar-Plate Method

One of the first methods to be used extensively for testing the effectiveness of preservatives was the agar-plate method. The procedures in this test were outlined by Schmitz and others ( 48 ). This method consists of preparing malt agar-preservative emulsions in a graded series of preservative concentrations. The media are poured into petri plates and inoculated. The radial growth on each plate is measured to determine the relative toxicity of the preservative con- cent rat ion us ed.

Agar-Block Method

Although the agar-plate method may provide a measure of the relative toxicity of preservatives to various fungi, information on the quantity of preservative which must be added to wood to prevent decay is not readily available. The exact quantities of preservative required to prevent decay in the field cannot be determined from laboratory testing; however, a better estimate of the relative quantities necessary is provided by application of the preservative to wood blocks. This test method has been described by Cartwright and Findlay ( 9 ). It involves the growth of the test fungi on a malt extract medium in Kolle flasks-- flat, glass flasks with a wide neck, having a ridge on the lower side of the neck to retain media in the flask. Wood blocks are then impregnated with a series of concentrations of the preservative to be tested. These blocks are placed on glass-rod frames above the test fungi in the flasks. The effectiveness of the preservative at each concentration is indicated by the extent of weight loss sustained by the test block.

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Soil-Block Method

The method of preservative testing in use at the Forest Products Laboratory is the soil-block method, ASTM D 1413-61 ( 1 ). In this method the preservative is added to a wood block, as in the agar-block method. However, soil rather than agar is used as the moisture-retaining medium, and the test fungus utilizes wood feeder blocks as a nutrient substrate, rather than nutrient agar.

This method is preferred over the agar-block method for a number of reasons. Using soil as the moisture-containing medium provides a longer period of controlled moisture content than does agar. Fungus growth is generally more vigorous on soil than on agar, with the result that differences between preservatives or treatments are emphasized. In addition, using soil it is possible to perform tests on highly volatile preservatives or upon woods with volatile fungitoxic extractives, whereas the use of such volatile substances in an agar culture may seriously inhibit fungus growth.

In the soil-block method, wood feeder blocks are placed upon moist soil and the apparatus is sterlized and inoculated with the test fungus. The test blocks are impregnated with various concentrations of preservative and placed directly upon the fungus mycelium growing on the feeder block.

Procedures are also outlined in this method for the laboratory testing of preservative resistance to weathering. The historical development of the soil- block method for the evaluation of wood preservatives was reviewed by Colley ( 11 ). Results of experimentation which led to the adoption of the soil-block procedure as an ASTM standard method are given by Duncan ( 16 ).

Testing of Natural Decay Resistance

Several variations of the soil-block method have been suggested for the application of this test to the study of natural decay resistance of wood. The altered test method is outlined in ASTM D 2017-62 T ( 2 ). The basic modifications of the soil-block method in this procedure are alteration in the size of test blocks, which are larger in cross-sectional area butthinner along the grain, and the incorporation in the test of reference blocks of a standard, nondurable wood. A measure of the decay resistance of the test blocks to various test fungi is given by the extent of weight loss sustained.

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Miscellaneous Methods and Equipment

Several methods, although not directly related to the study of microbiological deterioration of wood, provide valuable supplementary information for such studies.

Since heartwood and sapwood may differ considerably in their resistance to fungal attack, and since in many woods it is difficult to detect the presence of heartwood, a number of color tests for the differentiation of heartwood and sapwood have been developed. These tests, as applied to softwoods, were reviewed by Kutscha and Sachs ( 33 ). Several of these tests have been recom- mended for application to specific woods, including some hardwoods ( 51 ). The test recommended for application to Douglas-fir is alizarine red S, described previously. This method colors heartwood yellow and sapwood pink.

The benzidine test is recommended for application to pine wood. This reagent is prepared by dissolving 5 grams of benzidine in 23 milliliters of 25 percent hydrochloric acid, and 970 milliliters of water. For use this reagent is mixed in equal volumes with a 10 percent solution of sodium nitrite. The sapwood is colored yellowish-brown and the heartwood red by this method.

The test reagent recommended for application to oak is a 0.1 percent solution of methyl orange in water. Heartwood is stained red and sapwood yellow by this reagent .

Wide-Range pH Indicator

Since a change in pH is often associated with both chemical and microbiological deterioration of wood, an indicator capable of detecting changes in pH over a wide range of pH values can yield valuable diagnostic information. A common soil indicator has proven satisfactory for such application ( 33 ).

Detection of Oil-Borne Preservatives

A rapid staining test is suitable for the determination of the depth of pene- tration of oil-borne preservatives, such as pentachlorophenol. The test involves the application of an oil-soluble stain, which is dissolved and absorbed in the areas where oil is present. It was found at this Laboratory that, at least with pentachlorophenol, the distribution of oil as detected by this method and the presence of pentachlorophenol were well correlated.

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Differentiation of Heartwood and Sapwood

Gaseous Sterilization at Room Temperature

Occasionally it is desirable to avoid high temperatures in the sterilization of test materials, such as wood blocks. A sterilization method employing ethylene oxide in a nonflammable mixture with fluorinated hydrocarbons was discussed by Schley, Hoffman, and Phillips ( 47 ). The procedure recommended was the exposure of moist but not wet material to 300 to 500 milligrams per liter ethylene oxide for 6 hours at room temperature. In the procedure in use at the Forest Products Laboratory, the material to be sterilized is wrapped in paper and placed in a standard desiccator with a volume of approximately 9 liters. Since the ethylene oxide-fluorinated hydrocarbon mixture contains 12 percent ethylene oxide, the addition of 40 to 45 grams of the mixture to an evacuated desiccator provides the desired concentration of ethylene oxide. The material is allowed to remain in contact with the gas overnight. The desiccator may then be opened in a well-ventilated area and the material removed. A method of gaseous sterilization utilizing propylene oxide was described by Snyder and Hansen ( 50 ).

Inoculating Punch

An inoculating punch has been devised, and when used with a petri plate shield considerably accelerates transfer procedures ( 10 ). This punch is essen- tially an adaptation of the ‘cork-borer” method of transfer and rapidly provides inoculum of a uniform size. The punch is constructed of stainless-steel tubing with an inside diameter of 0.07 inch. It is fitted with a spring-loaded plunger for rapid removal of the inoculum.

Petri Plate Shield

The petri plate shield ( 10 ) consists of a wood base, which holds a petri plate in a vertical position, and a plastic sleeve which extends out from the petri plate and the wood base to prevent contaminating organisms from dropping upon the plate. This allows thelidofthe plate to be left off for long periods while making repeated transfers from it.

Fungus Growth Tube

A modification of the standard 25- by 200- millimeter test tube, to allow the preparation of horizontal strips of agar, was suggested by Scheffer ( 46 ). The tube is prepared by heating and drawing in a portion of the wall of the tube near the

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open end to provide a barrier which will hold back agar. This makes it possible to slant the tubes in a horizontal position, thus producing a long, narrow strip of culture medium of uniform depth, suitable for measurement of linear growth rate.

Growth- Tube Reader

A growth-tube reader was designed by Clark ( 10 ) for use with the growth tubes. The reader consists of a light box with a slot in the top; a sliding housing on fixed tracks which contains two glass microscope slides with blackened edges (serving as the cross-hairs for alignment with colony margins); and a scale with 0.5 millimeter divisions. This device makes possible the rapid measurement of linear growth rates.

Inoculation Tubes

A culture tube was designed by Basham ( 3 ) which is similar to the growth tube described above, except that it contains three invaginations instead of one, and has a small opening in the normally closed end of the tube. This tube is designed for the inoculation of wood strips by agar colonies of fungi. The com- partment at the closed end of the tube is filled with water through the small, cotton-plugged hole, the next compartment is left vacant, and the third compart- ment is filled with agar. A strip of wood is then placed on top of the invaginations, which hold it slightly above the surface of the agar. Humidity in the tubes is maintained at a high level by the presence of water in the end compartment.

Chisel Forceps

The chisel forceps designed by Hubert ( 24 ) to aid in the isolation of fungi from wood was described previously.

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Literature Cited

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PUBLICATION LISTS ISSUED BY THE

FOREST PRODUCTS LABORATORY

The following lists of publications deal with investigative projects of the Forest Products Laboratory or relate to special interest groups and are avail- able upon request:

Box, Crate, and Packaging Data

Chemistry of Wood

Drying of Wood

Fire Protection

Fungus and Insect Defects in

Logging, Milling, and Utilization of Timber Products

Mechanical Properties of Timber

Pulp and Paper

Structural Sandwich, Plastic Laminates, and Wood-Base

Forest Products Components

Glue and Plywood Thermal Properties of Wood

Growth, Structure, and Wood Finishing Subjects

Wood Preservation

Architects, Builders, Engineers,

Identification of Wood

Furniture Manufacturers, Woodworkers, and Teachers of Woodshop Practice and Retail Lumbermen

Note : Since Forest Products Laboratory publications are so varied in subject matter, no single catalog of titles is issued. Instead, a listing is made for each area of Laboratory research. Twice a year, December 31 and June 30, a list is compiled showingnew reports for the previous 6 months. This is the only item sent regularly to the Laboratory’s mailing roster, and it serves to keep current the various subject matter listings. Names may be added to the mailing roster upon request.