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Identification of fungi by the fluorescent antibody technique Johnson, Gary Clifford 1972

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IDENTIFICATION OF FUNGI BY THE FLUORESCENT ANTIBODY TECHNIQUE  BY  GARY CLIFFORD JOHNSON B.S.F., The U n i v e r s i t y  o f B r i t i s h Columbia, 1969  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE MASTER OF FORESTRY DEGREE  We a c c e p t required  THE  this  t h e s i s as conforming  to the  standard  UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1972  In p r e s e n t i n g t h i s t h e s i s an advanced degree at  in p a r t i a l  fulfilment  o f the  requirements  the U n i v e r s i t y of B r i t i s h C o l u m b i a ,  the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r  reference  1 agree t h a t and s t u d y .  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s  thesis  f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department by h i s r e p r e s e n t a t i v e s . of  It  this thesis for financial  gain shall  Department  7  or  i s understood that copying or p u b l i c a t i o n  written permission.  The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada  for  Columbia  not be a l l o w e d w i t h o u t my  ii ABSTRACT The fluorescent antibody technique was investigated as a means of f a c i l i t a t i n g the recognition and i d e n t i f i c a t i o n of the fungal components of a western red cedar (Thuja p l i c a t a Donn) heartwood f l o r a in situ. Fungi isolated from the heartwood were grown i n bulk and prepared for two different injection t r i a l s .  In one t r i a l the antigen was  the particulate matter of the c e l l that could be centrifuged into a p e l l e t after the hyphae were destroyed by a tissue grinder.  In the  second t r i a l the hyphae were ground up and ultrasonically disintegrated. Only the cytoplasm and small w a l l fragments were retained f o r injection.  After antisera collection the indirect staining method  was employed. Unlabeled specific antiserum was layered over the antigen, allowed to incubate and washed off before fluorescent sheep anti-rabbit globulin was applied to form the f i n a l layer. A l l attempts to detect s p e c i f i c antibodies to the fungal antigens failed.  This was probably due to not using antigens r i c h enough i n  protein.  Successful production of precipitating antibodies to fungal  antigens has been shown by other workers to be more l i k e l y when the antigen contains greater than 10 milligrams of protein per m i l l i l i t e r of antigen solution.  I t has also been found that i n some cases fresh  antigen must be prepared for each diffusion test and injection as i t can't be preserved even at -20°C. I t i s hoped that i f fresh, high protein antigen were to be used this study could be successfully completed.  iii  CONTENTS Page ABSTRACT CONTENTS  1  FIGURES  1  1  1  1  i  v  ACKNOWLEDGEMENTS  v  Introduction  1  L i t e r a t u r e Review  5  H i s t o r i c a l Perspective  5  F.A.T. Uses  8  Problems encountered w i t h t h e F.A.T Methods and M a t e r i a l s  12 14  Growth o f F u n g i i n B u l k  14  Antigen Preparation  15  Injection  17  B l e e d i n g and A n t i s e r u m  17  Antibody Detection  18  Indirect  20  S t a i n i n g Method  Microscopy  23  Results  23  Discussion  23  Glossary  28  References  29  iv FIGURES Figure 1  Page Thuja p l i c a t a Heartwood Zones and Fungi Commonly Present  2  2  Direct Fluorescent Antibody Methods  6  3  Indirect Fluorescent Antibody Methods  7  4  Typical Ring P r e c i p i t i n Test With Controls  19  5  Diffusion Plate Pattern  20  6  Hypothetical Explanation of Extra Sensitivity of Indirect Staining  21  7  Comparison of Direct and Indirect Methods  22  8  Typical Staining- T r i a l With Controls for One Fungus  23  V  ACKNOWLEDGEMENTS I wish to thank Dr. Bart van der Kamp, Assistant Professor of Forestry, for his helpful c r i t i c i s m and encouragement during the course of this project and i n the preparation of this thesis.  Votes  of thanks are also due to Dr. J . J . Stock, Associate Professor of Microbiology, for his worthwhile suggestions for the second t r i a l and to Dr. R.J. Hudson, Assistant Professor of Animal Science, for helping me to understand and use serological techniques.  To my wife,  Noni, I extend my deepest appreciation for her constant interest and encouragement.  1  IDENTIFICATION OF FUNGI BY THE FLUORESCENT ANTIBODY  TECHNIQUE  Introduction  C r o s s - s e c t i o n s o f b u t t s o f o l d ( a p p r o x i m a t e l y 200 y e a r s o l d ) w e s t e r n r e d c e d a r s (Thuj a p l i c a t a Donn) commonly e x h i b i t a c e n t r a l column o f decayed m a t e r i a l surrounded by s e v e r a l zones o f v a r i o u s l y s t a i n e d heartwood  (see F i g u r e 1).  The decay column i s e n c l o s e d by a  zone o f brown s t a i n e d , b u t sound wood. of  Outside of t h i s l i e s  a belt  red-brown o r p i n k heartwood which i n t u r n i s s u r r o u n d e d by a  straw c o l o r e d  zone.  Repeated i s o l a t i o n o f f u n g i from the heartwood o f T h u j a p l i c a t a has demonstrated t h a t t h e o u t e r s t r a w c o l o u r e d heartwood may be c o n s i d e r e d s t e r i l e b u t t h a t the red-brown and brown s t a i n e d heartwood zones a r e c o l o n i z e d by c h a r a c t e r i s t i c  fungi  (van d e r Kamp, u n p u b l i s h e d ) .  i s o l a t i o n work i s based on the assumption t h a t the l i v i n g  Such  organisms  p r e s e n t i n the heartwood w i l l grow on the media used f o r i s o l a t i o n . F u r t h e r m o r e , i n cases where two o r more organisms o c c u r t o g e t h e r i n the  wood i t i s n o t uncommon f o r one o f them to grow much f a s t e r on  the  i s o l a t i o n medium and i n f a c t e l i m i n a t e the o t h e r .  relative  Thus, the  f r e q u e n c i e s o f two o r more organisms i n the heartwood cannot  2  straw zone  heartwood zone  red-brown zone brown zone  sterile  decay colui  VI QQ  Figure 1 Thuja p l i c a t a Heartwood Zones and Fungi Commonly Present  be determined by the frequency of i s o l a t i o n .  Furthermore, i n the case  of western red cedar heartwood i t has proven impossible to separate the various fungal isolates by microscopic examination of variously stained  sections of heartwood.  Fluorescent antibody  staining offered the best p o s s i b i l i t y to overcome both these problems. The as yet unidentified fungi isolated from the stained heartwood of Thuja p l i c a t a may be viewed as forming stages i n a succession of fungi leading to decay.  Tests have shown that a l l the fungi commonly  isolated from the red-brown and brown stained heartwood zones are able to break down thujaplicin,a potent fungicide naturally occurring i n the heartwood of western red cedar. Naturally dark stained pieces of heartwood are much more susceptible to decay than l i g h t colored heartwood.  Test blocks of straw colored heartwood inoculated with  fungi isolated from the stained heartwood zone show a rapid decrease  3  in thujaplicin concentration.  In this instance, however, there i s no  p a r a l l e l decrease i n decay resistance. This suggests that the breakdown product of thujaplicin may be as toxic as thujaplicin itself.  This i n turn could mean that the further steps i n the breakdown  of thujaplicin are mediated by organisms not commonly isolated.  One  or more of the occasional isolates could actually be common i n the wood and essential for the loss of decay resistance. Fluorescent antibody staining, a technique used mainly i n medicine, appeared to offer an effective means of approaching this problem. According to Nairn (1962), the fluorescent antibody technique (hereafter known as F.A.T.) " . . . i s perhaps at i t s most useful when employed to determine the numerical or spatial distribution of microorganisms among a mixed population."^" Thus,the F.A.T. was investigated as a means of overcoming two specific problems. The F.A.T. i s based on the principle that specific antiserum can be precipitated when i t contacts i t s homologous antigen.  This fluorescent  complex could be readily viewed under u l t r a v i o l e t stimulation. I t i s important to note that the antigen(s) must be unique to one fungus i f a specific stain i s to be produced. This technique has the advantages of high s p e c i f i c i t y , s e n s i t i v i t y and rapidity (once developed).  The high degree of s p e c i f i c i t y i s  inherent i n the antibody which i s produced i n response to the entry of a foreign, high molecular weight compound into an animals  Ideally  this antibody globulin w i l l complex only with the antigen (usually protein or polysaccharide) to which i t has been prepared. ^ Nairn, R.C. 1962, Fluorescent protein tracing. London, p. 231.  Tagging  S. Livingstone,  4  antibody with some fluorochromes doesn't s i g n i f i c a n t l y affect i t s biological activity. sensitive. -18 1 x 10  In addition to being specific the F.A.T. i s also  I t i s possible by this technique to detect as l i t t l e as grams of dye (Eren and Pramer, 1966) and to identify a single  bacterial c e l l that contains 5 x 10 ^ milligrams of nitrogen (Coons, 1956).  Thomason et a l ; (1956) report no d i f f i c u l t y i n locating  s p e c i f i c a l l y stained bacteria i n mixtures containing ratios of contaminants to specific c e l l s as high as 10^:1. Once the technique has been developed i t can be used quickly to screen a large number of samples.  Staining and microscopic examination can be completed  in as l i t t l e as one hour.  Also, the presence of contaminants i s of  no concern i f proper controls are maintained and the technique i s equally useful for viable and nonviable organisms (Cherry et,al.,1965).  5  L i t e r a t u r e Review  H i s t o r i c a l Perspective In  the p e r i o d from 1930-42 s e v e r a l attempts were made to t a g  a n t i b o d i e s w i t h azo-dyes  ( R e i n e r , 1930)  (Creech and J o n e s , 1941). w h i l e the l a t t e r the  and l a t e r w i t h f l u o r o c h r o m e s  The former r e s u l t e d i n low  sensitivity  complexes were d e l e t e r i o u s t o the a n t i b o d y .  Until  e a r l y 1940's most workers had been p r e o c c u p i e d w i t h s t u d y i n g the  e f f e c t o f v a r i o u s r a d i c a l s on the i m m u n o l o g i c a l a c t i v i t y o f a n t i b o d i e s . Coons,  Creech and Jones  (1942) were the f i r s t  (1941) and Coons,  Creech, Jones and  Berliner  t o s t u d y t h e tagged a n t i b o d y from the p o i n t o f  view of u s i n g i t as a t r a c e r .  They used f l u o r e s c e i n  c o n j u g a t e d a n t i b o d y t o t r a c e pneumococcus  soluble  isocyanate-  polysaccharide  a n t i g e n i n t i s s u e s e c t i o n s o f mice i n f e c t e d w i t h pneumococcus. the  p e r i o d from 1950-51 a s e r i e s  o f papers on the F.A.T.'s  a s p e c t s were p u b l i s h e d by Coons and co-workers. and K a p l a n , 1950)  The  first  In  technical (Coons  d e t a i l e d t h e s y n t h e s i s o f the f l u o r o c h r o m e  f l u o r e s c e i n i s o c y a n a t e , and i t s c o n j u g a t i o n t o the a n t i s e r u m a l o n g w i t h i n s t r u c t i o n s f o r m i c r o s c o p e f i l t e r i n g systems, f o r t i s s u e powder removal of n o n s p e c i f i c s t a i n i n g and f o r Immunological p r o o f s o f s p e c i f i c i t y of s t a i n i n g .  This p u b l i c a t i o n along with four others  (Coons, Leduc and K a p l a n , 1951; Hill, the  Deane and Coons,  Coons,  Snyder, Cheever and Murray,  1950; K a p l a n , Coons and Deane, 1950)  t e c h n i q u e ' s p r i n c i p l e s , b a s i c mechanics  and  feasibility.  1950;  established  6  U n t i l the mid-1950's F.A. work was being done exclusively by the direct staining method.  In this case the fluorochrome i s conjugated  d i r e c t l y to the antibody (see Figure 2). This conjugate i s able to  I  Antibody  Fluorochrome Labeled y .j, antibodv  * P? - tt - - £ 5 3 * Antigen  X  Labeled antibody  o*  c  Antigen  X Specific fkiorescence(+)  Y  :K  —RP Heterologous Specific antibody fluorescence( —) Y*  X  f l u o r e s c e n c e( — ) Antigen  Figure 2.  Antibody  Antigen-antibody complex  X  Labeled antibody  Direct Fluorescent Antibody Methods (Kawamura, 1969)  7 adsorb  onto the homologous a n t i g e n to form a v i s i b l e s p e c i f i c  when e x c i t e d by u l t r a v i o l e t i r r a d i a t i o n . direct  technique t o be more s p e c i f i c  r e a s o n f o r t h i s i s not known. procedure  than the i n d i r e c t , though the  Deacon e t a l . (1957) v a r i e d  the  ( a l s o known as sandwich or a n t i g l o b u l i n ) method  homologous a n t i b o d y i s not tagged. globulin  E x p e r i e n c e has shown the  to i n d i r e c t l y detect a n t i b o d i e s i n unlabeled t e s t  the i n d i r e c t  stain  sera.  In  the  However, a n t i b o d y t o normal  yG-  (prepared i n a d i f f e r e n t animal) i s l a b e l e d and i t i s added  t o the homologous a n t i g e n - a n t i b o d y complex r e s u l t i n g i n s p e c i f i c fluorescence  e  .  (see F i g u r e 3 ) .  (J  +  .i n  I f t h e r e i s s p e c i f i c f l u o r e s c e n c e , one  — -  Antibody to Fluorochrome normal yG -globulin •  PP  Antigen  +  -aJ Antibody  fl*  .  Labeled antibody to normal yG-globulin  [S3  ««  +  Antigen-antibody complex  —  CS3[  Labeled antibody to normal yG-globulin  Specific fluorescence '  Figure 3  (+>  I n d i r e c t F l u o r e s c e n t A n t i b o d y Methods (Kawamura,  1969)  can i d e n t i f y the a n t i g e n when the n a t u r e o f the p r i m a r y a n t i b o d y one  t h a t i s not  o f the i n d i r e c t  tagged) i s known, or v i c e v e r s a .  (the  See methods f o r d e t a i l s  technique.  Numerous workers have i n v e s t i g a t e d the t e c h n i q u e s d e v i s e d by Coons and  co-workers and found  them t o be u s e f u l .  Many minor and  few major r e f i n e m e n t s have been made.  One  f l u o r e s c e n t a n t i b o d y a c c e p t a b i l i t y was  made by Riggs e_t a l . (1958)  a  important advancement i n who  8  described  the s y n t h e s i s and  fluorescein  (yellow-green  (orange-red). isocyanate  tions  fluorescence)  whose p r e p a r a t i o n  and  required  d e r i v a t i v e s of  tetraethylrhodamine  the use  of d i f f i c u l t - t o - h a n d l e prepared  a s t a b l e p r o d u c t t h a t c o u l d be  used to l a b e l p r o t e i n s w i t h no  ( u n l i k e the i s o c y a n a t e ) . Thus, w i t h  f u r t h e r chemical  e a s i l y h a n d l e d , a major d i f f i c u l t y  with  stored for manipula-  the development of p r o t e i n -  l a b e l i n g dyes t h a t were s t a b l e enough t o be marketed and  B  used u n s t a b l e f l u o r e s c e i n  i s o t h i o c y a n a t e d e r i v a t i v e was  thiophosgene and was  months and  of isothiocyanate  P r e v i o u s l y most workers had  gaseous phosgene. The liquid  use  of F.A.  commercially  t e c h n o l o g y was  overcome.  F.A.T. Uses  Initially  Coons et a l . (1942) d e v i s e d  m e d i c a l l y - o r i e n t e d problems.  T h i s continues  especially i n a diagnostic capacity. i d e n t i f y and  the major  t e c h n i q u e has  animal t i s s u e antigens  a l s o been used f o r the m i c r o s c o p i c  foreign antigens; and  The  to be  i n t r i n s i c antigens,  s p e c i f i c antibodies  use,  been used  study v i r a l , b a c t e r i a l , p r o t o z o a l , h e l m i n t h i c ,  amoebal, mycoplasmal and has  the F.A.T. t o i n v e s t i g a t e  to  fungal,  ( N a i r n , 1962).  It  i d e n t i f i c a t i o n of i n j e c t e d p r o t e i n s , enzymes, and  ( M e l l o r s , 1959).  hormones,  9  Some F.A.. t e s t s have proven so u s e f u l they are conducted i n diagnostic medical labs.  C h e r r y and Moody  regularly  (1965) have i n d i c a t e d  t h a t they c o n s i d e r the f o l l o w i n g t o be the most i m p o r t a n t d i a g n o s t i c a p p l i c a t i o n s o f the F.A.T. i n b a c t e r i o l o g y : •A S t r e p t o c o c c i , Treponema p a l l i d u m N e i s s e r i a gonorrhoeae  i d e n t i f i c a t i o n o f Group  (Schaudinn and Hoffmann) Schaudinn,  (Zopf) T r e v i s a n , Coasnebacterium  (Kruse) Lehmann and Neumann, Bordetel-la. p e r t u s s i s S a l m o n e l l a typhosus White, coli of  diphtheriae  (Pribram) Ldpez,  s e r o g r o u p i n g o f eh't'eropathogenic^Echerichia  (Migula) C a s t e l l a n i and Chalmers  and S h i g e l l a .  As a d i r e c t  the s u c c e s s o f r e s e a r c h e r s i n m e d i c a l b a c t e r i o l o g y , workers  result  i n other  f i e l d s a r e r e a l i z i n g the F . A . T . s p o t e n t i a l and are t r y i n g t o adapt 1  i t t o t h e i r p a r t i c u l a r needs. to  h e l p overcome problems  S e v e r a l r e s e a r c h e r s have used F.A.T.  o f working w i t h s o i l m i c r o o r g a n i s m s .  s t a i n i n g a i d s r e c o g n i t i o n o f the organism d i r e c t l y D i f f e r e n t problems a r e b e i n g a t t a c k e d w i t h the F.A. the  in i t s soil  R e l a t i o n s h i p s between microorganisms i n s o i l are r e v e a l e d by i n d i r e c t methods o n l y , and i t i s seldom p o s s i b l e t o e a s i l y d i f f e r e n t i a t e organisms o f p a r t i c u l a r i n t e r e s t from the m y r i a d o f o t h e r s t h a t comprise the m i c r o b i a l population of s o i l . 2  Even though i t may be shown t h a t a p a r t i c u l a r s p e c i e s e x i s t s i n a s o i l , c u l t u r a l experiments cannot prove whether the organism i s v e g e t a t i v e o r dormant, whether i t o c c u r s as s i n g l e c e l l s o r i n c o l o n y form, o r whether i t i s a s s o c i a t e d w i t h any one type o f p a r t i c l e , o r m i c r o h a b i t a t , i n the s o i l . - *  E r e n , J . and D. Pramer, 1966.  3  H i l l , I.R. and T.R.  Gray, 1967.  habitat.  as i n d i c a t e d  following quotations:  2  F.A.  S o i l S c i . 100(1), p. J . B a c t . 9 3 ( 6 ) , p.  39. 1888.  by  10  Schmidt e t a l . (1968) s t u d i e d s p e c i e s o f Rhizobium but  had d i f f i c u l t y  Paton  w i t h n o n s p e c i f i c a d s o r p t i o n by s o i l  i n the  particles.  (1960) showed the r e l a t i o n s h i p between Pseudomonas and the  free root surface of clover.  F.A.  s t u d i e s o f f u n g i i n the s o i l  been a b l e t o d e t e c t A s p e r g i l l u s f l a v u s L i n k ex F r . i n mixed (Schmidt et_ al_., 1962 to  soil  and 1965).  Eren and Pramer  soil have  culture  (1966) used F.A.T.  i d e n t i f y and measure the abundance i n s o i l o f A r t h r o b o t r y s c o n i o d e s  D r e c h s l . a nematode-trapping  fungus.  The F.A.T., when a p p l i e d t o f u n g i , has been m a i n l y f o r the purpose o f d i a g n o s i n g and i d e n t i f y i n g pathogens Kaufman, 1961>.  o f man  The t e c h n i q u e ' s advantages f o r f u n g a l  (Kaplan and identification  i n c l u d e r a p i d i t y and s e n s i t i v i t y as compared w i t h c o n v e n t i o n a l c u l t u r a l methods. Ochoa  Immuno-specific  s t a i n i n g e n a b l e d Kaplan and Gonzales  (1960) t o f i n d fungus elements o f Sporotrichum s c h e n c k i i  (. H e l e t .  and Perk.) de Beurm and Goug. i n smears from a human l e s i o n which proved n e g a t i v e by c u l t u r a l t e c h n i q u e s . v i t y though a study  T h i s i s one i n d i c a t i o n o f i t s s e n s i t i -  ( P o r t e r ejt a l . ,  1965)  of tissues infected with  H i s t o p l a s m a capsulatum D a r l i n g and Blastomyces d e r m a t i t i d - i s G i l c h r i s t and Stokes i n d i c a t e d t h a t h i s t o p a t h o l o g i c a l t e c h n i q u e s were more e f f e c t i v e than F.A.T., and both were b e t t o r than c u l t u r l n g .  This  latter  paper a l s o p o i n t s o u t the g r e a t d i v e r s i t y o f r e s u l t s from s t u d i e s i n v o l v i n g d i f f e r e n t methods and In albicans  o t h e r c a s e s Gordon (Robin) Berkh.  o t h e r genera  .  organisms.  (1958) used F.A.T. t o d i f f e r e n t i a t e  Candida  from o t h e r Candida s p e c i e s and from y e a s t s o f  A l - D o o r y and co-workers  (1963) were a b l e t o d i s t i n g u i s h  between C l a d o s p o r i u m c a r r o n i i T r e j o s and C_. bantianum  Borelli.  H i s t o p l a s m a capsulatum y e a s t c e l l s have been s e l e c t i v e l y s t a i n e d i n the  11  p r e s e n c e o f Blastomyces d e r m a t i t i d i s by Gordon a n t i s e r a t o C r y p t o c o c c u s neoformans on  (1959).  (Sanfelice) V u i l l .  96 i s o l a t e s o f t h i s organism and on a number  species  (Kase and M a r s h a l l ,  1960).  Conjugated has been t e s t e d  (23) o f h e t e r o l o g o u s  These workers r e p o r t e d  specific  s t a i n i n g o f 95 i s o l a t e s o f C_. neoformans and none o f the o t h e r  species.  In 1962 May used F.A.S. t o study and i d e n t i f y t h e s i t e s o f c e l l wall extension  o f Schizosaccharomyces pombe L i n d n e r .  Two y e a r s  later  Goos and Summers used a s i m i l a r t e c h n i q u e t o study the morphogenesis o f two  fungi.  They found t h a t w a l l m a t e r i a l o f t h e Candida  parent c e l l i s incorporated walls. and not.  i n t o the w a l l o f daughter c e l l s o r h y p h a l  C o n i d i a o f Fusarium oxysporum f . cubense  I n 1964 Paton made a s i g n i f i c a n t c o n t r i b u t i o n t o a i d p l a n t He d e s c r i b e d  e x a m i n a t i o n and i n c l u d e d v a l u a b l e  autofluorescence  the p r e p a r a t i o n  information  and n o n s p e c i f i c s t a i n i n g .  s t u d i e s o f p l a n t p a t h o g e n i c and s a p r o p h y t i c and A l l e n a specific The  (E.F. Smith) Snyder  Hansen s t a i n e d i n t e n s e l y b u t germ tubes emerging a f t e r s t a i n i n g d i d  g i s t s w i t h use o f t h e F.A.T. for  albicans  patholo-  of plant tissue  on c o n t r o l o f h o s t  I n s p i t e o f t h i s a i d F.A. f u n g i a r e n o t common.  Beno  (1964) used immuno-fluorescent s t a i n i n g t o r a p i d l y i d e n t i f y l i n e o f germinating Puccinia sorghi  (Schw.) u r e d o s p o r e s .  p r e s e n c e o f POlyporus tomentosus i n mixed c u l t u r e s , s o i l , and  i n f e c t e d p i n e r o o t s was d e t e c t e d  by F.A. (Kumar and P a t t o n ,  1964).  In 1965 some f a c t o r s a f f e c t i n g the a n t i g e n i c i t y o f the m y c e l i a o f 3 s p e c i e s o f Phytophthora F r . were r e p o r t e d found t h e . t y p e and c o n c e n t r a t i o n  of nitrogen  by B u r r e l l e_t a l .  They  s o u r c e , t h e amount o f  inoculum, and t h e age o f mycelium g r e a t l y a f f e c t e d the a n t i g e n i c e f f i c a c y o f the preparations.  Amos and B u r r e l l t h e f o l l o w i n g y e a r  found t h e F.A.T. t o be t h e most u s e f u l t e c h n i q u e  ( i n comparison  w i t h a g g l u t i n a t i o n and g e l d i f f u s i o n ) i n d i f f e r e n t i a t i n g among e i g h t  12 species of Ceratocysis.  In another study a fluorescent antibody  reagent for Botrytis cinerea Pers ex Fr. was able to distinguish spores and mycelium of that organism from those of three others (Preece and Cooper, 1969).  Price (1970) was unable to produce a fluorescent  antibody reagent specific for Sphaerothecia pannosa conidia. A large number of his comparative test species fluoresced along with S_. pannosa while other species did not. Problems Encountered with the F.A.T. The problems that can and do arise with the F.A.T. are manifold but most can be controlled to some extent.  One problem i s auto-  fluorescence of the organism being studied, of other contaminating members and of the organism's substrate or host. fluorescence i n particular masks the low intensity reactions.  Tissue autoimmune-specific  Autofluorescence can be minimized by prompt use of cut  sections and i s partly removable by f i l t e r i n g and counterstaining. Counterstains are useful i f b r i l l i a n t l y fluorescent and i f they have an emission maximum w e l l separated from that of the fluorescent conjugate (Hall and Hansen, 1961). Another problem i s nonspecific fluorescence which may result from the unreacted fluorochrome substance, from antibody with too strong a negative charge, from improper tissue f i x a t i o n , or from l e t t i n g the specimen dry out during the staining procedure (Kawamura, 1969).  Nonspecific staining may  be reduced or controlled by p u r i f i c a t i o n and fractionation of antigenic components, d i l u t i o n of antiserum, improved tissue  13 preparation techniques, removal of unconjugated fluorochrome and adsorption with tissue powders or homogenates. Thirdly, i t i s possible to obtain false positive staining reactions due to the antibody already being present i n experimental animals after natural infection.  This w i l l be i d e n t i f i a b l e i f controls are taken unless  animals are naturally infected after normal serum i s drawn.  Fourthly,  the same antigens i n heterologous organisms may result i n crossstaining.  F i f t h l y , fluorescent a r t i f a c t s may be produced by manipulat-  ing ( f i x i n g , etc.) the section to be observed.  Once stained, conditions  can be aLtered such that the antigen-antibody complex can be broken down. In addition Gooding (1966) points out that i n most cases i t i s necessary to work with fungal extracts which consist of multiple antigen systems which are d i f f i c u l t to analyze. He successfully isolated and used a crude DNA fraction from Fomes annosus Fr. i n double diffusion tests.  F i n a l l y , as Kaplan and Kaufman (1961) point out,  there i s a need for standardization of reagents, procedures and equipment so results w i l l be more comparable.-  Undoubtedly many  anomalies and discrepancies tp date are due to variation i n immunizing s t r a i n , immunization schedule, the fluorochrome used i n labeling, host r e a c t i v i t y , antibody t i t e r s , optical equipment used, etc.  14  Methods and Materials  Growth of Fungi i n Bulk  Three fungi commonly isolated from western red cedar heartwood were chosen to be studied.  They were a K i r s c h s t e i n i e l l a - l i k e species,  a V e r t i c i l l i u m - l i k e species and another unidentified species, each respectively known i n the U.B.C. Forest Pathology Collection as C4D4, VIQQ and IVTB. These fungi were transferred from storage slants to p e t r i plates containing malt agar where they were allowed to grow u n t i l approximately two-thirds of the plate was covered with an actively growing culture. Then the agar embedded mycelial mat was transferred into a Waring Blendor with 150 m i l l i l i t e r s of s t e r i l e water and ground up for 15 seconds.  Ten m i l l i l i t e r s of this slurry were added to each of  ten 250 m i l l i l i t e r shaker flasks containing 50 m i l l i l i t e r s of culture medium. This was repeated for each of the three fungi. medium was as follows:  The growth  15 0.75 g o f MgS0 *7H 0 4  0.75 g o f K H P 0 2  2  4  10.0 g o f y e a s t 20.0 g o f  extract  glucose  1000.0 ml o f water  The  two f i l a m e n t o u s  days u n t i l s u f f i c i e n t other and  f u n g i (C4D4 and IVTB)  grew f o r 9 t o 16  mycelium had been o b t a i n e d .  VIQQ,  u n l i k e the  two f u n g i , grew s l o w l y i n a y e a s t - l i k e f a s h i o n i n l i q u i d  as a r e s u l t  to h a r v e s t .  r e q u i r e d 27 days t o produce enough f u n g a l  The f u n g i were then c e n t r i f u g e d  Centrifuge)  culture  material  ( S o r v a l l SS-1 A n g l e  and washed t h r e e times i n phosphate b u f f e r e d s a l i n e (here-r  a f t e r known as P.B.S.) (Cherry  Antigen  and Moody,  1965).  Preparation  Numerous attempts were made t o d i s r u p t t h e f u n g a l c e l l s antigenic material while  a t the same time m i n i m i z i n g  F i r s t a Pyrex t i s s u e g r i n d e r with  denaturation.  a t o l e r a n c e o f 0.005 t o 0.007  was used, sometimes i n c o m b i n a t i o n w i t h g l a s s c h i p s . s a t i s f a c t o r y as o n l y about 20 p e r c e n t significantly  to expose  inches  I t d i d n o t prove  o f t h e m y c e l i a l c e l l s and  l e s s o f the y e a s t - l i k e c e l l s were b r o k e n up.  The second  attempt i n v o l v e d f r e e z i n g the organism i n l i q u i d n i t r o g e n and g r i n d i n g w i t h m o r t a r and p e s t l e .  I n a s i m i l a r case t h e f u n g i were f r o z e n  i n l i q u i d n i t r o g e n and s t r u c k r e p e a t e d l y cylinder.  The l a t t e r  i n a chilled  compression  attempts weren't any more s u c c e s s f u l t h a n the  f i r s t method and were much more d i f f i c u l t  t o complete.  The f o u r t h  time, t h e h y p h a l f u n g i were ground up by the Pyrex t i s s u e g r i n d e r and  16 then subjected to ultrasonic disintegration (Bronwill Biosonik I I I ) in short (12 second) bursts f°  r  a  t o t a l time of one minute while  being cooled by an ice-water bath.  Microscopic observation of the  treated tissue showed the hyphal fungi to be macerated but such was not the case with VIQQ u n t i l i t had been subjected to a further one minute of treatment.  This f i n a l and most successful treatment  ruptured at least 50 percent of the c e l l s . In the f i r s t injection sequence ( t r i a l one) the antigen used was basically the particulate matter of the c e l l .  Any c e l l component  that could be centrifuged into a p e l l e t at a R.C.F. of 6500 i n 20 minutes was used.  The fungus was macerated with the tissue grinder  only. . Fungal particulates were suspended at five milligrams per m i l l i l i t e r i n P.B.S. and frozen (-20°C) i n three m i l l i l i t e r l o t s . To this suspension an equal amount of Freund's complete adjuvant was added at the time of injection to .enhance antigenicity. In the second t r i a l the antigen was prepared by f i r s t grinding the fungus and then subjecting i t to ultrasonic disintegration. The heavy c e l l w a l l components were centrifuged and discarded.  The  supernatant containing cytoplasm and very small wall fragments were diluted to five.milligrams per m i l l i l i t e r .  An equal volume of  Freund's complete adjuvant was added and mixed vigorously just before injection. i n small v i a l s .  Prepared antigen, i n P.B.S., was stored at -20°C  17 Injection  Young, three to four pound rabbits were injected subcutaneously on the scapular region of the back using s t e r i l e disposable syringes. The syringes had a capacity of three m i l l i l i t e r s with one and onehalf inch long, 21 gauge needles.  Each of the three fungi were  injected into two rabbits for the f i r s t t r i a l and into three rabbits for the second t r i a l .  On injection day each rabbit received a t o t a l  of one m i l l i l i t e r of emulsion.  For the f i r s t t r i a l each rabbit  received a single injection once a week while the rabbits i n the second received half a m i l l i l i t e r i n each of two places every two weeks. Each t r i a l had four injection days. When two injections were given to each rabbit on. one day they were at least two inches apart. The Freund's sometimes caused open wounds which were quickly treated 4 with Cicatrin  a n t i b i o t i c powder and the subsequent i n j e c t i o n did  not contain the adjuvant. Bleeding and Antiserum  Two weeks after the last injection the rabbits were bled from the midvein of the ear.  Fifteen to 20 m i l l i l i t e r s of blood were  collected from each animal and l e f t overnight i n a cooler 4°C  for the  clot to form. Antiserum was decanted from the clot and centrifuged to remove any large debris.  A small amount (1.5 m i l l i l i t e r s ) of raw  antiserum was immediately frozen to -20°C. The remainder of the antiserum was cross-adsorbed with heterologous antigens to remove 4  C i c a t r i n , Calmic Limited, Toronto  18  n o n s p e c i f i c antibody. frozen  (-20°C) u n t i l  Antibody  Then the remainder was  divided into small v i a l s  used.  Detection  V i s i b l e p r e c i p i t a t i o n o r clumping o f p a r t i c l e s o c c u r s w i t h most antigens  as a r e s u l t o f the m u l t i v a l e n t (many bonding p o i n t s f o r  antibody) of  a n t i g e n and b i v a l e n t a n t i b o d y  a n t i g e n and  antibody.  The  two  forming  a g g r e g a t e s or  b a s i c antigen-antibody  a r e the p r e c i p i t i n i f the a n t i g e n i s i n a s o l u b l e form a g g l u t i n a t i o n i f the a n t i g e n i s p a r t i c u l a t e . d e t e c t i o n and s o l u t i o n and The prepared for  and  or a n t i b o d i e s i n  1968).  a g g l u t i n a t i o n r e a c t i o n was  used to t e s t  t o the p a r t i c u l a t e a n t i g e n o f the f i r s t  for  antibody  trial.  A prerequisite  the a g g l u t i n a t i o n t e s t i s a homogeneous s u s p e n s i o n .  o n l y t h e s m a l l w a l l fragments o f the 'hyphal A f t e r low  P.B.S.  Therefore  f u n g i c o u l d be  used.  speed c e n t r i f u g a t i o n t o e l i m i n a t e the l a r g e r w a l l p i e c e s  the s m a l l e r ones were d r i v e n i n t o a p e l l e t in  reactions  These r e a c t i o n s a l l o w  q u a n t i t a t i v e e s t i m a t i o n of a n t i g e n s t i s s u e s (Kabat,  T h i s was  t e s t as d e s c r i b e d by sheep r e d b l o o d  and washed s e v e r a l times  the a n t i g e n used i n the p a s s i v e Campbell e t a l . ( 1 9 6 4 ) .  c e l l s were washed and  Then they were coated w i t h  saline.  haemagglutination  In t h i s p r o c e d u r e f r e s h  then tanned w i t h  the a n t i g e n by m i x i n g  suspended i n normal serum and  Finally  tannic  together  the c o a t e d r e d  s t a n d a t room temperature f o r t h r e e to f o u r hours.  r e a c t i o n was  acid.  and  c e l l s were added to s e r i a l d i l u t i o n s of the serum, shaken and to  lattices  blood allowed  A positive  i n d i c a t e d by a compact g r a n u l a r a g g l u t i n a t i o n or a  d i f f u s e f i l m of a g g l u t i n a t e d c e l l s  c o v e r i n g the bottom o f the  tube  and  19 while a negative one appeared as a heavy ring of c e l l s or discrete smooth button of c e l l s i n the center of the tube. The yeast phase of the dimorphic fungus, VlQQ, was studied by the tube agglutination method of Cozad (1958).  To s e r i a l dilutions  of serum (0.5 m i l l i l i t e r s ) i n P.B.S. was added 0.5 m i l l i l i t e r s of the antigen suspension and the tubes were shaken vigorously.  The  tubes were immediately refrigerated (4°C) and l e f t overnight. The degree of agglutination was indicated by the size of c e l l aggregates and turbidity of supernatant. Ring and gel double diffusion tests were used to detect a n t i body to the cytoplasmic and fine fragments of fungi used i n the second t r i a l .  The ring or i n t e r f a c i a l test involved carefully over-  laying antiserum with a solution of antigen such that a sharp l i q u i d interface was formed. Antibody can be detected i n amounts as small as one microgram of protein.  An example of the test  including controls i s given i n Figure 4 .  A positive result (as i n Key  Tube Number  1  2  3  4  5  Top Layer Bottom Layer Observed Reaction  B Ab  B Ag  Ag N  B N  Ag Ab +  -  -  -  -  B Ab Ag N  = buffer = antibody = antigen = normal serum  Figure 4 Typical Ring P r e c i p i t i n Test With Controls  Figure 4) i  s  indicated by a fine l i n e or ring of precipitated particles  at the interface of the two complementary solutions while a negative one has no ring. The gel diffusion test was also used to detect antibody i n the second t r i a l .  In this case concentrations of antigen and antibody  20  d i f f u s e toward each o t h e r and i f o p t i m a l they form v i s i b l e bands o f p r e c i p i t a t i o n i n the s e m i s o l i d medium ( D i f c o N o b l e A g a r ) . d o u b l e d i f f u s i o n method (as d e s c r i b e d by Campbell et al., used.  T h i s t e s t r e s o l v e s components i n m i x t u r e s .  The  bands i n d i c a t e s the m i n i m a l number o f a n t i g e n - a n t i b o d y  Ouchterlony's 1964)  was  number o f systems p r e s e n t .  . F i g u r e 5 shows the p a t t e r n of a d i f f u s i o n p l a t e .  antiserum inner antigen well  Figure 5  Diffusion Plate Pattern  I n d i r e c t S t a i n i n g Method  The  i n d i r e c t method was  s e v e r a l reasons.  According  t o ten times as s e n s i t i v e .  s e l e c t e d r a t h e r than the d i r e c t f o r t o Kawamura (1969) the i n d i r e c t i s f i v e One  e x p l a n a t i o n f o r the d i f f e r e n c e s i n  s e n s i t i v i t y i s the a d d i t i o n a l combining s i t e s o f f e r e d by the molecule  sandwiched between the a n t i g e n and  antibody  tagged a n t i - r a b b i t serum  when u s i n g the i n d i r e c t method ( N a i r n , 1962).  In Figure 6 only  three  21  Figure 6  H y p o t h e t i c a l E x p l a n a t i o n of E x t r a S e n s i t i v i t y of I n d i r e c t S t a i n i n g ( i i ) N  a  r  n  s i t e s i n the a n t i g e n are a v a i l a b l e f o r a n t i b o d y  >  9  6  and  2  these  be the t o t a l number p o s s i b l e i f using- the d i r e c t method. i n d i r e c t method though the second l a y e r i s  two  fourfold.  dimensional  antibody.  A l s o , the i n d i r e c t method was  d i f f i c u l t y with  d i r e c t method and of improvement.  found  h i s weak immune-specific labelled  Thus, i n  i n d i c a t e d he  was  the  s w i t c h i n g to the i n d i r e c t F.A.T. i n hopes t h a t a u t o f l u o r e s c e n c e was reactions.  masking some of  Moreover, i t i s p o s s i b l e t o  a n t i - r a b b i t g l o b u l i n commercially  F.A.T. the c o n j u g a t i o n  antibody  chosen as the r e s u l t of a  the s e n s i t i v i t y of s t a i n i n g from  he was He  the  model the r e a c t i v e s i t e s have i n c r e a s e d  p e r s o n a l communication w i t h Dr. Yasu H i r a t s u k a who having  In  unconjugated  which behaves as an a n t i g e n f o r the conjugated Nairn's  t h r e e would  so i f one  uses the  buy  indirect  of f l u o r e s c e i n i s o t h i o c y a n a t e i s unnecessary.  22 See F i g u r e 7 f o r a comparison S t a i n i n g was  o f the d i r e c t and i n d i r e c t methods.  i n i t i a t e d u s i n g each fungus fixed to a s l i d e .  Fungi  f i x e d w i t h e t h a n o l and w i t h heat were d i s l o d g e d d u r i n g washing Haupt's a d h e s i v e w i t h p h e n o l was  used.  E v e n t u a l l y i t was  planned t o  study the fungus i n wood so each fungus, i n pure c u l t u r e , was growing  on western  r e d cedar heartwood.  a l l o w a more r e a l i s t i c fungus known.  trial  so  started  The i n o c u l a t e d samples  o f the s t a i n i n g  would  t e c h n i q u e , y e t w i t h the  At the same time i t would p r o v i d e a p l a c e where the "bugs"  ( f o r example, a u t o f l u o r e s c e n c e o f wood) c o u l d be worked out. Once the t i s s u e was Goldman (1968) was Step I  f i x e d t o the s l i d e  the s t a i n i n g p r o c e d u r e o f  f o l l o w e d as i n d i c a t e d below:  (a) A few drops o f u n l a b e l e d s p e c i f i c ( a g a i n s t each a n t i s e r u m were l a y e r e d over the a n t i g e n .  fungus)  (b) S l i d e s were i n c u b a t e d i n a m o i s t chamber f o r 60 minutes a t 25°C. (c) A n t i s e r u m drops were shaken o f f . (d) S l i d e s were immersed i n a s a l i n e , w i t h o c c a s i o n a l a g i t a t i o n f o r 10 m i n u t e s . (e) S l i d e s were r i n s e d i n tap water Step I I  and  dried.  ( f ) F l u o r e s c e n t sheep a n t i - r a b b i t g l o b u l i n was a p p l i e d t o the a n t i g e n f o r 60 minutes i n m o i s t chamber. (g) S l i d e s immersed i n P.B.S. and a g i t a t e d f o r 10 minutes.  occasionally  (h) S l i d e s were r i n s e d w i t h water and mounted w i t h b u f f e r e d g l y c e r o l (P.B.S.: g l y c e r o l = 1:9). D i r e c t F.A.T.  I n d i r e c t F.A.T.  - simpler procedure - more s p e c i f i c - each a n t i s e r u m must be labeled - uses g r e a t e r q u a n t i t i e s of s p e c i f i c antiserum  - more complex procedure - more s e n s i t i v e - o n l y one a n t i s e r u m must be l a b e l e d - uses l e s s s p e c i f i c antiserum  Figure 7  Comparison  o f D i r e c t and  I n d i r e c t Methods  23 Microscopy  P r e p a r e d s l i d e s were o b s e r v e d w i t h a L e i t z O r t h o l u x microscope equipped w i t h a h i g h - p r e s s u r e mercury vapor lamp. system i n c l u d e d and  a BG12 (4 mm)  a K430 m^ b a r r i e r f i l t e r  and UG1 (1 mm)  The f i l t e r  filter  as e x c i t i n g  ( C u l l i n g , 1963).  Antigen  Reagent f o r Step I  Reagent f o r Step I I  Homologous  - P.B.S. - normal serum -adsorbed s p e c i f i c antiserum - specific antiserum t o C4D4 -specific antiserum t o C4D4  - labeled  Heterologous (VI QQ)  specific antiserum t o C4D4  Figure  8  filters  - labeled -  labeled  -  labeled  Result  antiglobulin "  - . l a b e l e d normal serum  - labeled  T y p i c a l S t a i n i n g T r i a l With For One Fungus  antiglobulin  Controls  Results  I t was of the t r i a l s addition  n o t p o s s i b l e by any of the t h r e e methods n o r f o r e i t h e r t o demonstrate the  t h e r e was no f l u o r e s c e n t  p r o d u c t i o n of a n t i b o d i e s .  In  s t a i n i n g when the procedure o f  Goldman (1968) was c a r r i e d o u t .  Discussion  Researchers p i o n e e r i n g  the s e r o l o g y  o f f u n g i were o f t e n  frustrated  24 by the extreme variation i n the immunizing capacity of fungal antigens. For example, Coons and Strong (1928) and Nelson (1933) were not able to produce antisera, to several species of Fusarium, that was of s u f f i c i e n t l y high t i t e r for precipitin  tests.  Fusarium species produced a high t i t e r easily.  However, other Seeliger (1960)  reported that no r e l i a b l e method of immunization had been worked out, as of 1960, for the medically important fungal genus, Cryptococcus. Many experimenters working with fungi have worked with yeast or yeast-like forms or with spore or conidial suspensions which can be treated with techniques similar to those used for bacteria.  It i s  the hyphal fungi that have proven most d i f f i c u l t to handle. Unfortunately i t was not possible i n this study to produce antisera to the hyphal or yeast-like fungi. At the time the f i r s t t r i a l was being attempted the author had only a very cursory understanding of the principles and techniques of serology.  As such, two papers (Preece and Cooper, 1969; Eren and  Pramer, 1966) successfully applying F.A.T. to fungi were blindly followed with some minor modifications.  C e l l wall fragments were  used as the antigen because i t was the wall of an intact hypha that would be contacted by the conjugated antibody.  Gordon stated that  "the c e l l wall i s the exclusive s i t e of r e a c t i v i t y i n intact c e l l s of most fungus species."^ In spite of not being able to detect antibodies in the f i r s t t r i a l the indirect staining procedure was attempted because of the following statement by Paton:^ 5  Gordon, M.A., 1962. Differentiation and c l a s s i f i c a t i o n of yeasts by the Coon's fluorescent antibody technique, p. 214.  6  Paton, A.M., 1964. J . Appl. Bact. 27(2): 241.  25  On o c c a s i o n , a serum has been produced which showed no a g g l u t i n a t i o n t i t e r but p r o v e d t o be o f e x c e l l e n t v a l u e for t h i s technique. The o n l y v a l i d c r i t e r i o n 'of usef u l n e s s i s the s t a i n i n g r e a c t i o n i t s e l f . ^  No  s t a i n i n g was  Dr. R.J.  observed  however so e x p e r i e n c e d  Bandoni suggested  t h a t the c e l l  a i d was  solicited.  w a l l s o f the f u n g i b e i n g  s t u d i e d might be so s i m i l a r t h a t each fungus would c r o s s - a d s o r b  the  a n t i b o d i e s f o r the o t h e r a n t i g e n o r , i f not adsorbed, would form a nonspecific stain. s t u d i e d i t was antisera.  S i n c e o n l y the c r o s s - a d s o r b e d  a n t i s e r a had  been  p o s s i b l e t h a t a n t i b o d i e s might be p r e s e n t i n the  raw  T h i s proved n e g a t i v e a l s o .  The p l a n  and  r e s u l t s of t r i a l  one were taken  the U.B.C. Department o f M i c r o b i o l o g y . F.A.T. but had  He had  no e x p e r i e n c e  attempted some f u n g a l s e r o l o g y .  the c y t o p l a s m i c  f r a c t i o n as a n t i g e n  to Dr. J . J . Stock  He  suggested  (Hook e t a l . , 1967)  every two weeks i n two p l a c e s on the r a b b i t ' s back.  and  of  with using injecting i t  Antibodies  to  the s o l u b l e a n t i g e n would be e a s i e r t o d e t e c t than they had been i n the f i r s t  trial.  no s t a i n i n g  However, once a g a i n no a n t i b o d i e s were d e t e c t e d  observed.  S i n c e the second t r i a l attention.  and  an important  paper has  B u r r e l l , C l a y t o n , G a l l e g l y and  Lilly  come t o the  author's  (1965) s t u d i e d the  f a c t o r s a f f e c t i n g the a n t i g e n i c i t y of the mycelium o f t h r e e s p e c i e s o f Phytophthora. of  They subcutaneously  fourteen-day-old  injected soluble mycelial  c u l t u r e s i n t o r a b b i t s and  the p r e s e n c e o f p r e c i p i t a t i n g a n t i b o d i e s . p r e p a r a t i o n s were low  i n p r o t e i n and  wouldn't be s t i m u l a t e d .  suspensions  f a i l e d t o demonstrate  They h y p o t h e s i z e d  therefore antibody  t h a t the  production  B u r r e l l e t - a l . determined the f a c t o r s t h a t  i n f l u e n c e the n i t r o g e n c o n t e n t o f mycelium and  found  t h a t the p r o t e i n  26  content  p e r u n i t weight o f mycelium was  i n c u b a t i o n when ammonium s u l f a t e was n i t r o g e n source.  Rabbits  greatest after  used at two  If  time had p e r m i t t e d  c o u l d be measured by  s o l u t i o n c o n t a i n i n g 50 p e r c e n t (20 mg  cytoplasmic  of p r o t e i n .  injected  i n P.B.S.  and  in  conducted t e n days a f t e r the l a s t  thereafter u n t i l a s u f f i c i e n t l y high t i t e r would be  microOne  the form of a  50 p e r c e n t  of  Young r a b b i t s would  i n j e c t e d once a week f o r f i v e weeks (Pepys e t al.,1967) w i t h bleedings  Protein  Kabat e t a l . , 1961).  Freund's adjuvant  of protein/ml)  a  the E i u r e t r e a c t i o n or by  of a n t i g e n would be s u b c u t a n e o u s l y  the a n t i g e n  this high-protein,  the author would have p r e p a r e d  K j e l d a h l a n a l y s i s (Campbell e t a l . , 1964; milliliter  by  as  production.  antigen c o n t a i n i n g ten m i l l i g r a m s per m i l l i l i t e r concentrations  grams p e r l i t e r  immunized s u b c u t a n e o u s l y  s o l u b l e a n t i g e n y i e l d e d a good p r e c i p i t i n  t h r e e days of  i n j e c t i o n and was  t r e a t e d as p r e v i o u s l y d e s c r i b e d and  obtained.  specific  be  trial  weekly The a n t i s e r a  antibodies  d e t e c t e d on g e l double d i f f u s i o n p l a t e s . If i t may and  the f i n d i n g s of B u r r e l l et a l . (1965) are g e n e r a l l y a p p l i c a b l e be n e c e s s a r y  to p r e p a r e  each d i f f u s i o n t e s t .  preserve  antigens  a f r e s h a n t i g e n f o r each  injection  These workers were not even a b l e to  f r o z e n a t -20°C.  There was  a l o s s of  precipitating  a c t i v i t y w i t h i n a week.  I t would be hoped t h a t the use of a s o l u b l e  h i g h p r o t e i n a n t i g e n and  f r e s h a n t i g e n f o r each treatment  r e s u l t i n s p e c i f i c antibody may  production.  might  However, the a n t i b o d y  not be s p e c i f i c i n which case i t would be n e c e s s a r y  to f r a c t i o n a t e  the a n t i g e n complex u n t i l a s p e c i f i c a n t i g e n would produce a antibody.  formed  specific  27 Though the results of this project were not encouraging i t i s f e l t that new attempts, keeping Burrell et al.'s (1965) studies i n mind, could be successful.  Many years of medical mycology research have  shown the technique's f e a s i b i l i t y with pathogenic fungi of mammals. The basic methodology i s available and could be extremely useful i n plant pathology.  In addition to the uses envisioned i n this study  the F.A.T. could be used i n such i d e n t i f i c a t i o n situations as for spores on spore traps and for mycorrhizae.  Other p o s s i b i l i t i e s include  using the technique to study disease development, for example i n structures l i k e cankers.  28 GLOSSARY agglutination reaction - a reaction involving the clumping or aggregating of large particulate antigens (e.g., bacterial cells) by specific antisera; i n this case the particles are large enough to be seen under the microscope. antibody - a humoral globulin produced i n response to the parenteral introduction of an antigen into an animal; this globulin w i l l react with i t s homologous antigen. antigen - any substance which, when introduced parenterally into an animal w i l l cause the production of antibodies by that animal and which w i l l reaction s p e c i f i c a l l y with those antibodies. conjugated - a state i n which the antibody i s coupled to fluorochrome; synonymous with "tagged ,' 1  fluorochrome - a substance which fluoresces under u l t r a v i o l e t stimulation and can be conjugated to antibody. F.A. - fluorescent antibody abbreviation; indicates antibody conjugated with florochrome. F.A.T., F.A. staining, immuno-specific staining, immuno-fluorescent staining - terms used interchangeably to denote a staining method using fluorochrome-labeled antibody as specific staining agents for antigen. gel diffusion - antigen and antibody are brought together, through diffusion in. a semisolid, where they precipitate to form v i s i b l e bands. parenteral - other than by way of the intestines. p r e c i p i t i n reaction - a reaction occurring between large antigen molecules and antibody molecules resulting i n the formation of an antibody-antigen precipitate. tissue powder - a preparation used to eliminate nonspecific fluorescence. The powder i s usually from the tissue to be stained and i s produced by acetone precipitation of the tissue homogenate. t i t e r - the highest d i l u t i o n of an antiserum that w i l l show detectable agglutination or p r e c i p i t i n reaction; concentration or a c t i v i t y or potency of a serologically active substance.  29  REFERENCES  A l - D o o r y , Y. and M.A. Gordon, 1963. A p p l i c a t i o n o f the f l u o r e s c e n t a n t i b o d y p r o c e d u r e s t o the study of p a t h o g e n i c dematiaceous f u n g i I . J . B a c t . 8 6 ( 2 ) : 332-38. Amos, R.E. and R.G. Ceratocystis.  B u r r e l l , 1966. P h y t o p a t h . 57:  Serological differentiation i n 32-4.  Beno, D.W. and O.N. A l l e n , 1964. Immuno-fluorescent s t a i n i n g f o r i d e n t i f i c a t i o n o f P u c c i n i a s o r g h i germinated u r e d o s p o r e s . P h y t o p a t h . 54: 872-3. B u r r e l l , R.G., C.W. C l a y t o n , M.E. G a l l e g l y and V.G. L i l l y , 1965. 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