MICROBIAL ACTIVITY IN MAPLE THEE TAPHOLBS AND ITS RELATIONSHIP TO SAP YIELDS by Jack M a r s h a l l Sh eneman A THESIS S ubm itted to the Scho ol of Adva n c e d Graduate Studies of M i c h i g a n State U n i versity of Ag riculture and A p p l i e d Science in partial ful fil lme nt of the requ irem ent s for the degree of D O C T O R OF PH ILOSOPHY D e p a r t m e n t of M i c r o b i o l o g y and Publi c Heal t h 1957 ProQuest Number: 10008583 Atl rights reserved IN FO R M ATIO N TO A LL USERS The quality o f this reproduction is dep ende nt upon the quality o f the copy subm itted. In the unlikely event that the author did not send a com plete m anuscript and there are m issing pages, these will be noted. Also, if m aterial had to be removed, a note will indicate the deletion. uest ProQ uest 10008583 Published by P roQ uest LLC (2016). C opyright of the Dissertation is held by the Author. All rights reserved. This w ork is protected against unauthorized copying under Title 17, United States Code M icroform Edition © P roQ uest LLC. P roQ uest LLC. 789 East E isenhow er Parkw ay P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 MICROBIAL ACTIVITY IN MAPLE TREE TAPHOLES AND ITS RELATIONSHIP TO SAP YIELDS by Jack Marshall She neman AN AB STRACT S u b m i t t e d to the School of Advan c e d Graduate Studi es of M i c h i g a n State U n i v e r s i t y of Agricul tur e and Appl i e d Science in partial fulfil lmen t of the req uir ements for the degree of D O C T O R OF P H I L O S O P H Y D e p a r t m e n t of M i c r o b i o l o g y and Public H e a l t h 1957 Approved b v ( ,/v f<2i£/^A /T) - ABSTRACT The influe nce of m icrobial a c t i v i t y in the maple taphole on sap yield has b e e n studied durin g three seasons of sap flow. Sap yiel d s and m i c r o b i a l popu lations w ere f oll o w e d and l U ^ isola t e s of m i c r o o r g a n i s m s w e r e obta ine d by fr equent sampling of sap from 398 tappings. Va rious dates and pr oc edur es of t a p ­ ping and v a r i o u s treatments of the tapholes w e r e investigated. M icr o b i a l a c t i v i t y in the taphole w a s foun d to be r e ­ sponsible for prema tur e dec line in sap yields. teria, yeasts, Although b a c ­ and molds w ere all capable of causing d e creased y iel d s in inoculated tappings, ba cteria and yeasts wer e the most pr eval ent types of or gani sms fo und in n a t u r a l l y infected tappings. Corre l a t i o n s b e t w e e n mi crobial a c t i v i t y in the tap- hole and de clin e in sap yields w e r e found to be h i ghly s i g n i f ­ icant in c o m p ariso ns of ’’aseptic," type tappings. regular, and inoculat ed E a r l y tapping dates wer e shown to increase the inci den ce of prema tur e decline in sap yield because of the long er peri od of time al lowed by this technique for the d e v e l ­ opment of m i c r o b i a l acti v i t y in the taphole. Of 8 types of treatments used in a n i n v e s t igation of meth o d s of c o n trolling the grow th of m i c roorga nisms in tapholes, me rcu ric iodide and p araformaldehy de pellets inserted into the tapp i n g s w e r e the most successful. However, because of the r esi d u a l c o n c e n t r a t i o n s of these co mpounds in the fin ished sirup, they cannot be recom m e n d e d for practical application. 1 On i d e n t i f i c a t i o n of the b a c t e r i a from maple sap, it w a s fo und that gram ne gative rods c o n s titut ed more t han twoth ir ds of the total number; and of these, the P s e u d o m o n a s w a s by far the mos t f r e q u e n t l y e n c o u n t e r e d genus. The species P s e u d o m o n a s geni c u l a t a ap peared to be the mos t prevalent species, tions. amoun t i n g to t h ree-fourths of all p s e u d o m o n a s i s o l a ­ All yea sts present w e r e fo und to be n o n - f e r m e n t a t i v e , a n a s c o s p o r o g e n o u s types. The y eas ts w ere f o und to represent 5 d i ffere nt gene ra and 8 species. Twe nty -fi ve precent of the yeast cu ltures w e r e placed in the genus C r y p t o c o c c u s : and about 20 percent w e r e cl ass ified as T r i c h o s p o r o n p u l l u l a n s . The largest grou p of mold cu ltures (nearly one-third) were u n i d e n t i f i e d and con sidered to belong to class B A S I D I O M Y C E T E S . The rem aining two-t hir ds w e r e d i s t ributed among 9 genera. 2 ACKNOWLEDGMENTS The author wishes to ex pr es s his sincere a ppreciation to Dr. R. N. C o s t i l o w for his interest and constant guid anc e d u ring this investigation. He is also grat e f u l to Prof e s s o r P. W. Robbi ns of the D e pa rtment of F o r e s t r y for his m any he l p f u l suggestions and gen er ous assistance during the three sap seasons in the map le woodlot, and to Dr. E. S. Beneke of the D e p a r t m e n t of Bot any and Plant P a t h o l o g y for his as sistance in the m o l d i d e ntification studies. the three graduate students assi gne d to this project by the De p a r t m e n t of Forestry, Messrs. and B. M c L e m o r e . A p p r eciation is due to J. E. Douglass, The f i nancial support provided by a r e ­ sear ch grant f rom the U n i t e d States De partme nt w h i c h made this acknowledged. G-. H. Rogers, of Agricul tur e investi gati on possible is also grat e f u l l y TABLE OB COT. TENTS P a G-E LIST 07 F I G U R E S . . . . . . . . . . LI S T OP' TABLES ............ . . . . AOLTOBLEDGLLNTS ................... .. . iittroductiot • • f r* * . . . . . . . . . . HE V I E W OF L I TERATURE * - . • * • • * * • 20 * 20 ..................... Sar:oling L e t h o d s ......... .. 1 e . . . . . . . . . B U P E R I E E 1'’TAL K E T HCDS ATI) PROCEDURES Ta p p i n g Methods * 21 . . . . lie a aa ,reno n t ana Cel le cticn of S a a * • » 23 C^uant 1 tat I B o c t er icleg i c&l r.ethods ' ■ . . . . . - « 23 o * • • * • • • eg froci I an a a U a -aa in In.vs s 11 ga t lug too hoi 3 of organ is* is in Prer.,a turn Decline of a a ; o iovr « • • * . oo . c . * * 26 IT ant If icaa tion Methods Proc c o.■'.v e s of RESULTS . . . , . . • * . the Tag ho le Tro?. t n rt 3 1 :n . ClC" <■ . . . . . . . . . . . . . 33 M i c r o h i a l Populations end ’I I 31 c.s of M A sop 1"* " and hegnla^ Tapp In'; s . . . . . . . . Taphole IrecolatIr^ Taphole Trertoe^t 3 dofa. a Studies I d £>n 1 1f l e c t i o n S t m i a s . . . . . . DISCUSSION . . . StTfEART ............................. . . . pi . „ . . * #> * • • l,o* T * . . ?a 66 . . . . . . B I B L I O G R A P H Y .............. APPENDIX - - • . . . . . . 33 . . . . . . . . . " . . * . . . - * —r1 77 LI3T OF TABLES TABLE I. F AO Identity of culture s none in tree incoalat ion studies II. III. IV . V. Aver a; r o C l t j of (It;"6 sers(u) . ........................... 27 of yields in r e ondocd srt. 9g t ;•g ~ and tupping: (1956 sea s o u ) ................. 53 Aver°:'gs of yields in pounds of saL ey treatment and Inoculctic ' (1957 s e n s o r ) . . . . , . . . . . Origin of crltures i sole ted. fr or: no ole ta ohole s (1956 su. u O . ..... ........... . Licrocrg?ui sms isoiul fro.. . VII. VIII. TV -i-Jw ft In to rhole s (1955 58 Lae ter i'" isolated fror Yeas t s isolate a fro-. :iaglc tag'holes tagholes Ilclds isolated free: ra ole t: edoles Microerg a l e g 55 57 sec son.) VI. xlj iso la. (1955 season). (13 55 sea s on) (1955 se :.s:u) ... Tree: .eagle ta:: .sirs (1958 . 60 . 62 . 63 LIST OP FIGURES FIGURE 1. 2. 3« Lj.. 5i ^6 . P AGE M i c r o b i a l a c t i v i t y in sap fro m "aseptic" and regul ar tappings during the 1 9 5 5 s e a s o n ..... 3 I4. Average yields for "aseptic" and reg ular tappings according to date of tapping and percent increase In y i e l d prod uce d by the "aseptic" tap pin g • • 39 A c t i v i t y of bacteria and yeasts in sap f r o m n a t u r a l l y infected reg ular tappings during the 1955 season ........................................ I4.O Cum ula tive yields of sap from "aseptic," re gular and inoc u l a t e d tappings for two ta pping dates duri ng the 1 9 5 6 season ........... Ij.2 Aver a g e yields of sap f rom "aseptic," regular and y'inoculated, tappings during the 1 9 5 6 season for trees having yeast, bacte ria and m o l d in oculations M i c r o b i a l ac tivity in sap f r om "aseptic," regular and inoculated tappings of trees havi ng three types of tree inoculations . . <................. . C o r r e l a t i o n of differences In m i c r o b i a l c o n t a m i n a ­ tion w i t h di fferences In yields of sap f r o m "aseptic" and i noc ul ated tappings of the 1 9 5 6 season ............ ....................... .. 9« 10. 11. 12. 36 C o r r e l a t i o n of di ffe ren ces in m i c r o b i a l c o n t a m i n a ­ tion w i t h differe nce s in yields of sap f r o m "aseptic" and regular tappings of the 1 9 5 5 and 1956 s e a s o n s ............. .................. 7. 8 . Ip8 M i c r o b i a l a c t i v i t y in sap fro m inoc ula ted and u n ­ in oculated tappings: para for maldehyde treatments M i c r o b i a l a c t i v i t y in sap f rom inocu lat ed and u n ­ inoculated, treated and u n t r e a t e d tappings: m erc u r i c iodide and sterilite powder treatments • M i c r o b i a l a ctivity In sap f r o m ino cul ate d and u n ­ i n oculated treated and un tre ate d tappings: 0-SIl ver solution and plastic tubing treatments.. Ave rag e yields of sap from treated and u n t r e a t e d tappings of i n o culated and unino c u l a t e d trees ac cor ding to treatment . . . . . Iplp . 50 5 l 52 53 1 INTRO D U C T I O N The art of producing maple the sap of the sugar maple syrup and m apl e sugar f r o m tree has long been know n by i n ­ h a b i t a n t s of the n o r t h e a s t e r n part of the N o r t h A m e r i c a n con­ tinent • primit ive Indian knowledge of this process is I n d i c a t e d by the tapping and sap col lec tion devices w h i c h have been f o u n d in map le forests know n to have been f r e quented by the Indians. Also, tap marks in the saw logs from an cient maple trees in these areas have ofte n g iven mute evidence of the r e d man's e a r l y ac tivity in this industry. Brown (1919), early re cords have p r o d u c i n g ma ple A c c o r d i n g to shown that the India ns were products as early as 1673- tlers found the In di ans using maple E arly whi te set­ sugar for trade pur pose s as w e l l as for their own consumption. The settlers learned the art as practic ed by the Indians, qu ickly and use d their primitive meth o d s w i t h little change In practice. A slash on the side of a tree w i t h a hatch et served as a taphole, and a piece of w o o d drive n into the slash became a spile. C o l l e c t i o n cont aine rs for the sap were often h o l l o wed-out pieces of logs. press ion s stones The Indians po ured the sap into h o l l o w d e ­ in rocks, or into w o o d e n bowls, into the sap to evaporate and d r o p p e d h e ated the water from the sap. most impor tant c o n t r i b u t i o n of the white settlers to this The 2 pro cess was the use of a large iron pot over an open fire for an evaporator* of the sugar bush, 3 ap the sap was was pou red into the kettle. suppor ted f r o m a tripod Here, in a centra l part c ontinuously boil ed d o w n as n e w The syrup was r e m o v e d f r o m the kettle on ly at the end of the day w h e n all of the coll ect ed sap h a d been evaporated. U n t i l the latter part of the 19th century, of syrup an d sugar p r o d u c t i o n chang ed but little. was bored w i t h a bit, wood, inser ted into the techni que s The taphole and a cone -shaped spile, u s u a l l y mad e the hole. of The collection containers were w o o d e n bu ckets inst ead of h o l l o w logs. The bo il ing process still r e q u i r e d the use of the iron kettle u n t i l the i n t r o d u c t i o n of the first open pan evaporator, ab out i860* Since this time, the w o o d e n spile has been r e p l a c e d by m etal spiles of va rious designs, and the w o o d e n buckets have g i ven way to m e t a l buckets and plastic col lection bags. The area of the U n i t e d States of maple products tivity includes in w h i c h the p r o d u c t i o n is a r e l atively important agri c u l t u r a l a c ­ ten states and extends along the n o r t h e r n part of the U n i t e d States f r o m New E n g land on the M i n n e s o t a on the west, and to K e n t u c k y on the accor d i n g to Will i t s and P o r t e r east to south. Althou gh, (195>0)> m a p l e products do not constit ute a m ajor a g r i c u l t u r a l co mmo dit y in any of these states, there are areas in each of these states where maple pr oducts are an importa nt part of the economy. maple In 1955* p r o d u c t i o n In the U n i t e d States a m o u n t e d to l5l>000 3 p o u n d s of sugar and 1 ,6 5 7 , 0 0 0 at •?_}_ m i l l i o n dollars Service, gallons of syrup, and wa s valu ed (Michig an Crop and L i v e s t o c k R e p o r t i n g 1955)* Since the p r o d u c t i o n of ma ple produc ts on the f a r m o c ­ curs during the w i n t e r and early spring f a r m acti vit ies are at a minimum, seasons w h e n other the o p eration of a woodlot for this purpose gives the f a r m e r an o p p o rtunity to use o t h e r ­ wis e u n p r o d u c t i v e time in tne p r o du ction of a cash crop. cording to Moore, A n d e r s o n and Ba ker (1951), Ac­ the average p r oduction cost per g a l l o n of syrup in Ohio for the period 1924-6 to 1924-9 was $2,924-, while producers was $2i.fe9* the average price r e c e i v e d by This left an average net income of $1.75 per gallon. Ea r l y investigation s into the p r o d u c t i o n of maple products were concerne d primar ily w i t h techniques of e v a p o r a ­ tion of the sap and w i t h methods of improving the sugar b u s h s ilv iculturally in order to increase In r e cent years, however, the prod ucti on of sap* an incre ase d interest in this i n d u s ­ try has stim u l a t e d r e s e a r c h into m a n y aspects of syrup and sugar production. R e s e a r c h has been initiated into the chemistry of the flav or and color components of the syrup determine their origin. The to des ign of the evapo rato rs and the source of h e a t for the e v a p o r a t i o n process has also been investigated. Spile des ign and me thods of tapping the trees and coll ecti ng the The m a p l e sap has been gi ven careful consideration. tree taphole itself has been in vestigated f r o m the k stand poi nt of compass p o s i t i o n of the taphole, tapping, de pt h and diame ter of the taphole, heig ht of and. best tapping date • Producers some maple of maple tappings products have long o bserved that stop p r oducing sap relat i v e l y early in the sap sea son during some years, while other tappings con­ tinue to produce h e a v y runs of sap u n t i l the v e r y end of the season. Also, tapholes w h i c h show this early decline in produ c t i o n often are found to produce cloudy sap w h i c h has sharp or u n p l e a s a n t flavors and odors. Such sap is k n o w n to require m u c h longer boiling periods using m u c h gr eater volumes of sap to produce of clear, the sweet sap* same q uant ity of syrup than is r e q u i r e d The syrup p roduced f r o m such cloudy sap is often d ark and off-flavored, and is therefore of a m u c h lower quality than n o r m a l syrup. The presen t this study was in iti ated to determine whether premature decline in sap prod uct ion f r o m the taphole is the result of m i c r o b i a l ac tivity w i t h i n the taphole during the sap season; hole; the sp ecific m i c r o f l o r a present and to de ter mine produc ing premature the imp ort ance of the taphole stoppage. in the tap- m i c roflora Finally, in metho ds of control lin g or preve nti ng m i c r o b i a l inf ect ion of the taphole were c o nsidered in an attempt to find a practic al means of controlling m i c r o b i a l activity, and thus, and quantity of sap produ ced by the tree. increase the quality R E V I E W OF L I T E R A T U R E Map le during sap is a sweet clear fluid n o r m a l l y pr esent late w i nter and early spring in the tissues species of trees of the genus A c e r . the car bohydrate Edson (1910) of certain reported content of the sap to be k to 3 percent sucrose w i t h traces of invert sugar. In addition, small amounts of pr otein material, m i n e r a l matter, m a i n l y calc i u m and potassium, to be present. and of acids, The h y d r o g e n ion c o ncentration of sap was d e ­ termined by Holg ate and D u g a l pri mar ily mal ic acid, wer e found (19ip0). (1950) confirming pr evious w o r k by Bois The extreme range d e t e r m i n e d f r o m various samples w a s pH 6.0 to 7-3 w i t h an average of pH 6.6; Holgate however, (1950) r e p o r t e d that in only a few instances were the values of p H 6.5 to 7.1 exceeded. No significant dif fer enc es in pH were app are nt between aseptically co llect ed sap and u n ­ spo ile d regul ar sap. teristics make Edson (1910) co nclu ded that these c h a r a c ­ the sap a good m e d i u m for the g r o w t h of m i c r o ­ organisms p r o v i d e d suitable temper atu res are maintained. The seaso n dur ing w h i c h maple sap flows in the tree occurs only w i t h i n the three or four w e e k period immedi ate ly precedi ng the annu a l unfol d i n g of the leaf buds in the spring. The sap flow is not continuous but occurs in short periods or runs de pend ing u p o n w e a t h e r conditions. F r e e z i n g nights 6 f o l l o w e d by m o d e r a t e l y w a r m days charac teri ze di tion s for sap flow a c cording g a t h e r e d early in the flavor. As the to E d s o n the best (1910). sea son is transparent, The con­ sap w i t h a sweet cl ea n sea son progr ess es and the te mp er atur es c o m m e rcially g a t h e r e d sap u ndergoes m a r k e d changes, increase, bec omin g cloudy and d i s c o l o r e d w ith the dev elo pme nt of u n p l e a s a n t flavors and odors r e s u l t i n g f l a v o r e d sirup. in lower quality, darkly colored, off- E d s o n r e p o r t e d that such spoiled or " s o u r ” sap rapi d l y d e t e r i o r a t e d u p o n storing for only a few hours. M i c r o o r g a n i s m s in Maple Sap Sap w i t h i n the tissues of mapl e aseptic tapping techniques, 1955)* ob tained by has been proven to be es sent ial ly sterile by a number of workers Nagh ski and Willits, trees, (Edson, However, 1910; Holgate, contami nat ion and g r owth of m i c r o o r g a n i s m s may occur as the sap flows f r o m the ti ssues into the tap hole and through the spile, the co llec tion or hand ling of the 1950; tree or during sap prior to its f i nal p r o ­ cessing into f inis hed sirup. The po pula tio ns of m i c r o o r g a n i s m s spoiled saps were d e t ermine d by Edson, in spoiled and u n ­ Jones and Carpent er C o l l e c t i o n of sap under very sanitary but not sterile r e s u l t e d in a con side r able r e d u c t i o n of contamination. ations of m i c r o b i a l popul ati ons under these 5 to 500 or ganisms pre sent per m l of sap. (1913)* conditions Determin­ conditions r evealed C o m m e r c i a l l y collected 7 u n s p o i l e d sap, however, e x h ibited a m u c h higncr degree of co n t a m i n a t i o n w i t h po pula tio ns of llpO to 1 ,0 0 0 , 0 0 0 per ml. The gr eatest popul a t i o n s o c c u r r e d in sap col lected late in the sea son during warmer w e a t h e r conditions. sp oiled after storage for a short time, and u n d e s i r a b l e flavors. This sap became de vel oping cloudiness Samples of spo iled saps exhibiting various degrees of cl ouding and d e c o m p o s i t i o n were examined, and the n u m bers of org anis ms direct r e l a t i o n to range present were found to bear a the degree of d e composition noted. The of c o ntaminat ion of the spoiled sap was f r o m 3 2 0 , 0 0 0 to llpl,Il20,000 or ganisms per i7il. A se quence of organisms was n o ted by Edson the season of 1909. (1910) during The pred omi nati ng organisms during the early part of the season were found to belong to the yeast group; while bacteria, w h i c h were few in numb er at first, became of importance tions by Edson, as the Jones and Carpente r this observation. (1913) Later o b s e r v a ­ did dot co nfi rm A l t h o u g h few organisms of any type were f o und early in the season, dominating type. season advanced. Yeasts ba cteria ge nerally were the p r e ­ app ear ed in important nu mbers only w h e n the season was well advanced. The specific types of organis ms occu r r i n g in u n s p o i l e d sap have not apparently been determined. p roba ble that m a n y of the various However, it is types of bacteria, yeasts, and mol ds pr esent in spoiled sap are also present as c o n t ami­ n a ting organis ms of u n s p o i l e d sap. Thus, spoiled sap probably r e s u l t s f r o m m u l t i p l i c a t i o n and increas ed m e t a b o l i s m of organ ism s pr esent v a n c e d state in u n s p o i l e d sap resulti ng in a more a d ­ of deco mposition. Types of S p o i l e d Sap ana Organisms Respon sible for Spoilage Edson (1910) studied Ip types of spoiled by v a r i o u s m icroorganisms . and C a r p e n t e r organisms (1913) A later repo rt by Edson, Jones atte m p t e d to describe and ident ify the obs e rved in the spo iled saps. spoiled sap, sap inf ect ed F r o m eac h type of org ani sms were isola ted and r e i n o c u l a t e d into u n ­ spoi led sap. The resu l t a n t sap spoilage was obser vea and the effect of the spoiled saps u p o n the qu ality of the f i n i s h e d sirups was determined. A type of spoiled sap not commonly encountered in these stu dies was true stringy sap, a stringy consistency, tion, cha rac te rize d by a m i l k y color, an acid r e a c t i o n w i t h slight gas f o r m a ­ and a strong ch ara cteristic odor suggestive of yeasts. The clarity and flavor of the finished sirup was affec t e d as a resu lt of this type of spoilage. e x a m i n a t i o n r e v e a l e d swarms of actively motile no yeasts were found. of the cultures Mi croscopic bacteria, but Plating procedures r e v e a l e d a p r e p o n ­ de r ance of bacteri al colonies of a single type. of pure seriously I nocul ation into u n s p o i l e d sap re sulted in the production ch aracteristic type of spoilage. was d e s c r i b e d as a new species, The o r g a n i s m respo nsi ble Bacillus a c e r i s , later changed t° A c h r o m o b a c t e r aceris accordi ng to Br eed et a l , (1914-^3) • This o r g a n i s m was i n dicated to be g r a m negative, a c t i v e l y motile, and rod shaped; w h e n g r o w n in carbohydrate media, it had a slight capsulation and appe are d to derive its n o u r i s h m e n t f r o m the u t i l i z a t i o n of sucrose A more common spoiled sap, sap but prese ntin g a more In the maple somewhat rese m b l i n g lumpy appearance, the close of the comme rci al sap season. Buskirk of bacte ria growing This (1935) together. f ound that rop y maple a cti v i t y in the in fected sap was stringy spoilage was a s ­ yeasts, and F a b i a n and sirup r e s u l t e d f r o m con­ t a m i n a t i o n of sap by A e r obacter a e r o g e n e s . o r g a n i s m was not found viable sap. was ob served after soc iate d w i t h the presence of filame nto us fungi, va rious types non-sp orul ati ng, A l t h o u g h this In the fini she d sirup, its in dica ted by in cre ased acidity of the sap. A sec ond type of spoiled sap studied by Edson, and C a r p e n t e r (1913) Included those g r e e n i s h or g r e e n i s h - b r o w n color, b i t t e r flavor. samples possessing a a cloudy appearance, This g r e e n sap w a s the mos t spoilage observed. Jones and a common type of M i c r o s c o p i c examination freq uen tly i n d i ­ cated the presence of several types of mic ro orga nis ms. Platin g t e c hniques always r e v e a l e d several types of colonies present. M o l d s and yeasts were oft e n present along w i t h bacteria, the b acteria were always m u c h more abundant. o b s e r v e d colony, The most frequently often 90 percent or more of the sessed a g r e e n floures cen t character. but total, pos­ These organisms wer e of 10 the P s e u d o m o n a s group and appeared to be more or less closely r e l a t e d to the liqu efy ing and n o n - l i q u e f y i n g strains of Pseudomonas that these fluooescens. isolates were of the m a p l e responsible sap. indic ate d capable cf producing g r een clouding A second type of o r g a n i s m fou nd to for this parallelus. R e i n o c u l a t i o n experim ent s type spoilage was a new species, be Bac ill us This o r g a n i s m was d e scribed as a n o n - f l o u r e s c e n t or f e e b l y f l o u rescent o r g a n i s m somewhat similar to the F s e u d o ­ monas type of o r g a n i s m isolated. The g r e e n sap organisms were thought to be n o u r i s h e d by the traces of protein material in the sap, leaving or g a n i s m s was the sugars unchanged. Sap infect ion by these cons idered to be initi ate d as a result of e x t r a n ­ eous c o n t a m i n a t i o n of the sap in the bucket tree, snow, rain, by bark from the or dust f r o m the air. A type of spoil ed sap possessing a r e d dish brown sedi ­ ment was also o b s e r v e d by Edson, a few samples. Jones and Carpent er (1913) in This red sap was found to be es p ecially r i c h in yeasts, ye ast-l ike organisms w h i c h formed red colonies, yea sts w h i c h formed grey colonies, a l r eady described. and floure sce nt organisms These va rieties of yeasts were obser ved to be present in m a n y samples of other types of s p o i l e d saps, and o f t e n in u n s p o i l e d saps as well. to ferme nt com mon sugars including The yeasts were una ble sucrose. Reinoculation ex p e r i m e n t s y i e l d e d inconc lus ive results due to diffi cul tie s in techniques. It was observed, however, that yeasts and molds proba bly de veloped in in cubated sap too slowly to be of 11 import anc e. The tw een these possibility of a sti mulatin g r e l a t i o n s h i p b e ­ organisms and spoilage bacteria was expressed* such a r e l a t i o n s h i p were p r o v e n to exist, If yeasts w o u l d then n e c e s s a r i l y be r e g a r d e d as important factors in sap spoilage because of the impetus g i v e n to bacte rial developm ent as well as because of the changes bro ught about by their own m et ab oli sm. The yea sts w e r e not further P i n k co lonies were f r o m sour i d e n t i f i e d .or described. frequently found in plates poured sap pr od uced late in the season. these were due mentioned, M i c r o c o c c u s roseus was also present. Edson, to the yeast or yeast-like A l t h o u g h some of Jones and Carpent er (1913) organisms previous iy also descr i b e d a m i l k y type of spoil ed sap possessing a ch ar acte ris tic ance of a pale m i l k y hue, and the unpleasant, f l a v o r of the g r e e n type of sap. pearly white Inoculation spoilage character, cloudy a p p e a r ­ slightly bitter Col oni es were largely of a different from those alr eady mentioned. of u n s p o i l e d sap produced char acteristic sap similar to the spoiled sample. A m ilky appe ar ance wa s o bs erved in a few hours and a bitter but not acid flavor developed. N o m a r k e d odor was detected. d e s c r i b e d as spore forming bac ter ia of the hay bacillus group. O ther samples of m i l k y sap spoilage the col lec tiv e The organisms were appeared to be caused by action of many different types of organisms. C o n s i s t e n t l y assoc iate d with the other types of organisms prese nt in the sp oiled sap were mol d spores. A l t h o u g h seldom found in grea t numbers, molds belonging to the genera P e n i c illiu m 12 end Aspergillus were isolated. I n o c u l a t i o n studies i n d i c a t e d that the m olds m a y be respo n s i b l e Tor sugar i n version in the A n o t h e r type of sap affec ting the quality f i n i s h e d sirup has b een c a l l e d "buddy sap." condi t i o n w h i c h occurs later sid ered by E d s o n (1910), has of the A l t h o u g h this in the seas on has been con­ and Edson, Jones and C a r p e n t e r to be p r imarily the res ult of micro b i a l activity, (1950) sap. (1913) Holgate shown f r o m studies of aseptic sap that this c o n d i ­ tion is due to p h ys iological changes in the tree and the com­ p osi tion of the sap itself. A definite increase in the or ganic n i t r o g e n content was fo und to bring about und esirable changes maple in the character of the products. increa ses These sap, m a king it uns uita ble for changes are coincident with the seasonal in air and soil temper atu res and also w i t h the r e ­ sultant r enewal of vegetati ve u n f o l d i n g of the leaf buds* r esp onsible activ ity as ev idenced by the The for the prod ucti on of same conditions of temperature "buddy sap" by the tree are also favor abl e for the m u l t i p l i c a t i o n of micr oor gani sms . "buddy sap" Thus, pr oduced under comm erci al conditions may also be h e a v i l y c ontamin ated w i t h such organisms. M i c r o o r g a n i s m s as a Cause of Pr ema tur e It has long been o bserved that many maple "dry u p ” or cease sap season, Stoppage to produce of Sap Pl ow tap holes sap during w a r m e r weather of the e s p e c i a l l y if the trees were ori gin all y tappe d 13 ea r l y in the season* r e s u l t f r o m the w all s This drying is popularl y b e l i e v e d to physical action of' the air or wind upo n the of the tap hole 1952). However, pre mat ure increase itself, (Barraclaugh, Naghsk i and Will i t s tap hole drying was and not w i t h physical drying. sterile clos ed condition. organis ms Cope, indic ate d that tap hole and in the sap This was de monstrated tap holes and m a i n t a i n i n g t h e m in a All inoculat ed tap holes sap after the bacteria l B (1955) 1937; co rrelated closely w i t h the of m i c r o o r g a n i s m s w i t h i n the by inoculating of 10 1 9 5 2 5 Bryan, stopped producing content of the sap r e a ched the range per ml. These results were compared w i t h other n a t u r a l l y infec ted tap holes w h i c h were not m a i n t a i n e d in a closed system. The and of sap fl ow stoppage conditions. Premature pa tte rn of g r o w t h of m i c r o o rgani sms appeared to be similar in the two stoppage duce the yield of sap by 2 0 to 7 5 by u n c o n t a m i n a t e d free fl owing produce sap thro ugh out the a p p arently i n f lue nced by of sap flow was found to r e ­ percent of that produ ced tapholes wh ich were able season. Taphole to drying was not compass location of the tap hole or by the date of tapping. Naghs ki n umb e r s cause and Will i t s (1955) also found that large of m i c r o o r g a n i s m s w i t h i n the infected tap hole can c o n t a m inat ion and ferme n t a t i o n of the flowing sap r e ­ sulting in the pro duc tio n of spoiled sap fro m the This o b s e rva tion confi rme d previous work by Edson, tap hole. Jones and Ik Carpenter (1 9 1 0 ) w h i c h indicat ed that h i g h l y co ntaminated s poi l e d sap was produ c e d f r o m in fected tap holes, P o s t - S e a s o n I n fe ction of the Tap Hole Post sea son Infect ion of the old exposed tap holes after r e m o v a l of the (1951)-) • A fungus, spile was re ported by Spros t o n and Scott Val sa l e u c o s t o m o i d e s , was i s o l a t e d and d e ­ t e r m i n e d to be the princip le cause of cone-shaped, dry, discolored, d e c ayed areas in the w o o d around old tap holes. Ninety- four pe rcent of tap ped trees were foun d to be infe cted w i t h this organism. It was s u ggested that protective metho ds be d e v e l o p e d to prevent Infection. Recommen d a t i o n s were made that old tap holes be spray painted at the end of the season, and that pr odu cers tap on the di ag onal or up and down the butt to avoid u nproductive tap wood. Ch e m i c a l and P h y s i c a l Changes in Sap and Sirup Due to M i c r o b i a l Ac tivity Cha nges microorganisms products, occurrin g in the sap due to the ac tivity of are foun d to affect the quality of the finis hed r e su lting in dark colored, ropy or o f f - f lavored sirups. H a y w a r d and P e d e r s o n (1914-1) have r e p o r t e d that bac ter ial a c ­ t i v i t y in sap causes in cre ased alkalinity and inversion of sucrose w h i c h resul ts in dark colored sirup. earlier w o r k by Edson, Jones and Ca rp ente r This confirmed (1913) who obs erve d that b a c t e r i a l g r owth in sap causes grade of the sirup. Holgate (195b) b a c t e r i a l p o p u l a t i o n increases, increases In crease and sucrose the a decrease in the color also found that as the Invert sugar content content decreases. Al tho u g h no m a r k e d in a l k a l i n i t y was obs erved in re gular sap as com­ pared to ase ptica n y sathere d sap, exh ibit incr e a s e d alkalinity. badly spoiled saps did F a b i a n and Bu skirk (1935) fou nd that c o n t a m i n a t i o n of sap w i t h Aero bacter aerogenes causes an increase in the a c i dity of the of m aple sirup. N eutr a l i z i n g the acidity of the f e r m e n t e d sap r e d u c e d the ropy The sap and r esulted in a ropy type condit ion of the resu lti ng cause of 11buddy" sirup. flavor of the late season sirup was foun d to result f r o m previou sly m e n t i o n e d phy sio log ica l changes In the sap itself, and not from m ic robial activity. This fla vor was app arent in sirups pre pared from both sterile and r e g u l a r saps co llected late in the season. Holg ate c onc l u d e d that the m os t common forms of mi cro organisms in maple (icBo) present sap have a greater effect upo n the color than the fl a v o r of the sirup. seriou sly affect A l t h o u g h some types of organisms may the flavor, for the c h a racteristic the tree itself is responsible "buddy" flavor of late season sirup. C o n trol of Microorg a n i s m s In Sap The pri mary m e t hods of control of sap spoilage o rga ni sm s are based u p o n efforts by m i c r o ­ to mini miz e conta min atio n at 16 the various to r estrict steps in collecting and ha ndling of the sap, and the activity of the organisms in the sap to the sh ortest p e r i o d of time. Cleanlines s has been the most ef­ fectiv e me ans of c o n t ro lling c o n t a m ination of sap in spiles, bu cke ts and storage tanks* Edson, Jones and Carpen ter (1913) f o u n d that c o l lection of sap under sanitary conditions using sc alded spouts and buckets, procedures, and a daily bucket covers, careful tapping sap collection routine of replaci ng e a c h bucke t w i t h one w h i c h h a d been scalded, w o u l d reduce c o n t a m i n a t i o n greatly. C o n t a m i n a t i o n resulting after a p p l i ­ cation of these proced ure s was 5 to $00 organisms sap, per ml of compared to co nt amination of commerc ial ly col lec ted sap r e s u l t i n g in the presence of lipO to 1,000,000 organisms M e t a l u t e n s i l s were found to be preferable were nore re adily cleaned. to woo d because they C o v ered buckets were found to aid in preve n t i n g the entrance of extraneous and r a i n and snow. per ml* cont ami nat ed m a t e r i a l It was r ecommen ded that sap be con centrated as soon as pr ac ticable after col lec tio n to shorten the time dur ing w h i c h organisms may be active in the sap. A n o ther m e t h o d commonly advoc ate d Bryan, 1937; Cope, 1952) r e a m i n g of the tap hole (barraclaugh, 1952; to prevent contam ina tion of sap is to remove accumula ted organisms and d ebri s w h i c h infect the sap and produce drying of the tap hole. Nagh sk i and Wi llits (1955) r eported that the yield of sap f r o m dried infec ted tap holes after reaming was negligible. 17 This o b s e r v a t i o n confirmed reports of producers N a g h s k i and W i l l i t s (1955) sults f r o m the use of this Methods after use of the plastic sap Lane (1953) technique* The in the sap been repo r t e d by some workers* latter workers of su nlight was effective (1955) appears to be mos t found that the st eri lizi ng power in r educi ng the b a c t e r i a l content of sap expo s e d in plastic bags. The bags were found to be slig h tly tr ans parent to the longer ge rm i c i d a l wave ultra-violet Factors determ inin g light. The collection bag r epo rted by S p r o s t o n and and Naghs ki and Willits promising* maple w h i c h i n dicated disc ouraging r e ­ to co ntrol or des troy organis ms c o n t a m i n a t i o n have cited by lengths of the e f ficiency of the su nlight to act as a sterilizing agent under these conditions inclu ded temperature, the radi ati on* time of exposure, Ev idence was ob tained that sunli ght during cool we ath er became and Lane (1953) and the intens ity of sap exposed to bright practi cal ly sterile. Sproston indic ate d that a g e r m i c i d a l factor may be present in the plastic of the bag w h i c h may be activ ated by sunlight and, thus, exert g e r m i c i d a l activity upon the contents of the bag. The use of an tis eptics to control organisms were r e p o r t e d by Holg ate were (1950). in the sap A large numb er of compounds c o n s i d e r e d but all were fou nd to be ob ject ionable since they either imp aire d the quality of the sirup or were found to be toxic for h u m a n consumption. of 3 0 A di lution of 1 to 7,500 percent h y d r o g e n peroxide as an inhibit ory agent was 18 most nearly satisfactory♦ to be quite critical. undesirable fla vo r to the sirup while w o u l d not inhibit light has in storage tanks. organ i s m s of maple this dilution was found S lig htly greate r amounts the organisms Ultra-violet spoilage However, sap sl ightly smaller amounts satisfactorily. been used in attempts to prevent Acco r d i n g to a d i s c u s s i o n on the (Naghski, that ster ila mps w e r e effecti ve stored in out door tanks. imparted an 1953)> one producer in preventing spoilage of sap In the same d i s c u s s i o n it was cated that in order for these lamps to be effective, w o u l d have indicated to p e netrate the liquid. Thus, indi­ the rays either constant a g i t a t i o n of the stored sap w o u l d be req ui red to expose the liquid to the ra diat ion s, the sap or it w o u l d be n e cessary to have f low arou nd the lamp in a relat i v e l y t hi n film. The M i c r o b i o l o g y of Maple Sirup and Maple Sugar Because of the h i g h sugar conce ntr ati ons in finis hed m a p l e products, stances. menting yeas ts few organisms are However, the able to grow in these yeasts and mo lds sugars. F a b i a n and Hall can cause sub­ spoilage by f e r ­ (1933) fou nd seven groups of of the g e nera Saccharomyces and Z y g osaccharomyces were pr e sent in fermented sirups. The moisture content of sirups showing visible signs of f e r m e n t a t i o n was 32.7 to 3 ^1 * 6 percent, while the water content of fr eshly pro duce d sirups was 2 6 . 3 cent. High moisture to 3 6 . 5 per­ content of the finished sirup and the absorption 19 of m o i s t u r e by the sirups f r o m a m o i s t environment w e r e factors f a v o r i n g d e v e l o p m e n t of yeast f e r m e n tations in the sirups. Fellers (1933) in a study of the cause of spoilage of m a ple sugar stor ed in tubs f o u n d that off flavors were p r o ­ d u c e d by molds, the enzyme, sugar, tion p r i n cipally A s p e r g i l l u s . The mo lds pro du ced inverta se w h i c h h y d r o l i z e d the sucrose in the and cause d it to become moist or semisolid. Hayward (19lp6) r e p o r t e d that for maple sirup storage was a the most favorab le c o n d i ­ dry, cool environment. He r e c o m m e n d e d that the cans of sirup be f i lled and sealed while hot to d i scourage yeast and m o l d contamination. d i s c o l o r a t i o n of sirup due to oxidation, To prevent it was also r e c o m m e n d e d that the h e a d s p a c e in the cont ain er be kept at a minimum. 20 EXPERIMENTAL METHODS AND PROCEDURES A l l e x p e r i m e n t a l w o r k of this in ves tigatio n was out during a thre e- year p e riod w h i c h included the 1955> and 1957 sap p r o d u c t i o n seasons* A ma ple 20 acres in size contai nin g about 200 maple not p r e v i o u s l y been tap ped was chosen for sam plin g studies. All carried 1956 sugar bush of about trees w h i c h had the tapping and trees great er than 15 inches in diameter were n u m b e r e d and the trees sel ected for each season's study were c h osen f rom this group by r a n d o m selection. Tapping Methods Two general techniques of tapping were employed during this investigation with modifications of these techniques being used for specific studies of taphole infection and for studies of methods of treatment for control of infection. The commercial or normal technique involved boring a hole through the bark into the sapwood of the tree to a depth of about ij, inches using a bit 7/l6 inch in diameter, and driving a commercial type spile tightly into the hole. The second technique, “aseptic11 tapping, was used during the 1955 and 1956 seasons. Not all of these tappings were originally aseptic and none remained in an aseptic c o n d i t i o n t h r oughout the season. w ere u s e d to exclude micro b i a l All pr ec auti ons possible contami nat ion and thereby r e ­ duce the level of m i c r o b i a l act ivity in the tappings. fore, “a s e p t i c 1* as u s e d in this study refers There­ to this specific t a p p i n g pro cedure and not to the us ual b i o logical def ini tio n of this term. The technique u s e d for the “a s e p t i c 1* tapping stu dies was a m o d i f i c a t i o n of the and Will i t s (1955)* procedure used by Naghski The outer layers of bark were first r e ­ m o v e d f r o m an area of about if square Inches around the site of the taphole usi ng a d r a w k n i f e . w i t h alcohol, Whi le ignited, the alco ho l was still burning, Upon withdrawing spile was im mediate sap f r o m the and spile was sat urat ed and all owed to burn about 2 minutes. d ept h of about Ip inches Into inch bit. T hen the area was the a taphole was bored to a sapwood, the bit, using a sterile 7/l6 a sterile closed-type tightly driven into the taphole. The condu cted through a completely enclosed s y s t e m of rubbe r and glass tubing into a sterile 5 gallon can f i t t e d with a 2 holed rubber stopper w i t h a cotton stoppered U- tu be to equalize m ent u s e d for the the air pressure wi thin the can. All e q u i p ­ "aseptic** tapping was sterilized by auto- claving at a pressure of 1 5 pounds for 20 to 3 0 minutes. Sampling Methods Samples of sap were taken at intervals throughout the three seasons of sap flow studied, usually during the major sap runs of each season. were D u r i n g the 1955 season most taken f r o m sap colle ctio n containers and c o m mercial type tappings. of bot h " a s e p t i c 11 Sterile pipettes were u se d to tran sfe r 10 ml samples of sap from buckets, sap cans to were sterile samples sample vials. Some plastic bags and samples, however, taken dire ctl y f r o m the runni ng tapholes by h old i n g the sample via ls at the spiles u n t i l 2 or 3 ml of sap were collec ted D u r i n g the 1956 and 1957 seasons, d i r e c t l y f r o m the run ning tapholes. all samples wer e To a c c o mplish this, pint m i l k bottles were fitted w i t h wire hooks, to be hung u p o n the spiles of the taken half­ allo win g them co mm ercial type tappings. The mo u ths of these bottles were of a size w h i c h w o u l d admit the rubb er ing the stoppers of the "aseptic" same bottles collection systems, to be u s e d to obtai n samples dire ctly and asep t ically frorn both com mercial and "aseptic" Before taking samples, all bottles were foil caps and s t e rilized at 15 pounds minutes. allow­ Samples wer e tappings. covered w i t h a lumi n u m steam pressure for 3 0 taken by removing the foil caps and h a n g i n g the bottles by the wire hooks on the commercial type spiles, or by tran sfer rin g the rubber stoppers of the "aseptic" colle c t i o n systems f r o m the sap co llection cans to the mou th s of the bottles. The openings of the cans were the a l u m i n u m foil caps f r o m the bottles while wer e being after w h i c h the rubber "aseptic" taken, covered w i t h the sap samples stoppers of the collecti on systems were ret urn ed f r o m the bottles to the cans. The bottles were re moved f r o m the tappings after 15 or 20 m l of sap had been collected in each and sterile paper bottle caps were pressed over the bottle openings. Tree number and type of tapping was noted on each cap for identifi­ cation of the sample. Measurement and Collection of Sap The sap produced by the commercial type tappings was measured by weighing to the nearest tenth of a pound each day on which a sap flow occurred. The yield of sap from the "aseptic1* type tappings was determined on days of sap flow by weighing the sap in the container without removing the can from its tube connection with the taphole. Periodically, the sap in the "aseptic" containers was collected by replacing the nearly filled cans with empty sterile cans, or by dis­ connecting the can from its collection tube assembly, emptying it and rinsing it thoroughly with a germicidal solution of Poccal in a concentration of 1250 ppm before reconnecting the can to the collection tube. Quan tit ative B a c t e r i o l o g i c a l Methods P o p u l a t i o n s of bacteria, mined in all samples using yeasts and molds were d e t e r ­ standard plating and counting t e c h ­ niques. D u r i n g the 1955 and 195& seasons, u s e d for the enumeration of the bacteria, nu trient agar was and dextrose agar c o n t a i n i n g 0.1 pe rce nt yeast extract and acidifi ed w i t h 2 ml of 5 p e r cent tartaric acid solution per 100 ml of m e d i u m was u s e d for yeast and mold was 3*8* the During counts- the 1957 The final pH of this m e d i u m season total m i c r o b i a l popul ati ons samples were deter m i n e d using nutrient peptone a m e d i u m consist ing of agar w i t h 0*1 per cen t yeast extract, and 2 percent dextrose. All incuba tio n of plates was done at r o o m temper atu re for three the 1955 and 1956 seasons, to five days. repres ent ativ e plates of the highe st dilutio ns were tubes During colonies from transferred to culture con tai ning approp ria te media to preserve or ga nisms for future 0*1 percent the m i c r o ­ i dentification and study. Ident i f i c a t i o n Method s The were cu ltures divided iso late d during the 1955 and 1956 seasons into the three maj or groups of microorg ani sms , yeasts, m o l d s and bacteria, on the basis of colonial appea ran ce and m i c r o s c o p i c morp hology. The yeast isolates were further e x a m i n e d and I d e ntified using ide ntification metho ds and keys d e s c r i b e d by Lodder and K r e g e r - V a n HIj were (1952). The bacteria exami ned accordin g to procedures found in Manual of M e t h o d s for Pure Culture te r i o l o g i c a l Technique, 19lp6). Study of Bacteria (Committee on B a c ­ Society of A merican Bacteriologists, Spec ifi c i d e n tification was accomp lis hed using keys found in the 6th edit ion of B e r g e y ’s Manu al of De terminative B a c t e riology (Breed et al. , 19lp8) . The m o l d cultures were e x a m i n e d only for m o r p h o l o g i c a l ch aracteristis and were iden t i ­ f i e d to genus using Barnett (1955) and Beneke (1956) as refer enc e 25 P r o c e d u r e s U s e d in I n v e s t i g a t i n g the Role of M i c r o o r g a n i s m s in P r e m a t u r e D e c l i n e of Sap Flo w To inve sti gat e m atu r e de cline the re lationships exist ing be tween p r e ­ in sap prod uct ion fro m the taphole and m i c r o b i a l a c t i v i t y w i t h i n the t a p h o l e , studies were 1955 and 1956 seasons in w h i c h m i c r o b i a l po pul atio ns and sap yiel ds of "aseptic" and regul ar were compared. conduct ed during the tappings on the same trees The relationship b e t ween various dates of t a p ­ ping and m i c r o b i a l a ctivity in the taphole was also studied dur ing the 1955 season. Te n trees were approximately tapped on each of 5 dates spaced at 15-day in tervals be ginning on January 19, 1955* The dates were cho sen so that the first three dates w o u l d fall before the n o r m a l tap ping peri od for the Fast Lansing area, the f o u r t h date durin g this period, the n o r m a l tapping time. "aseptic" and a regular and the last date after E a c h tree was tapped w i t h an tapping six inches apart on the same q uad rant of the tree accordi ng to compass tions of m i c r o o r g a n i s m s were position. Popula­ in sap f rom both types of tappings dete r m i n e d at interv als during the season and me asurements of yields of sap during the season were recorded. D u r i n g the 1956 season 15 trees were tapped on each of two dates, 7 and F e b r u a r y 29. of the tree Fe bruary E a c h tree had 3 tappings on the same half spaced at approximatel y 6-inch intervals. One tapping was a c c o m p l i s h e d in the regular commercial manner while the ot her two w e r e One of the latter, tapped according however, to the "aseptic" technique. was inocul ate d w i t h 2 ml of a cul­ ture of a specific m i c r o o r g a n i s m or gr ou p of mi cr o o r g a n i s m s just prior to ins ert ing the "aseptic" The cultures chos en for the ino culations r e p r e s e n t e d the five m a j o r groups of bacteria, yeasts, durin g spile. the five majo r grou ps of and one of the groups of molds w h i c h were isola ted the previous season and subseq uen tly identified. The o r ganisms u s e d and the number of trees in ocul ated w i t h eac h are shown in Table X. Bacte ria and yeast cultures were p r e ­ p a red for the ino cula t ions by allowing them to gro w in a broth m e d i u m for five days at r o o m temperature. terial cultures were tained popul ati ons incub a t i o n period. The b a c ­ i n o culated into nutr i e n t br oth and a t ­ of 10^ to 10^ organisms per m l after the The yeast cultures were g r own in dextrose b r o t h c o n taining 0.1 percent yeast extract and reached popu­ lations of about 10* or ganisms per ml. The mold Ino cul ati on sus pensions were prepa r e d by wash i ng well sporulated slants of the mol d culture w i t h sterile saline solution. in di cated populat ion s of b e t ween 10 ml. the 3 and 10 ^4- Plate counts mold spores per A l l in ocul ations of tapholes were acc omplished by injecting suspe nsi ons into the taphole us ing sterile 2 ml syringes. 27 T A BLE I I D E N T I T Y OP CULTU R E S U S E D IN TREK I N O C U L A T I O N STUDIES C ulture Numb e r of Trees Inoc ula ted P s e u d o m o n a s p;eniculata k Pseudomonas putre^aciens 2 F l a v o b a c t e r i u m solare 2 Achromobacter sp. 2 varians 2 Micrococcus 2 C a n d i d a sp. Rhodotorula Torulopsis 2 r-lutinis 2 aeria laurentii 2 T r i c h o s p o r o n pullulans 2 P e n i c i l l i u m so. 2 Cryptococcus cultures 2 M ixture of yeast Mixture of b acteria cultures 2 Mixture of ba cteria 2 and yeasts T o t a l nu mber of trees inoc ulated 30 28 P r o c e d u r e s of the Taphole Tre atme nt Studies In order to evaluate the effects of various t r e atments and m e c h a n i c a l methods consi der ed to be of possible value In controll ing taphole infection, out during the seasons 1956 and 1957 n a t u r a l tapholes chemical studies were carried In w h i c h a r tificial and tre ate d in va rious ways were com pared with u n t r e a t e d control tappings. M e a s u rements of m i c r o b i a l p o p u ­ lations and of sap yields were made. Preliminary c o mpleted during pings per Three studies of three taphole trea tme nts were the tree were 1956 season. subjected to five diffe rent treatments. tap holes on each tree were treat ed as a control. The Ten trees h a v i n g four t a p ­ treated and one r e m a i n e d u n ­ treatments included two different sized pel lets of sorbic acid w h i c h were placed into hole just prior to the i n sertion of the spile. different taphole rinses, syringe at w e ekly intervals throughout the The fifth treatment was a solution of calcium, h y p o ­ The results these two the solutions being injected from a two-ounce chlorite u s e d In a manner reason, Also, concent retions of aureomycin solutions were use d as v e t e r i n a r y dosage season. the tap- of these similar to the aureo myc in solutions. studies were inconclusive* and because of certain objections treatments, For this to the nature of they were not used during the succeeding inve s t i g a t i o n s . Before the 1957 sap season, trea tments were ev aluated under three ad dit io nal types cf laboratory conditions using 29 six artific ial , glass tubes, w h i c h had the taphole, tapholes.” ^ These consi ste d of glass inside dimensions of a norm al com mercial f i t t e d to stopcocks enabling the rate t h r o u g h the tapholes to be adjusted. n e c t e d to r e s e r v o i r s of sterile, a s y s t e m of syphons. long and i A The stopco cks were con­ a rtificial sap by m e a n s of A block of m aple w o o d about 2 inch square was placed in the artificial along w i t h the organisms. of flow of sap inches taphole chemical used for treatment and an in oc ulum of m i c r o ­ The flow of ar tif i c i a l sap th rough the tapholes was a d j u s t e d to a rate of about 0.5 ml per min ute and all tapholes and sap rese r v o i r s were plac ed under r e f r i g e r a t i o n temperature, ip° C. The a r tificial sap con tai ned 2 pe rcent sucrose w i t h 0 .0 5 > percent ye as t extract and 0.05 percent p e p ­ tone. The treatm ent s u s e d in cluded two tapholes w i t h m e r c u r i c iodide, two w i t h paraf o r m a l d e h y d e in an agar pellet, para f o r m a l d e h y d e taphole. In three weeks flow per day, taphole. in a plaster pellet, six hours of about 20 liters of sap had fl owe d th rough each Samples of sap were in dicated that formaldehyde and one u n t r e a t e d control of sap flow w i t h about taken at intervals and p o p u l a ­ tions of organisms were determined. studies one w ith The results of these the merc u ric Iodide and the para - treatments kept the tapholes sterile, ■^The a r t ificial tapholes were supplied bj the Eastern U t i l i z a t i o n R e s e a r c h Laboratory, U. S. D e p a r t m e n t of A g r i c u l ­ ture, P h i ladelph ia, Pa. wh i l e the con trol taphole became high ly a short c o ntamina ted w i t h i n time. A p r a c t i c a l d e m o n s t r a t i o n of the e f f iciency of the three t r e atments m e n t i o n e d above and of three others in contro lli ng the m i c r o o r g a n i s m s w i t h i n the taphole was trie purpose of the studies c ond u c t e d during the 1957 season. The trea tme nts u sed were as follows: P a r a f o r m a l d e h y d e treatments: A gar pellets - S uff ic ient w a r m 10 percent agar s o l u ­ tion was adde d to paraform a l d e h y d e (t r i o x y m e t h y l e n e ) to make a th ick slurry. This was drawn into glass tubing of the d e s i r e d diameter of the f i n i s h e d p e l ­ let. A fter the mixtu re was a l l o w e d to h a r d e n in the tubing, it was forc e d out f orming a long c y l i n ­ d r ical roll. This was cut into pellets of about 2j inches in leng t h and all owed to dry. The pellets wer e laid in the tapholes just before the spile was inserted. P l a s t e r pellets - Su ffi ci ent wa ter was added to a m i x ­ ture of~~li0 percent pa ra formaldehyde and 6 0 percent plaster of paris by weight in order to make a w o r k ­ able paste mixture. This was m o l d e d into pellets approximately inches long and ~L/hr inch square. These pellets were allo wed to harde n and then i n t r o ­ duced Into the tapholes just before the i nsert ion of the spiles. Sterilite powder treatments: S ter il it e powder, a p r e p aration of colloidal carbon coated w i t h co lloida l silver, was obta ined f r o m the S h e l l m a r - B e t n e r Flexib le Packa g i n g D i v i s i o n of C o n t i ­ n e n t a l Can Company, Inc., Mount Vernon, Ohio, owners of the Ste ril ite process patent. The powder was a p ­ plied to the tapholes using a w o oden spatula. Ab out one g r a m of powder was placed In each taphole and p r e s s e d onto the surface of the taphole wi th the spatula prior to placing the spile in position. Mercuric iodide treatments: P e l l e t s consisting of 50 percent plaster of paris and 5 0 percent me rcuric iodide by weight were pre pare d in a manner similar to the preparation of the plaster oellets or paraformaldehyde. These were similarly i n ­ serted into trie taphole just before the spiles were plac ed in position. 31 0-Silver treatments: 0-Silver Aqueous, a germicidal suspension of oligo­ dynamic silver in water, was obtained from the Chloramine Company, 5U- West loth Street, New fork, 11, N. Y. The tapholes were stuffed with cotton and the 0-Silver solution in a concentration of 80 ppm of silver was forced into the taphole from a polyethlene squeeze bottle, saturating the cotton and the surfaces of the taphole with the solution. The s^ile was driven into the taphole immediately after this treatment. Plastic tubing treatments: Tygon plastic tubing in lengths of one foot were con­ nected to ’'aseptic” type spiles and the tubes with the attached spiles were allowed to soak immersed in a solution of 1 2 5 0 ppm of the germicide, Roccal, for 214hours. Before the spiles were inserted, each taphole was rinsed thoroughly with two ounces of the Roccal solution which was forced into the taphole from a twoounce veterinary dosage syringe. Seven of the lip plastic tubes used in thus study had previously been treated on the inside by allowing a silicone prepara­ tion to run through the tubes. This preparation was Tow-Corning 200 fluid, obtained from the Dow-Corning Corporation, Midland, Michigan. A group of lip trees was tapped for each of the six t reatments, each tree having a treated and an untreated tapping. Except for those trees having tygon plastic tubing treatments, seven trees of each group were inoculated with a culture of microorganisms in both tappings while the remaining seven trees remained uninoculated. The inoculation used was 2 ml of a standardized broth culture isolated from maple (10^ cells/ml) previously sap and identified as Pseudomonas reniculata. The lip tygon treated trees were sub-divided into two groups of 7 trees, one group of vhich had silicone coated tygon tubing on treated tappings while the other group had uncoated tygon tubing. All tappings having tygon tubing were rinsed w it h two ounces of a solution of a germicide, Roccal, before 32 the spile with the tubing attached was inserted into the taphole. Data on populations of microorganisms in the sap was recorded at intervals throughout the season for all tappings and data on yields of sap was kept for all tappings except those which were in trees having treatments with mercuric i o d i d e .^ ^Yield data was not recorded for the mercuric iodide treated trees because the sap was allowed to run onto the ground from the taphole to prevent people from drinking it. 33 RESULTS Microbial Populations and Yields of "Aseptic11 and Regular Tappings Examination of the data on populations of sap from the "aseptic" and regular tappings of the 1955 season revealed that although most of the "aseptic" tappings remained rela­ tively free of microbial contamination until the end of the season, some of the early "aseptic" tappings had become highly contaminated early in the season. The microbial activity within these tappings was reflected by the low total yields as compared with those tappings which had remained "aseptic." In order to make a true comparison of the populations and yields of "aseptic" tappings with regular tappings it was necessary to omit the trees having contaminated "aseptic" tappings from the final analysis. Therefore, the data for both "aseptic" and regular tappings was omitted for 2 of the 10 trees tapped on January 10, 2 of the trees tapped on January 25 and one of the trees tapped on February 10. Figure 1 shows the averages of logarithms of the total populations of microorganisms in the sap from "aseptic" and regular tappings determined for the trees tapped on each of the five tapping dates. The January 10, January 25, and February 10 tappings indicate relatively early attainment of (.Pace) & £ TAPPING «nt ASEPTIC Cl < O CM i_: o S O _d l£ 'V V o 0* & oft in OJ GO LlI ii OJ w a V 2 — __ w.' Cl tf\ LfS 4*^ /r\ 0*4 0 #J"S I:1* HI |'r;1 P 0’ «r? €3 S4 a o OJ ■ I I o dvs j o "iw add iNnoo nvioi d o oon • o 2 c4 O OJ .Q '■»< i $ «'H 'I "5* 35 h i g h populations of microrganisms in the sap from the regular tappings as compared to the ’’aseptic.11 The difference in microbial activity between the two tappings was much less in the February 25 tappings, and was insignificant for the Ma r c h 10 tapping date. The weather conditions were such that the micro­ organisms developed rapidly in both types of tappings during the last of March and the first part of April. Higli microbial activity within the taphole was accom­ panied bj decreased average yields of sap per tree produced by regular tappings as compared with the averages produced by the relatively uncontaminated ’’aseptic” tappings. Figure 2 shows the percent increase in yields produced by ’’aseptic” tappings (A) as compared to the regular (R) and, also, a com­ parison of the average yields from the two types of tappings for each tapping date. Progressively smaller percentage dif­ ferences in yields resulted as the date of tapping became later in the season. The relatively small average yields for both ’’aseptic” and regular tappings indicated for the February 10 tapping was due to the presence of several small or defective trees which were fcund to be in this group. The small yields indicated for the March 10 tapping date resulted from the fact that this tapping occurred after a portion of the total sap run for the season had already been collected for the other tapping dates. The relationship between tapping date and yields from ’’a s e p t i c ” and regular tappings was determined statistically BY "ASEPTIC" TAPPING YIELD IOO F 50 - 0 JAN. 10 JAN.2 5 FEB.IO FEB.25 MAR.IO TAPPING DATE □ "ASEPTIC" □ REGULAR 225 - 150 - AVERAGE YIELD PER TREE - ( POUNDS) PERCENT INCREASE IN (Page) 36 JAN. 10 JAN. 25 FEB.IO FEB. 25 MAR.IO TAPPING DATE gtW%M 2. Average fields i?or "Aseptic" Asd aofralas Cappings Aooordlng; « ;jste Ot faspla® And x>ereent laoreese i ® XAeXA -retosod By ■‘/..septlo" Tapping 37 by Douglass is based* (1955) using the data upon which the present study It was shown that the difference between ,!aseptic,f and regular tappings for the January 10 tapping date was sig­ nificant at the 1 percent level, the differences for the January 29 and the February 10 tapping dates were at the 5 percent level, significant and the differences for the February 25 and March 10 tapping dates were not significant. Douglass (1955) also compared all ,fasepticn and regular tappings statistically, regardless of tapping date, and found that there is a significant difference between the two types of tappings at the 1 percent level. Further studies comparing "aseptic” and regular tappings during the 1956 season Indicate the existence of a similar r e ­ lationship, although the differences were not as pronounced* The difference in yields between the "aseptic" and regular tappings was not quite significant at the 5 percent level but there was a 22 percent greater yield from the "aseptic" tap- holes than from the regular* The data for this will be con­ sidered in the next section since it is to be compared with yield data from inoculated tapholes. Xn order to obtain a correlation between the increased microbial activity within the taphole and the decrease in yield resulting from this activity, it was first necessary to establish a measure of the degree of contamination of sap considered to represent a condition of incipient decline in yield from the taphole. Examination of the microbial p o p u l a t i o n data i n d i c a t e d that a pop ula tion of 10^ organisms per m l In the sap could, be considered, to repre s e n t a hig n level of m i c r o b i a l acti v i t y in the taphole w h i c h w o u l d e v e n ­ tually r e sult was in stoppage of sap flow. A correla tio n analysis c o m p u t e d u s i n g all data for "aseptic" and regular yields and m i c r o b i a l p opulations from both 1955 and 1956 seasons* The per cent i n creased yield prod uce d by the "aseptic" as c o m p a r e d to the regular days di ff ere nce per ml of sap indica tes tapping was corr elat ed w i t h the b e t w e e n dates in the of att ainment of 10^“ organisms "aseptic'1 and regul ar tappings. Fig ure 3 this co rr elation w i t h lines showing the esti mat ing e quation of the cor re l a t i o n and the standard error of estimate. A c o r r e l a t i o n coefficient of 0*66 was "t" test this 1 percent tapping calculated. Using the c o r r e l a t i o n was found to be signi fic ant at the level. The r e l a t i v e activity of ba cteria and of yeasts and molds in n a t u r a l l y infe cte d regular sea son is indic a t e d in Figu re Ip* logarithms of counts tappings during the TYie increase 1955 in ave rag es of per ml of sap for 30 reg ular tappings f r o m the Jan ua ry 10, January 25 and Febr uary 10 tapping dates is shown for the b a c t e r i a and for the yeasts and molds as the s e ason progressed. D u r i n g the early part of the season the counts of the b act eria and of the yeasts and mo lds were not high. as the season pr ogre ssed increasing populations or ganisms were observed. The However, of these counts of the bacteria at the (Page) 39 ( |ui/^oi = H dDS Ul | U 1 / 6J 0 O l 40 9 4 o a )-(|O i / ^ o i =V 4U 9U I U I D U V U| 9400 ) 90U 9J 9^|0 sAoq q: q. ifrom T?,V3aptie% pnd. InaouX&iel Tapping ';?or Owo Z&BninO Pato® "hzx*±nc th® 1956 :>ea&an k3 T A B L E II A V E R A G E S OF Y I E L D S B Y T Y P E AN D D A T E OF TAPPING (1956 Season) Date of Tappi ng Average Y i e l d s Gr a n d Average in Po unds of Sap by Type of Tapping "Aseptic" Reg ular Inoculated Feb. 7 152. k 1 2 6 .7 57. 3 112.7 Feb. 114-9.8 121.1 77.9 116.3 151.1 123-9 67 •S’ SHc 1114..3 29 Gr and Average "^'There is a sig nificantly smaller yield f r o m the inoc ulated tappings than from either "aseptic" or regular tappings. The significance of the regular vs. i n o culated tappings was d e t ermined by an alyses of va r i a n c e of the yields A p p e n d i x I). one percent "aseptic" vs. regul ar and the from the three types of tappings A l t h o u g h a h i ghly significant difference fo u n d b e t w e e n the regular ho significant difference was and "aseptic" yields by date of The abse nce ol a significant difference m b e t ween these h i g h deg ree at the level was shown to exist b e t ween the yields of r e g u l a r and i n o culated tappings, tapping. (see tappings m a y be explained yielus by the lack of any of m i c r o b i a l activity in the re gular tappings r e s u l t i n g f r o m the effect of the cold wea ther of the 1956 sea son w h i c h dis couraged microbi al activity in the taphole. Of partic ular Interest among these results, was the fact that all micr o o r g a n i s m s tested reduced sap flow (Figure 6). □ "ASEPTIC" □ REGULAR 200 150 PER TREE - (POUNDS) 0 3 INOCULATED AVERAGE YIELD 100 50 0 YEAST BACTERIA MOLD TYPE OF INOCULATION j?ICPTiia 6 * Average fieIJta t 't g *3ap Troa "Asajptiio" * 15©f3ilej? A»i XnocuXased Tappings •luriafs the 1956 ->©aso» for Trees Having Inoculations Bacteria /*al --sold 1*5 E v e r y i n o c u l a t e d tapping h a d either* co mpletely stopped p r o ­ d u c i n g sap or h a d d i m i n i s h e d in flow appr ecia bly at least two w eek s before sap y i e l d due the n o r m a l end of the season. The decrease in to i n o c ulation av eraged aoout 60 percent of the yield s f r o m the "aseptic" tapholes and r a n g e d f r o m 30 to 70 percent r e d u c t i o n in yield. Fi gure 7 shows the average per m l of sap for the regular, on trees i n o culated w i t h pure molds. logarithms of total counts ’'aseptic" and inocu l a t e d tapholes cultures of uacteria, yeasts and The yea st and b a cterial inocu lat ions were ap pa re nt in the h i g h p o p u l a t i o n s of m i c r o o r g a n i s m s season. in the sap early in the On M a r c h 1, the yeast inoc ula tion s avera g e d about 10-^ organisms per m l and b a c t e r i a inocu lat ion s avera g e d abou t 10^" per ml. This m i c r o b i a l acti v i t y in the sap in c reased through7 out the s e a s o n u n til a m a x i m u m average count of 10 per ml d was r e c o r d e d for the bact e r i a and about 10^ per m l for the yeasts. mold The plate counts of sap f r o m tapholes spores r e m a i n e d low t hro ug hout the season. tappi ngs was f ound to b e compl etel y sterile sap p r o d u c e d f r o m the in o culated tapping. and swab samples of these m o l d infection. in ocu lated w i t h One of these in res pect to the However, ream ing tapholes revealeu. a h i g h degree of The absence of molds in the sap is at tributed to the failure of the molds to sporulate w i t h i n the tapholesj the m o l d s as a m y c e l i a l mat on the surfaces probably e x i s t e d of the taphole. 7 - BACTERIA INOCULATED TREES i.a 6 5 "a s e p t ic " o—— 3 REGULAR □— .— md INOCULATED •— i ***** 4 a** 3 2 I O 6 YEAST INOCULATED TREES i.^Q 5 4 3 — 2 I O 6 MOLD INOCULATED TREES 5 Reamings from inoculated hole (molds per gram ) 4 -=*X >> v *p 3 2 ___________ I _________________________ O MAR. 10 FiaiL'S 7. MAR. 2 0 MAR. 3 0 APR. 9 m-aroblal Activity la From .ad Inoculate! trappings 'V.tri Havter; £ypes Of Zaoeulatlona h7 A c o r r e l a t i o n analysis of the ’’a s e p t i c ” and inoculated tapp5.ngs of all ba cterial and yeast inoculated trees involving yi eld s an d m i c r o b i a l populat ion s of the sap is in dicated in Figure 8, The percent inc reased yie ld produ ced by the ’’a s e p t i c 11 t apping as co mpared w i t h the inocula ted tapping was co rrelated w i t h the days di ff er ence bet wee n date of atta inm ent of 10^* o r g a n i s m s per ml in the sap of the two types of t a p p i n g s • The lines of the of the estimating equ at ion of the co rre lat ion and st andard error of estimate are shown. c oef ficient at the The co rrelation c a l cul ated to be 0.58 was found to be significant 1 pe rcent level using the ” t ” test. Taphole Treat m e n t Studies P r e l i m i n a r y investigations of treatment techniques duri ng the 1958 season in volved testing pellets of sorbic ac id and rin ses of aureomycin and calc i u m h ypo ch lori te taphole. in the The first two treatments were not further studied be caus e of the inconclusive re sults obtai n e d and because of the fact that both sorbic acid and aure omy cin were f o u n d to have effect only on definite groups in the taphole and not hypochlorite of microorganism s present to have effect on other groups. The tr eatment was dis cont in ued because of its very t e m p o r a r y limited effect in the presence of the large amount of org an ic taphole. tissue of the ( |w/pi = 1 dDS u i | u i / 6j o 01 •*oa)-(l'%OI = V 9 4 0 a) i° ju a u iu iD u v uj ao u eja^iQ s^dq 14-9 Of the six treatments used in discou rag ing m i c r o b i a l a c t i v i t y in the taphole during the 1997 season, d i c a t e d by p o p u l a t i o n studies of the running two were sap to have sidera ble effe ct in control ling m i crobial infection. in­ con­ As in­ d i c a t e d in F i g u r e s 9 and 10, tapholes treated w i t h pellets of p a r a f o r m a l d e h y d e with mercuric iodide were very effective t a m i n a t i o n of the tappings* (t r i o x y m e t h y l e n e , (HCHO)^ ), in agar and in red uci ng sap f r o m both inoculated and unin o c u l a t e d I n fact, nea rly all of the treated tappings r e ­ m a i n e d sterile th ro ug hout the season while un treate d tappings r a p i d l y i n c r e a s e d in levels of m i c r o b i a l activity. runs occ ur red later in the treat men ts seaso n than in u n t r e a t e d tappings. The taphole of par aform a l d e h y d e in pellets of plaster of paris pellets the average trea tmen ts ev en w ith the contamina tion of the sap f rom The effect of the r e ­ in reduc ing m i c r o b i a l act iv ity in the tap- is indic ated in F i g u r e s s olu t i o n and plastic st erilite powder in reducing m i c r o b i a l However, the tap holes was r e d u c e d considerably. hole sap to two weeks acti vit y as we re the agar pellets. maining Also, in tr eated tappings g enerally one were a p p a r e n t l y not quite as effective plaster the con­ tubing 10 and 11. A l t h o u g h the 0-Silver treatments had little effect, the did redu ce m i c r o b i a l activity in sap from b o t h i n o culated and u n i n o c u l a t e d tappings. Fi gur e 12 r e p r e s e n t s the average yields of treated and u n t r e a t e d ta ppings of both inocul at ed and u ninoculated trees. 3 Inoculated Control □ Inoculated Treated Uninoculated G< Uninoculated Tr .o PARAFORMALDEHYDE IN AGAR 6 5 <3 4 JO 3 o 2 I 0 6 PARAFORMALDEHYDE IN PLASTER 5 ►o 4 O' .o1 3 o 2 I □< MAR. 10 20 3« liaTO'bial ..•activity to top Fr©& Xnocul&t \n■ ■■ ■■ ■ Uninoculated Treated LOG. OF TOTAL COUNT PER ML OF SAP MERCURIC IODIDE 6 S TE R ILITE " POWDER 5 4 3 2 0 30 20 MAR. 10 FIGOms 10. aiorobi&l Activity In. 'Jap From Inoculated And Uninoeulated CDapplngs: Mercuric Iodide And "dterlllte" Powder 'Treatments (1 —o Control Control Tubing Silicone Treated TUBING MAR. 10 o * ~ 0 30 Inoculated Control Uninoculated Control — a Inoculated Treated Uninoculated Treated O -S IL V E R SOLUTION LOG. OF TOTAL COUNT PER ML OF SAP PLASTIC Tubing not Treated MAR. 10 PXOTAA XX* m e p o M a l Aotlvtty la B'rom Xuooulated mml Uaioooulated rc&ppiags* ^laotdLo ’ Xubing Aa& lfc>*AAXuei*T* Solufcioa t r e a t m e n t * ( a :;e ) 300 250 200 150 100 50 0 300 UNINOCULATED TAPPINGS 250 200 150 100 50 0 (HCHO)3 in Agar fir. u'ss (HCHO)j in plaster s t e r il it e powder "o - s il v e r " solution p l a s t ic tubing 12* Average 3fl®lda Of J-ai> Svoa Treated 'nd ITsfe r o u t e d ‘I'a yp ifig sa v<£ I n o c u l a t e d ttninoculatod ‘ freea Aooordi&s -'o Treataont <'n d 53 5k These avera ge valu es are also pr esented in Table III. quite e v i dent formaldehyde that the paraformald ehy de in plaster, in agar, and the Sterilite It is the pa ra­ powder treatments p r o d u c e d s i g n i f icantly greater yields from inoculated tappings t h a n was p r o d u c e d by the control tappings. are a ppa rent These differences in the u n i n o c u l a t e d tappings also, are not as great. Analy ses of variance of these show that the paraf orm ald ehyd e although they treatments in agar and the Sterilite powder tre atments were signif ican t at the one percent level, the p a r a f ormaldehyde the 5 pe rcent and plas tic level in plaster treatment was significant at (see Appe n d i x XX). tubing treatments while The 0-Silver so lution showed no significant differences b e t w e e n yield s from treated and untrea ted tappings. Since no yield data were kept for the mercu ric iodide treated tappings, the signif ica nce tically. tap ping s du ced of this treatment was not determin ed statis­ However, f rom the popul atio n studies of these it can be assumed that this treatment wou ld have p r o ­ signif ica ntly higher yields than w ould be produced f rom control tappings* I d e n t i f i c a t i o n Studies A total of 1273 cultures was Isolated f rom samples ta ken f r o m 5>0 trees during the the i d e n t i f i c a t i o n studies. tak en f r o m each group of 1 0 1955> season, and preserved for The number of times samples were trees rang ed from 1 0 times for the 55 TABLE III A V E R A G E S OF Y I E L D S IN P O U N D S OF SAP BY TREATMENT AND I N O C U L A T I O N D U R I N G 1957 SEASON ( 2 8 ta ppings for each treatment) T rea t m e n t Inoculation Treate d Untr e a t e d G r and Av erage 105.1 6 I4..O 212.4 206 .7 Paraformaldehyde In agar Uninoculated Inoculated 3 1 9 .8 G r a n d Ave rage 334.6 Uninoculated Inocu l a t e d 271 .Ip 305*8 191*0 75*6 231*2 190.1 G r a n d Average 283.5 132.9"'" 210*6 349.4 &k-5'y~'‘c 208.5 Paraformaldehyde in pl ast er Sterilite powder Uninoculated Inoculated 2l_p3 •3 260.9 202.7 116.1 223*0 186.5 G ran d Average 252.1 159*5"*' 205*8 0-Silver s olution Plastic Uninoculated Inoculated 138.7 99*5 172*5 87*5 155*1 93*5 G r a n d Average 118.6 129*9 ^ 125*3 tubing Silicone coated 191*1 Uncoated 257*7 G r a n d Average 224-4 ':HfY i e l d f r o m tr eat ed gr e a t e r than y i e l d percent level. '“Y i e l d f r o m treated gr eater than yield percent level. 154.0 190.9 172.5 1 172.6 2 2 k -3 198.5 tappings found to be significantly f r o m untreate d tappings at the one tappings found to be significantly f r o m u n t r e a t e d tappings at the 5 hfleld f r o m treated tappings not found to be significantly gr eater than yield from u n t r e a t e d tappings. 56 trees t a p p e d earl y in the t a p p e d later on M a r c h 10 taphole sea son to 5 times for the (Table IV). trees The total num ber of samples o b t a i n e d for eac h tapping date rang ed f r o m 200 samples f r o m the early tappings to 100 samples f r o m the latest ta pping giv in g a to ta l of 8 2 0 o b t a i n e d du ring the season. platings of all samples taphole samples of sap Iso lat ion s were made from in wTh i c b microorganisms were found to be present. During the 1956 season an addi tio nal 173 i solat ion s were o btained in a similar man ner from 10 trees, each tapped w i t h Ip tapholes, ma king a total of LpO tapholes sampled. Two samples were taken from each taphole during the season for a total of 80 samples. Table V gives molds the numbers of the bacteria, i s o l a t e d during the 1955 season. yeasts and The bacteria were i sol a t e d f r o m nu trient agar plates and the yeasts and molds from a c i d i f i e d dextrose nu mber s agar plates. Therefore, of the three groups have no significance w i t h respect to their f r e q u e n c y of occurrence in maple However, the relative since all cultures were tree tapholes. selected at ra ndom from platings of the highest di lu tion s of the samples, q uenc y of oc cur rence g e n e r a and species the f r e ­ in h i g h populations of the individual is estim ate d by the percentage of the total is olat ions w h i c h each group represents. Table n ega tive rods VI shows the types of bacteria isolated. const i t u t e d the largest group, G-ram accounting for 2/3 of 57 TABLE IV O R I G I N OP C U L T U R E S I S O L A T E D F R O M M A P L E TAPHOLES (1955 Season) Tapp i n g Date Trees T a pped Tapholes Sampled Samples Taken D u r i n g Se a son Number of Isolate s* Ja nuary 10 10 20 200 320 Janua ry 25 10 20 200 311 F e b r u a r y 10 10 20 180 317 F e b r u a r y 25 10 20 340 226 M a r c h 10 10 20 100 99 50 100 820 1273 Totals '''"isolations w e re made f r o m all samples in wh ich micro o rga ni sm s were found. 58 TABLES V M I C R O O R G A N I S M S I SOL A T E D F R O M M A P L E T APHOLES (1955 Season) Type of O r g a n i s m Num be r of Iso lates Bacteria 469 Yeasts 412 Mo l d s 370 Strep t omycetes Totals 24 1273 59 the t otal b a c t e r i a l cultures* posit ive spore types, rods, both were foun d G-rain positive cocci and gram forming and non-spore forming in much, smaller numbers. The genus 1 seudo- inonas a c c o u n t e d for 71 percent of the total g r a m negative rods w i t h 3/Uother these identifie d as Pseudomonas g e n i c u l a t a * species of P seudomonas were identified including P g . p u t r e f a c l e n s , Ps> m e p h i t i c a, and P s . s t r i a t a . Achromobacter 26 pe rcent rods, Achromobacter The genera and F l a v o b a c t e r i u m together represen ted about of the w i t h one Three total number of isolates of the g ram negative species of each genus being identified, superficiale and F l a v o b a c t e r i u m s o l a r e . Genus Kicrococcus accounted for most of the gram positive isolates* cocci, with K. varlans having the largest number of K. luteus, M. conglomeratus and M. candidatus were also present in smaller numbers. The largest group of iiicro- coccus indicated as miscellaneous consisted of cultures having many varying characteristics which would not allow them to be placed in a single species or even in a group of species. Sarcina was also r e p r e s e n t e d by a small group of isolations. The group of cultures of gram positive spore forming rods was found, to consist almost entirely of Bacillus circulans and Bacillus cereus in nearly equal numbers of isolates. The non— spore forming gram positive rods were found to be a heterogeneous group with widely varying morphological and physiological characteristics. belong to the genus B a c t e r i u m . Kany of these probably 60 TABLE VI B A C T E R I A I S O L A T E D P R O M M A P L E *T A P H O L E S (1955 Season) —— r ■, Number of Isol ate s Type and Spe cies GRAM NEGATIVE RODS PSEUDOMONAS P s , geniculata Ps. pu trefaciens Ps* m e p h i t i c a Ps. s t r iata Miscellaneous Perce nt of G r o u p 8 .6 2.5 2.9 12 3 .8 FLAVOBACTERIUM PI. solare Mi seellane cus 46 36 1 4 *6 11.5 3.1 ACHROMOBACTER A. superf icl ale Miscellaneous 36 9 27 11.5 2.9 8.6 9 2.9 UNCLASSIFIED 78 73 G R A M P O S I T I V E COCCI MICROCOCCUS M. varians M. luteus M* conglomer atus M. Cand ida tus Miscellaneous 18 15 9 7 24 5 SARCI NA G R A M P O S I T I V E R ODS (SPORE FORMING) BACILLUS B. cereus B* circulans Mi s c e l laneous 25 25 12 10 3 3 0 .8 6. Ip 5 >3 100.0 48.0 4 0 .c 12.0 1 1 .1 lj-69 10C.0 00 TOTAL 16 .6 93-6 2 3 .O 19.2 11.5 9.1 m G R A M P O S I T I V E RO DS (NON-SPORE FORMING) Percent of Tot al 71.0 53.2 27 8 9 10 .. 6 7 .0 31 4 223 167 . "'^This was a very h e t e r o geneous group of cultures probe bly b e l o n g i n g to the genus B a c t e r i u m , 61 Table VII Indi cat es the ge nera and i d e n t i f i e d f r o m the 1955 Isolations. were isola t e d w i t h the Nine species of yeasts species of yeasts largest numb er being ide nti fie d as Trlchosporon pullulans. Sp ecie s of R h o d o t o r u l a , C a n d i d a , C r y p t o c o c c u s and Torul o p s i s as indicated were also identified. The three species of the genus Cr ypto coccus ac counted for the largest single gro up of yeasts, cent of the total yeast amounting to nearly 2 5 per­ cultures. Nine g e n e r a of mo lds were iden tif ied among the 1955 isolations isolates, (Table VIXI). The largest number of identified about 2 2 percent of the total, were members of the genus P e n i c i I l i u m . Phoma, Hormodendrum, Fusarium, Alternaria, C e p h a l o s p o r i u m , R h i z o c t o n i a , A s p e r g i l l u s , and C o niothyrium were also Identified. lates, cause The largest single group of mold Iso­ n e a r l y 30 percent of the total were not Identified be­ they did not sporulate and produce typical fruiting s truct ure s on labor ato ry media. m e m b e r s of the class, 1956 in the season, of these are probably BASIDI0MYCETE5• Table XX gives during the Many season. a bre akdown of microorganisms is olated Since these samples were taken late w h e n bact e r i a l and yeast populations were r e l a t i v e l y high, no m o l d s were isolated from the plates of the h i g h e s t dilutions. Ei ghty -fi ve percent of the bacteria is olates w ere m e m bers of the genus P s e u d o m o n a s . e n c o u n t e r e d during the previous season, coun t e d for about Ij. percent of the A genus not C h r o m o b a c t e r i u m , ac­ isolates, and the genera 62 TAB LE VII Y E A S T S ISOL ATE D F R O M M A P L E TAPHOLES (1955 Season) Species Numbe r of Isolates Percent of Total TRICHOSFOROK FULLULANS 81p 20 R H O D O T O R U L A OLU TINIS 75 1 8 .2 C A N DIDA sp. lj.8 1 1 .6 .Ip 1 0 .2 TORUL O P S I S AER IA C R Y P T O C O C C U S LAURE NTI I 36 8.7 C R Y P T O C O C C U S DIFFL U E N S 32 7.8 C R Y P T O C O C C U S ALBIDUS 32 7.8 CANDIDA CURVATA 23 5*6 8 1.9 32 7.8 1+12 1 0 0 .0 TO R U L O P S I S CANDIDA Unclass ifled To tals 63 T ABL E VIII M O L D S ISOLA T E D P R O M M A P L E TAPHOLES (1955 Season) Genus N u m b e r of Isolates Percen t of Total PENICILLIUM 83 22 .k PHOMA 60 1 6 .2 HORMODENDRUM 36 9.7 PUSARIUM 26 7.0 ALTERNARIA 16 i|.ip CEP HALO SP O R I U M 13 3.5 RHIZOCTONIA 11 3.0 ASPERGILLUS 10 2.7 6 1 .6 CONIOTHYRIUM U n i d e n t i f i e d -' Totals 109 29.5 370 100 .0 '“'This g r o u p co nsisted of cultures whi ch did not sporulate and pro duce t y p ical fruit ing structures on laboratory media. M a n y are pro bably member s of the class BASIDIOMYCETES. 61+ TABLE IX MICROORGANISP1S I SOLAT ED PRO M M APLE TAPHOLES (1956 Season)''*' G e nus Numbe of Isolates Percent of BACTE RIA Pseudomonas Chromobacterium Flavobacterium Baci H u s Uncla s sifled 101 81; .9 1 ± .2 5 3 l 9 2.5 0 .8 7.6 119 Totals 1 0 0 .0 YEASTS 59.2 16.7 9.3 7.k 74 12 Candida Cryptococcus Rhodotorula Trichosporon Un cla s s ifled 9 5 k k Totals '‘Trees tapp ed = 10 N u m b e r of tapholes = IpO Tot al numb e r of isolates = 173 5k loo.o 65 F l a v o b a c t e r i u m and Bacil l u s wer e also present. G e n e r a isolated d u r i n g the 1955 seas on but not a p p arently present in si gnif i­ cant n u m b e r s in the 1 9 5 6 samples include A c h r o m o b a c t e r » M i c r o ­ coccus and Sarcina, A m o n g the yeas t isolates, p r e d o m i n a t i n g genus, total* Candida was found to be the accounting for nearly 6 0 percent of the C r y p t o c o e c u s was second in number of isolates r e p r e ­ senting about 17 percent, wer e also present. for about 1 2 and Rhod o t o r u l a and T r i c h ospo ron The genus T o r u l o p s i s , w h ich accounted percent of the total yeasts in 1955# was not f oun d du rin g the 1956 season. 66 DI S C U S S I O N High, m i c r o b i a l acti vit y w i thin the maple result sap. in prematu re Thes e r e s u l t s (1955)* stoppage taphole will of sap flow and low yields of agree w i t h those of Naghski and Willits The popula r belief that the physical effect of air or w i n d u p o n the wall s of the tapping is responsible for taphole dr ying Is refuted. It should be noted that in every case of g r e a t l y d i m i n i s h e d yields from regular as compared wi t h ’’a s e p t i c 1* tappings in these studies, h i g h levels of m i c r o b i a l a c t i v i t y were evident regardless of whether the tapping was n a t u r a l l y or a r t i f icially contaminated. versely, In every case of ver y low mi crobial activity due to ’’a s e p t i c ” tapping, reasons, Con­ taphole treatment, or other unexplained the yiel d was high compared w i t h highl y infected tappings• F o u r factors were obse rve d to influence rate tent of m i c r o b i a l a c t i v i t y w i thin the tapping and, the degree contamination conditions, therefore, of r e d u c t i o n of total yield from the optimum w hich wo u l d be pr od uced by an u n c o n t a m i n a t e d tapping. were and e x ­ of the tapping, and an tis eptic These factors date of tapping, weatner treatment of the tapping. H i g h level o rigi n a l contamination of the tapping during the tap ping procedure, as in dic ate d by studies of ’•aseptic,” 67 re gular, and i n o culat ed tappings, w a s fo und to be of pri mar y im portance tappings, yields in causing stoppage of sap flow. I n inocu lat ed such c o n t a m i n a t i o n caused a great r e d u c t i o n in in spite of the w e a t h e r co nditions during the 1956 seaso n w h i c h te nded to disc our age n a t ural m i c r o b i a l act ivi ty in the reg ula r tappings. Other con tam ination subsequent to the tappi ng pro cedures may be sufficient to cline cause early d e ­ in y i e l d but this w o u l d be dependent on the amount the in oculation, of and on how early in the season the c o n t a m i n a ­ tion occurred. The during season were about 10^ yeasts or 10^ bacteria the 1956 per taphole, while levels of in ocu lat ion u sed in t & p pings those in oc ul ated tappings of the 1957 season r e c e i v e d 10^ bacte ria per tapping. or iginal inoc ula tio ns were All of these sufficient to result in low sap yield• N a t u r a l taphole contamin ati on of a commercial type tapping may result f r o m poor sanitation practices in the storage and use of tapping equipment. portant source of the tree of mapl e of 1 itself. 20 M i c r o b i a l plate the tapping ta ppings Other include counts of the outer bark season indicated populations m i l l i o n organisms per gram. bark may easily be pus hed into the procedure. the most im­ of con tam inat ion is probably the outer bark trees during to However, probable Bits of this taphole during the tapping sources of co nta mina tion of nat ural rain w a t e r w a s h i n g down the bark ol the tree into the taphole, n a t i o n of the w i n d b l o w n dust, rain and snow, spile and taphole by Insects, and contami ­ birds and small animals• The d i f f i c u l t y In maintaining the taphole "aseptic*1 conditions in in the envir onme nt of the woodl ot is indicated by the fact that about 10 percent of the "aseptic" tappings be­ came c o n t a m i n a t e d at an early date and re sulted in low yields. P r a c t i c a l l y all of these tappings became season. The infected late In the source of cont ami nati on of these tappings was probably the bark of the trees during the tapping procedure, a l t h o u g h water ma y have cracks enter ed the in the w o o d around The dev elopment taphole and the tappings through minute the "aseptic" spile. of microorgani sms in the maple subsequent stoppage of sap flow appears to be a r e l a t i v e l y no n-specif ic b iol o g i c a l etiology. condition in regard to m i c r o ­ This is indicated by the fact that eve ry o r g a n i s m used in the inoculation studies nificant d e c r e a s e in sap yield. organism can cause premature on its abili ty condition. stoppage apparently depends only A l t h o u g h in the inoculation studies, stoppage, indicated that the are Thus, whether a particular and b a c t e r i a were all eminently capable causing taphole monas caused a sig­ to g r o w in the chemical and physical en viron­ ment of the taphole. yeasts, m o lds tree of the study of nat ura l contamination p s ychrophil ic bacteria of the genus Pseudo- prob a b l y most prevalent in bringing about this N a t u r a l b acterial populations in tapholes showing 69 premat ure dec lin e in sap flow were always at high levels while yeast p o pulations one tapho le in the tappings woul d vary c o n s i d e r a b l y , showi ng relat ive ly hi gh yeast contamination in co m b ination w i t h h i g h bacter ial counts while another wou ld exhibit little or no yeast activity* pings showing no decline type ear l y in the in sap flow had few organisms of any season. A l t h o u g h bacteria were present in higher p o p u l a t i o n s than yeasts during season, Sap from normal tap­ the latter part of the both types of organisms were found in numbers wh ich indi cat ed si gnif ica nt act ivi ty in the taphole. This o b s e r v a ­ tion co nfirms results of a population study by hason, and C a r p e n t e r (1913)* low th rough out to indicate holes. the Jones The fact that mold populations were season in all regular tappings wo uld appear these organisms had little activity in the tap- However, the lack of mold sporulation exhibited in the m o l d inoculated tappings indicates that it would be dif­ ficult to eval uate the importance of molds in natural taphole infecti on ent irely on the basis of popu lat ion studies of the sap because this estimation would give little evidence of increasing m o l d a c t i v i t y in the taphole. The rate of increase in numbers w i t h i n the n a t u r a l l y in fected tappings a ffe cted varied and weather considerably, conditions. of micr oorganisms in which the yield was depending upon date of tapping However, it was found tnat when a level of m i c r o b i a l a c t i v i t y i n d i c a t e d by a population of 10^ o r g a n i s m s per m l of sap was a ttained a definite reduction in sap f low u s u a l l y occurred, n e a r l y c omplete stoppage w h i c h g e n e r a l l y was f o l l o w e d by of flow w i t h i n one week. be poi nted out that the n u m bers in the sap of m i c r o o r g a n i s m s obse r v e d proba bly r e p r e s e n t e d only a small percentage the po pulations occurring yi eld is that this provides which greater microbial may occur and develop in causing decline in fection of the exp ose d t&pholes to the level w h i c h will reauce season, However, the it is consid ere d desirable tap early in order to be certain of collecting season. in longer periods of time during In comme r c i a l practice runs of the of in the tissue ar ound the taphole. The effect of early tapping yield. It should the early to sap as demonst rate d by the 1955 if ex trem ely early tapping dates were use d in com­ m e r c i a l operations, of early this technique would insure collection sap runs at the in yield. However, risk of causing early decline if tne early tapping was combined with techni que s w h i c h would prevent taphole infection, this practice w o u l d be ideal for obtaining m a x i m u m production of sap f r o m the tapping. the The study of the 1953 season in w hich "aseptic*1 tappings on early tapping dates produced high yields d e m o n s t r a t e s the practicability of combining tap ping date w i t h a teciinique de signed to minimize early taphole contamination• The effect of w e a t h e r conditions during the season should not be decli ne ignored in as ses sin g in sap yield. the factors involved in early Since microo rga nis ms flourish in warm 71 weath er, u n s e a s o n a b l e w a r m weath er early in the season could be r e s p o n s i b l e for a h i g h incidence of premature Conv e r s e l y , dur ing the cold w e a t h e r 195& late into the season, stoppage. such as occurred season, w o u l d discourage activity of m i c r o ­ or ga nisms r e s u l t i n g in little premature stoppage of sap flow. Other ef fects of the weather wind, r a i n and snow in incre asi ng co nt amination of the open tappings. Also, r a d iant include the activity of the sunlight on the taphole side of the tree cou ld increase tempera ture s of tne tapholes in freezing w e a t h e r enough to encourage microb ial activity. A l t h o u g h it is imprac tica l for the commercial operator to r e s o r t to " a s e p t i c 1* techniques of tapping, chemical trea t m e n t s of the taphole the use of and sanitary measures duri ng the ta pping procedure could do m u c h to reduc e premature decline in yield. R e c o m m e n d e d sani tat ion measures would include the use of antiseptic rinses for al l tapping equi p­ ment and ca reful h a n d l i n g of this equipment during the tapping procedures to m i n i m i z e ori gi nal co ntam ination of the taphole. Remova l of the outer layer of bark before drilling the taphole is also desira ble . The ideal m e t h o d for in the taphole after the controlling m i crobial activity tapping procedure w o u l d involve using a che mical substan ce w i t h i n the taphole hav ing the follo win g ch aracteristi cs: unt il the end of the tivit y (a) continual germicid al activity seas on in the taphole in small conce ntr a t i o n s environment, in preser vin g the (b) a c ­ collected sap 72 w h i c h flows f r o m the tapping, (c) lack of toxieitv or r e o o i h%) r e n d e r e d n o n — toxic during the ev apo rat ion process tion or v o l a t i l i z a t i o n of the g e r m i c i d a l agent, less to rea ctions flavor and color by d e composi­ and (d) h a r m ­ in the evap orat ion process during which cha rac ter isti cs of the finished sirup are produced. Of the five in taphole types of treatments studies during f o r m a ldehy de treatments the investigated closely 1957 season, only the p a ra­ show any promise of meeting some of these r e q u i r e m e n t s of a good taphole antiseptic. hyde Paraformalde­ (t r i o x y m e t h y l e n e ), the trimer of formaldehyde, is active against bacteria, yeasts and molds when in solution as formalin. In a pellet as in agar, form, slowly r e l e a s i n g microorganisms the sap, the antise pti c throughout the it exerts In quantities active against period of the sap season. spoilage of the stored sap. In the sap appears the flavor or color of the finished substance may be r e d u c e d from the sirup dur ing product, little effect on sirup. Altho ugh this the ev apo r ation process, further work Since formaldehyde is toxic to be used as a preservative for a food it would have final product. to have in concentra tio n or possibly eliminated is n e c e s s a r y to e s t a b l i s h this. and not permit ted In a residu al antiseptic activity which w o u l d at least t end to r e t a r d Paraformaldehyde it can be made to disintegrate to be com pl etel y removed from the 73 The effe c t i v e other treat men ts w h i c h appe ared to be significantly In m a i n t a i n i n g the taphole in an antiseptic cond i­ tion were m e r c u r i c iodide and sterilite powder* H o w e v e r the (colloidal silver) toxicity of these substances ana the d i f f i c u l t y in eliminating them from the fin ish ed sirup w o uld make their use undes ira ble* Th e resu l t s agree w i t h similar sap by Edson, most of the id ent ific ati on studies in general studies of micr oor gan ism s in spoiled maple Jones and Carpenter (1913)* They found that the c o m m o n l y encou n t e r e d micr oor ganisms of spoiled sap were m emb e r s of the genus P s e u d o m o n a s . Also found to be active in sap sp oilage wer e bact eri a w h i c h were apparent ly of the genera A c h romobac ter and Microc occus * w hic h w e r e not identified further, Red and grey yeasts and molds belonging to the ge ne ra E r o t i u m and P e n i c i l l i u m were also found* present study, In the or gan isms of all of these groups were id enti­ fied except for the mold, E r o t i u m . M any other genera and species of m i c r o o r g a n i s m s not desc rib ed in the previous were also identified. study 7k SUMM A R Y A s tudy of* the m i c r o b i o l o g i c a l a c t i v i t y a s s o c i a t e d w i t h maple tree ta pholes and its effect on sap yields has sented. Microbial populations during three and sap yie lds w er e r e c o r d e d sap se asons f r o m a total of 39& tappings. dates and m e t h o d s treatments of tapping, m i c r o b i a l ino cul ations of the tapholes were for premature However, and other decline taphole was foun d to be in yield of sap. yeasts and molds were all capable of pr oducing sap flow. Var ious investigated. The m i c r o b i a l activ i t y in the respon sib le been p r e ­ ba cteria and yeasts were Bacteria, stoppage of the most pr eva len t types of m i c r o o r g a n i s m s found in n a t u r a l l y inf ect ed tappings. " A s e p t i c , ” regular and inocu late d type tappings were compared in order to e s t a b l i s h a correlat ion between the m i crobial a cti vity w i t h i n the taphole and the decline in sap yield resulting f r o m this activity. The pe rcent difference in sap y iel d b e t w e e n ’•aseptic'1 and regu lar tappings was correlated w i t h days d i f fer ence in date of attainment of lcA organisms per ml of sap. A correl ati on coefficient of 0.66 was c a l c u ­ lated and foun d to be h i ghly significant. tion coefficient, tappings, high ly deter m i n e d for "aseptic" and inoculated was foun d to have a value of significant. A similar co rrela­ This was also 75 The on the treatme nt spiles and studies in volved the use of plastic tubes the i n t r odu ction of 7 chemical substances into ta pholes in an attempt the taphole. The c hemical substances included calcium h y p o ­ chlorite, iodide, sorbic acid, Sterili te were merc u r i c aureomycin, powder, su c c e s s f u l of these to control m i c r o b i a l activity in paraformaldehyde, me rcuric and 0-Silver solution. The most treatxuents in controlling micr o b i a l activity iodide and paraformaldehyde. However, due to r e s i d u a l concentrations of these compounds in the finished sirup they cannot be recommended for practical application. E a r l y tapping dates were dence of prematur e decline period of time shown to increase the inci ­ in sap yield because of the longer all owed by this technique for micro bia l a c ­ tivity to d e v elop w i t h i n the tapping. "As ept ic11 methods of tapping w e r e found to be effective in r e d u c i n g microbi al act ivity and in pr od ucin g greater sap yields than regular tappings• A study was made of HpLj.6 cultures isolated from llyO map le tree tap hol es durin g the first two seasons of this investigation. The g r a m negative rods constituted more than two-thirds of the b oth seasons, total number of ba cterial isolations during and of these, the genus P s e u domonas was by far the m o s t f r e q u e n t l y e n c o u n t e r e d group, cent during the 195& season. amounting to 71 p e r ­ 3.955 season and nearly 85 percent during the The species P s e u d o m onas g e n iculata appeared to be the most commonly e n c o u n t e r e d species, amounting to 76 t h r e e - f o u r t h s of all the P s a u d o m o n a s 1955 season. Al l yeast s coun t e r e d ge nus the present were f o u n d to be non- fermentative, anascosporogenous gener a isolated, isolatio ns dur ing types. Of the five yeast C r y p t o c o c c u s was the most frequently e n ­ durin g the 1955 season, percent of the total. However, pullu lan s was prevalent the most amount ing to 2 5 the single species T r i c h o s p o r o n of the eight species i d e n t i ­ fied, r e p r e s e n t i n g over one-fi fth of the yeast isolates. D u r i n g the genus, 1956 season, Candida was the most common yeast a c c o u n t i n g for nearly 60 percent of the yeasts. Nea rly o n e - t h i r d of the mol d isola tes were u n i d e n t i f i e d and considered to be m e m b e r s of the were identified, class B A S I D I 0 M Y C E T B 5 . Nine m o l d genera of w h i c h P e n l c i I l i u m and Phorna were the most promine nt in number of isolations. 77 BIBLIOGRAPHY Barnett, H. L. 1955 I l l u s t r a t e d G e n e r a of Imp erfect P u m i . Bu rgess P u b l i s h i n g Co., He w York, N. Y • Barraclaugh, K. E. 1952 in N e w Hamp shir e. sion Bull,, 1 0 3 * Beneke, Bois, E, S, 1956 M a ple sirup and sugar p r o d u c t i o n U n i v e r s i t y of N e w Hampshire E x t e n ­ P e r s o n a l communication. E. , and Dugal, L. C. 19lt-0 La seve d'er able et son Le N a t u r a l i s t e Canadian, 67,, I3 7 -II4.I. pH. Breed, R. S., Murry, E. G. D., and Hitchens, A. P. 1 9 I4.8 B e r g e y 1s M a n u a l of Deter m i n a t i v e B a c t e r i o l o g y , oth ed. The W il liams and 'Wilkins Company, Baltimore, Kd. Brown, N. C. Use. 1919 Fo rest Products, Their Man ufacture and John W i l e y and Sons, N e w York, N. Y. Bryan, A. H., Hubbard, Win. F. and Sherwood, S. F. 19lf7 P r o d u c t i o n of maple sirup and sugar. U.S.D.A., P a r m e r Bull., 1 3 6 6 . Committee on Bacteriological Technique, Society of A merican Ba cter iol ogi sts, M a n u a l of Methods for Pure Culture St udy of B a c t e r i a . Biotech. Pub., Geneva, N. Y. Cope, J. A. 1952 Ma ple Bull., 3 9 7 • sirup and sugar. Cornell Ex te nsio n Dougl a s s , J. E. 1955 The effect of date of tapping on the y i e l d of m a ple sap f r o m sterile and non-sterile tapholes. U n p u b l i s h e d M. S. thesis, M ichigan State Univers ity , East Lansing. Edson, H. A. 1910 Buddy sap. Vermont A g r . Exp. Sta. Bull., 251Edson, H. A., Jones, C. H., and Carpenter, C. W. 1913. M i c r o o r g a n i s m s of map le sap. Vermont Agr. Exp. Bull., 1 6 7 . Sta. Fabian, F. W . , and Buskirk, H. A. 1935 Aerobacter aerogenes as a cause of ropin ess In maple sirup. Ind. Eng. C h e m . , 27, 31+9* 78 F abi an, F. W., and Hall, II. H. 1933 Y e a s t s found in f e r ­ m e n t e d m a p l e sirup. Zent. fur Bakt. Abt II, _89, 31-1*7. Fe llers, C. R. 1933 Spoilage J. Bact., 2£, 67-68. of maple products by molds. Hayward, F. W. 19lp6 The storage of maple State Agr, Exp. Sta. Bull., 7 1 9 * sirup. New Y o r k Hayward, F. W* an d Pederson, C. S. 191-1-1 Some factors causing d a r k co lor ed ma ple sirup. N e w Y o r k State Agr. Exp. Sta. Bull., 718. Holgate, K. C. 1950 Changes in composi tion of maple sap dur i n g the tapping season. New Y o r k State Agr. Exp. Sta. Bull., 7^2. Lodder, J . , and lireger-Van Rij, N. J. W. 1952 The Yeasts: A T a xonomic S t u d y . Inte rsc ienc e Publishers, Inc., N e w York, N. Y. M i c h i g a n Cro p and L i v e s t o c k R eporting Service, U. S. Departme nt of Agriculture, Agric ult ure Ma rketing Service, A g r i ­ culture Es timates D i v i s i o n and M i c h i g a n State D e p a r t ­ m ent of A g r i c u l t u r e , May IB, 1955• Moore, H. R . t Anderson, W. R. , and Baker, R. H. 1951- Ohio m a p l e sirup; some factors influencing production. Ohio Agr. Exp. Sta. R e s e a r c h Bull., 7 l S . Nagh ski , J. 1953 THe organisms of maple sap; their efx*ect and control. Repor t of Proceedings, Second Conference on Maple Products, Nov. 16-18, 1953* Ea stern Regional R e s e a r c h Lab oratory, Philadelphia, 18, Pa. Na ghski, J. and Willits, C. 0. 1953 Maple sirup. VI. The sterilizing effect of sunlight on maple sap c o l ­ lected in a trans par ent plastic bag. Food Tech., 7, 81-83. Naghski, J. and Wi llits, C. 0. 1955 Mi cr oorg ani sms as a cause of premature stoppage of sap flo w f rom maple tap holes. J. Appl. Microbiol., lLj-9-l5l. Sproston, T. J., and Lane, Susan. 1953 Maple sap c o nt amina­ t ion and m a p l e sap buckets. Vermont Agr. Exp. Sta., Pamphlet 2 8 . 79 Sproston, T. J* and Scott, W. W. 195U Vais a le u c o s t o m o i d e s , the cause of decay and d i s c o l o r a t i o n in tapped sugar maples. Phytopath, ijlj., 12-13* Willits, C. 0. and Porter, W. L. 1950 Mapl e sirup. I. R e s e a r c h p r o g r a m on maple products at the E a s t e r n R e g i o n a l R e s e a r c h Labor ato ry. Bure au of Agricult ure and I n d u s t r i a l Chemis try , A g r i c u l t u r a l R e s e a r c h A d ­ m i nistratio n, U • 8. D. A . , Philadelphia, Pa. 80 APPET3DIX 81 APPENDIX I A N A L Y S E S OP V A R I A N C E OP Y I E L D D A T A - 1956 SEASON R E G U L A R VS. Source T o tal "ASEPTIC" TAPPINGS D. F. M e a n Squares 59 Date 1 T app i n g 1 D x T 1 3k Error 56 3,053 21+21 11 ,1214. 2 •4?here Is no significant difference between yields of F e b r u a r y 7 and F e b r u a r y 29 tappings by type of tapping ^There is no significant difference between yields of the re gul ar and "aseptic tappings • R E G U L A R VS. INOCULATED TAPPINGS Source T ota l D. F. Mean Squares 59 Date 1 T app ing 1 D x T 1 Error 56 si+u 1 1+7 ,14.33 * * 2,571+ ** 1+8 ^There is no significant difference be tween yields of F e b r u a r y 7 and F e b r u a r y 29 by type of tapping. ‘"""'A h i g h l y significant difference is indicated. (1 percent level) 82 APPENDIX II A N A L Y S E S OF V A R I A N C E OF Y I E L D D A T A OF TAPHOLE T R E A T M E N T STUDIES - 195? SEASON T R E A T M E N T - P A R A F O R M A L D E H Y D E IN AGAR Source T ota l D. F. M e a n Squares 27 Treatment 1 Inoculation 1 234 1 T x I 1 8,743 ':Hf Er ro r 214- '‘''A h i g h l y significant difference is indicated* 437,750 ** 1,305 (l percent level) ^There is no significant difference between yields of inoculated and u n i n oculated tappings according to the treatment of the taphole* T REA T M E N T - PARAFORM A L D E H Y D E IN PLASTER Source Total D. F. Mean Squares 27 Treatme nt 1 169,14-88 * Inoculation 1 11,717 1 T x I 1 39,863 Er ror 214- 3,439 *'A si gnif ic ant difference (5 percent level) is indicated. *4?he.re is no significant difference between yields of inoc u l a t e d and u n i n o c u l a t e d tappings according to t rea tm en t of the taphole. “^ A h i g h l y signi fic ant difference (1 percent level) is Indicated. 63 T R E A T M E N T - S T ERILITE POWDER Total t • Source M e a n Squares 27 T r eatment 1 Inoculation 1 8,335 1 T x I 1 18,995 2 Error 22p ^ ‘‘A h i g h l y significant difference Is Indicated. 60,162 5 ,138 (1 percent level) There is no significant difference between yields of inocu lat ed and u ninoculated tappings according to the treatment of the t a p h o l e . p “^There is no significance indicated in the inter­ a c tion b e t ween treatment and inoculation. T R E A T M E N T ■- o.-SILVER SOLUTION Source Total D. F. M e a n Squares 27 T rea tm en t 1 Ino c ulation 1 26,666 T x I 1 3,793 Error Pip 2,916 89k 1 ^There Is no significant difference between yields of t r e ated and untre a t e d tappings in either inoculated or u n i n o c u l a t e d tappings* h i g h l y si gnificant difference (1 percent level) is indicated. 2There is no significance in the interaction between t r eatment and inoculation. TREATMENT - PLASTIC TUBING WITH AND WITHOUT SILICONE COATING Source Total I). F. Mean Squares 27 Treatm ent 1 13,91? Goat ing 1 18,777 T x C 1 1,537 Error 2b 10,260 ■'"There is no significant difference between yields of treated and untreated tappings* p There is no significant difference between yields of treatments with silicone coating and treatments without silicone coating.